ECB4 Media Guide

7/26/2019 ECB4 Media Guide

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Bruce Alberts

Dennis Bray

Karen HopkinAlexander Johnson

Julian Lewis

Martin Raff

Keith Roberts

Peter Walter

The teaching and learning resources for instructors and students areavailable online. The instructor’s resources are password protected andavailable only to qualified instructors. The student resources are avail-able to everyone. The resources can be accessed from:

MEDIA GUIDE

FOURTH EDITION

ESSENTIAL

CELL BIOLOGY



2

INSTRUCTOR RESOURCESwww.garlandscience.com/instructors

The Art of Essential Cell Biology , Fourth Edition -mats. The images have been optimized for displayed on a computer. Thesearch feature on the website allows the figures to be searched by figurename, figure number, or by keywords in the figure legend from the book.

Figure-Integrated Lecture OutlinesFor instructors who would like a head start creating lectures for theircourses, the section headings, concept headings, and figures from the texthave been integrated into PowerPoint presentations.

Animations and VideosThere are over 130 animations and videos available to both students and -mats: WMV and QuickTime. The WMV-formatted movies may be used byinstructors who wish to integrate the movies into PowerPoint presenta-tions on Windows® computers; the QuickTime formatted movies may be

used in PowerPoint for Apple computers, or in Keynote® presentations.The movies can easily be downloaded to your computer using the “down-load” button on the movie preview page.

Question BankThe question bank, written by Linda Huang, University of Massachusetts, formats. These formats include multiple choice, fill-in-the-blank, true-false, approximately 60-70 questions, a number of which will be suitable for usewith personal response systems (that is, clickers). The text of the questionbank is available in Microsoft Word® format. The question bank is also

available pre-loaded into computerized test-generation software. The testgeneration software can also be used to upload the contents of the ques-tion bank into a learning management system.

References -erence and suggested reading assignments. They have been adapted fromthe detailed references of Molecular Biology of the Cell and organized by thetable of contents for Essential Cell Biology. is available on both the instructor and student websites.

Medical Topics Guide

This document highlights medically relevant topics covered throughout thebook, and will be particularly useful for instructors with a large number ofpremedical, health science, or nursing students.

Blackboard® and LMS IntegrationThe movies, book images, and student assessments that accompany - garland.com.



Adobe and Acrobat are either registered trademarks or trademarks of AdobeSystems Incorporated in the United States and/or other countries.

PowerPoint and Windows are trademarks of Microsoft Corporation in the UnitedStates and/or other countries.

Mac OS X , QuickTime, and iPod are registered trademarks of Apple Inc.

STUDENT RESOURCESwww.garlandscience.com/ECB4-students

Animations and Videos

There are 130+ movies that cover a broad range of cell biology topics.The movies review key concepts from the book and illuminate the cel-lular microcosm.

Student Self-Assessments

The website contains a variety of tools designed to help students assesstheir understanding of the material covered in the book:

comprehension.

number of animations and respond to specific questions about theconcepts and molecular details..

available in the book.

of conceptual understanding or to think from an experimentalperspective.

Cell Explorer

This application contains interactive micrographs that highlight impor-tant cellular structures.

Flashcards

The key terms presented at the end of each chapter can be reviewedusing the flashcards application.

Glossary

The complete glossary from the book is available on the website; termsin the glossary can be searched or the entire glossary can be browsed.

References

A set of references is available for each chapter for further reading andexploration.



4

INTRODUCTION

The Essential Cell Biology Student Websitewww.garlandscience.com/ECB4-students

As never before, new imaging and computer technologies have increasedour access to the inner workings of living cells. We have tried to capturesome of the excitement of these advances on the Essential Cell Biologywebsite. The site contains over one hundred and fifty video clips, ani-mations, molecular structures, and high-resolution micrographs—alldesigned to complement the material in the individual book chapters.Nearly all items are accompanied by a short narration that introducesand explains key concepts. Our intent is to provide students and instruc-tors with an opportunity to observe living cells and molecules in action,

dynamics of the cellular and molecular world.One cannot watch cells crawling, dividing, segregating their chromo-somes, or rearranging their surface without a sense of wonder at themolecular mechanisms that underlie these processes. We hope that the Essential Cell Biology website will motivate and intrigue students whilereinforcing basic concepts covered in the text, and thereby will make thelearning of cell biology both easier and more rewarding. We also hopethat instructors can use these visual resources in the classroom to illu-minate, not only the course material, but also the beauty and wonderof this microcosm. We designed animations to bring to life some of themore complicated figures in the book. Many of the videos provide vis-ual demonstrations of topics that can be difficult to appreciate, such asmembrane fluidity, and the high-resolution micrographs allow studentsto explore some magnificent cell images in detail. We have also createdthree-dimensional models of some of the most interesting molecules,presented in short tutorials.

The contents of the Essential Cell Biology website represent the work ofnumerous laboratories around the world that provided video clips fromoriginal research, animation segments, micrographs, and moleculardata. We are deeply indebted to the scientists who generously made thismaterial available to us.

Using the Movie Callouts

-

outs” that directly link the content of the book to movies on the EssentialCell Biology website. The movie callouts are integrated throughout thebook to indicate when relevant movies are available on the EssentialCell Biology website. The callouts are similar to the callouts for figuresin the text, except that they indicate a particular movie is available. Themovie callouts and figure callouts are in different colors and are easy todistinguish.

The website contains a field where you can enter the movie number field, that particular movie will play. You can also browse all the media bychapter, but we hope this feature will allow students to easily integratethe media resources on the website into study with the book.

ESSENTIAL CELL BIOLOGY WEBSITE VIEWING GUIDE



ECB4 WEBSITE TABLE OF CONTENTS

Chapter 1 The Fundamental Units of Life 11

1.9 Chlamydomonas

Chapter 2 Chemical Components of Cells 18

2.2 Palmitic Acid 182.3 ATP 18

Chapter 3 Energy, Catalysis, and Biosynthesis 21

Chapter 4 Protein Structure and Function 24



6

Chapter 5 DNA and Chromosomes 33

Chapter 6 DNA Replication, Repair, and Recombination 36

6.7 Holliday Junction 39

Chapter 7 From DNA to Protein: How Cells Read the Genome 42



Chapter 8 Control of Gene Expression 49

Chapter 9 How Genes and Genomes Evolve 52

Chapter 10 Modern Recombinant DNA Technology 54

Chapter 11 Membrane Structure 57

11.8 The Lazer Tweezers 60

Chapter 12 Transport Across Cell Membranes 63

12.1 Aquaporins 6312.2 Na+ - K+ 12.8 Action Potentials 67 12.10 Optogenetics 68



8

12.11 Neurite Outgrowth 6912.12 Neuronal Pathfinding 69

Chapter 13 How Cells Obtain Energy From Food 72

Chapter 14 Energy Generation in Mitochondria and Chloroplasts 78

Chapter 15 Intracellular Compartments and Protein Transport 85

15.9 Phagocytosis 90

15.13 Pancreas: View 1 91



Chapter 16 Cell Signaling 94

16.8 Lymphocyte Homing 98

Chapter 17 Cytoskeleton 100

in vivo 101

17.5 Organelle Movement on Microtubules 10217.6 Kinesin 102 17.10 Myosin 10517.11 Listeria Parasites 10517.12 Heart Tissue 106

Chapter 18 The Cell-Division Cycle 110

18.8 Mitosis 11318.9 Apoptosis 113



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Chapter 19 Sexual Reproduction and the Power of Genetics 116

19.1 Meiosis 116

Chapter 20 Cell Communities: Tissues, Stem Cells, and Cancer 119

20.1 Wound Healing 119 20.3 Drosophila 20.5 Megakaryocyte 121

20.9 Angiogenesis 122



1

1.1 Developing Egg Cells

This frog egg cell has been fertilized and starts dividing. The first cell

same time divide and develop in almost perfect synchrony.

After a day or two, embryonic development is completed and tadpoleshatch from the eggs.

1.2 Keratocyte Dance

Keratocytes, found on the scales of fish, are specialized for very rapidmotility in order to heal scratches.

Video editing and concept: Justin Reichman

Music: Freudenhaus Audio Productions (www.fapsf.com)

1.3 Crawling Amoeba

This single-celled amoeba crawls around by using actin polymerizationto push out pseudopods, or false feet, to explore new territory. At the

same time, organelles move in complex patterns within the cell.

Reproduced by copyright permission of CELLS alive!, CDROM and Video Library,1998–2001.

From "From Egg to Tadpole"

Jeremy Pickett-Heaps and JuliannePickett-HeapsCytographics (www.cytographics.com)

Mark S. CooperUniversity of Washington

CELLS alive!www.cellsalive.com



12

1.4 Cytoplasmic Streaming

Elodea densa, chloroplasts andmitochondria move in a constant stream along cytoskeletal tracks.Although the complex network of the cytoskeleton cannot be seen, afew of the cytoskeletal tracks are visible.

This circulation of the cytosol and organelles within the cell is calledcytoplasmic streaming. The rate of streaming is affected by exposure to

light, temperature, and pH. many of the cells.

1.5 Cytoplasmic Crowding

E. coli , wesee the cell wall surrounding the cytoplasm; the cytoplasm itself, which

concentrated in the lightly stained region. electron microscopy cannot clearly resolve the individual molecules ina densely packed cell, nor show them in motion, computer simulationshave been developed to visualize the molecular environment.

Zooming in to a small portion of the cytoplasm, this computersimulation shows 50 different types of the most abundantmacromolecules in E. coli with one another, and are swept to and fro by random thermal motion. bump into other molecules. The larger molecules stay in place relativeto the smaller ones, but continuously move as they are pushed around

and interact with other molecules in this densely packed microcosm.

motion in the cytoplasm; in real time, it would be impossible to followthe slowed-down movements shown here.

The empty spaces in this environment are filled by water moleculeswhich were omitted from the simulation.

Kristina Yu © Exploratoriumwww.exploratorium.edu

Adrian ElcockUniversity of Iowa Carver College of Medicine



1

Richard E. TriemerRutgers, State University of New Jersey

Doug BrayThe University of Lethbridge, Canada

Brian Oates and Cyprien LomasThe University of British Columbia

1.6 Swimming Eutreptiella

flagellate, which uses both flagella and pronounced cell shape changesto swim.

1.8 Plant Cells

1.7 Beating Heart Cell

the cell contracts, it pulls on the substratum which becomes wrinkled.Although individual heart cells can beat with their own rhythms, theyare coordinated in an intact heart so that all cells beat synchronously.

Reproduced from: CELLebration, 1995, edited by Rachel Fink, produced anddistributed by Sinauer Associates, Inc., by copyright permission of Barbara Danowski.

Barbara DanowskiUnion College

Kyoko Imanaka-YoshidaMie University

Jean Sanger and Joseph SangerUniversity of Pennsylvania School ofMedicine



14

1.9 Chlamydomonas


1.10 Liver Cell: View 1


reticulum







1












16

1.15 Quiz: Chapter 1

1.16 Concept Questions: Chapter 1

One theory is that an ancestral eukaryotic cell was a predator that fedby capturing other cells...

1.17 Challenge Question: Chapter 1

characteristic of life. The first Mars probe analyzed soil samples...

Image courtesy of Ira Herskowitz andEric Schabatach.



1

1.18 Flashcards: Chapter 1

archaea

environments such as hot springs or concentrated brine. ( Seealso bacteria.)

bacteria (singular bacterium)

disease.

1.19 References: Chapter 1

Science

carrying information from genes to ribosomes for protein synthesis. Nature 190:576–581.

archaeonbacteriumcellchloroplastchromosomecytoplasm

cytoskeleton

KEY TERMS



18

2.1 Glucose

A glucose molecule is a six carbon sugar, consisting of a total of

hydrogen bond.

hydrogen in white.

2.2 Palmitic Acid

This molecule of palmitic acid contains a long tail of 16 carbons bonded overall hydrophobic character.

very polar.

2.3 ATP

ATP molecules store and supply energy for cellular processes. An ATPmolecule contains three building blocks: the flat purine ring system

containing multiple nitrogen atoms shown in blue, the ribose sugar inthe middle, and the three phosphate groups with the phosphorus atomsshown in yellow.



1

2.4 Noncovalent Interactions

Molecules in solution undergo random thermal movements and mayencounter each other frequently if the concentration is sufficiently high. bonds will form between them. Thermal motion of the molecules rapidlybreaks these bonds apart, and the molecules separate.

will form between the two. The bonds hold the molecules together for a

Tightly bound molecules will spend most of their time associatedalthough they will go through cycles of association and dissociation.The affinity of the two molecules for one another is a measure of therelative time they spend bound together.

Storyboard and Animation: Sumanas, Inc. (www.sumanasinc.com)

2.5 Quiz: Chapter 2

1. What is the smallest particle of an element that still retains its


bonds, covalent bonds, hydrogen bonds, van der Waals attractions,

another.



20

2.7 Challenge Questions: Chapter 2

32 3.

A. How many 12-oz (355-mL) bottles of 5% beer could a 70-kg ethanol, and assume...


acid

dissociation generates hydronium (H3O+) ions, therebylowering the pH.

amino acid

carboxyl...


Atkins PW (1996) Molecules. New York: WH Freeman.

acidamino acidatomatomic weightATPAvogadro’s numberbase

KEY TERMS



2

3.1 Analogy of Enzyme Catalysis

react in four different ways.

To undergo any of these reactions, the molecule must overcome anactivation energy barrier that is of a characteristic height for each of thepossible reactions.

This can be achieved, for example, by putting more energy into the

molecule will enter many different reaction paths by overcoming similaractivation energy barriers.

An enzyme, in contrast, reduces the activation energy barrier of onlyone specific reaction path. An enzyme therefore allows reactions toproceed at normal temperatures and directs them into one desiredpathway.

3.2 Random Walk

allows them to diffuse through cells in a random walk. Our simulationshows the degree to which a small sugar molecule on the left and alarger protein on the right explore the interior space of a cell, hereshown as a cube with a 10 micrometer side. The animation representsone second in real time.

Note that the paths of the molecules in the second simulation aredifferent from those in the first, thus showing the randomness of thismotion.

Final composition: Blink Studio Ltd. (www.blink.uk.com)

3.3 Quiz: Chapter 3



22


example of each.

generate energy for the cell; anabolic pathways use energy to drive thesynthesis of…


The organic chemistry of living cells is said to be special for tworeasons: it occurs in an aqueous environment and it accomplishes some different from the organic chemistry carried out in the top laboratories


transferable acetyl group to many metabolic reactions,including the citric acid cycle and fatty acid biosynthesis; the

acetyl CoAactivated carrieractivation energyADP, ATPanabolismbiosynthesiscatabolism

KEY TERMS



2




24

4.1 Viewing Proteins: SH2

Protein structures can be displayed in many different ways. A small the different ways in which a protein structure can be displayed. Thebackbone view shows the path of the polypeptide chain. The chain is

The ribbons view accents helices and sheets. These secondary

structure elements determine the fold of most polypeptide chains. and helices are shown as twisted cylinders.

wireframe presentation, the covalent bonds between all of theatoms in the polypeptide are shown as sticks.

A spacefill view depicts each atom in the polypeptide as a solid sphere.The radius of the sphere represents the van der Waals radius of theatom. The coloring scheme follows the same rainbow spectrum used

blue, oxygen red, and sulfur yellow.

Molecular modelling and animation: Timothy Driscoll, Molvisions

Chime conversion and QuickTime production: Sumanas Inc. (www.sumanasinc.com)

4.2 Helix

The helix is one of the most common secondary structures in backbone that forms the core of the helix. The chain is stabilized in this

conformation by hydrogen bonds between the backbone amino groupof one amino acid and the backbone carbonyl group of another aminoacid that is four positions away. These interactions do not involve sidechains. Thus many different sequences can adopt an helical structure.

helices are regular cylindrical structures. One full turn occurs every3.6 residues and extends the length of the helix by approximately0.5 nm.

ribbon follows the path of the peptide backbone.



Source: Glactone (www.chemistry.gsu.edu/glactone)

PDB ID number *: 1SHA

PDB ID source: Glactone



2

4.3 Coiled-Coil

helices wrap around each other to form astable structure. One side of each helix contains mostly aliphatic aminoacids, such as leucines and valines, while the other side contains mostlypolar residues. Helices containing distinct hydrophobic and polar sides aligned so their hydrophobic sides snuggle tightly together in the center,with their polar faces exposed to the solvent.

A triple coiled-coil is another stable structure formed by this case, three amphipathic helices twist around a central axis. Thehydrophobic sides of all three helices face the center of the coil, creatinga stable hydrophobic core.

in blood clotting. The fibrous nature of this protein is intimately relatedto its ability to form clots.



4.4 Sheet

The an helix, it is formed by hydrogen bonds between backbone atoms strands. These

interactions do not involve side chains. Thus, many different sequencescan form a sheet.

A sheet is a regular and rigid structure often represented as a series of the example shown here the two middle strands run parallel—that is, inthe same direction—whereas the peripheral strands are antiparallel.

The amino acid side chains from each strand alternately extendabove and below the sheet, thereby allowing each side to havedistinct properties from the other. sheets are usually twisted and notcompletely flat.



PDB ID number *: GCN4 leucine zipper(2ZTA); Trimeric coiled-coil domain ofchicken cartilage matrix protein (1AQ5);Native chicken fibrinogen (1EI3)

PDB ID number *: 1PGB



26

4.5 Oligomeric Proteins

Many proteins are composed of multiple polypeptide chains, or small sheets—one from each subunit—zipper up and form a larger sheet.

The enzyme neuraminidase is composed of four identical subunits

to-tail fashion, by repeated use of the same binding interaction. Thisbecomes clear when the polypeptide chains are colored in a rainbowpattern, so that the same regions of each subunit have the same colors.All subunits adhere to each other through contacts between the orangeand light-blue regions.

composed of two subunits and two closely related subunits. Oxygen subunit can sense whether neighboring subunits contain bound oxygen.The protein subunits therefore communicate with one another throughthe interfaces that hold them together.

The tumor suppressor protein p53 is a tetramer of four identical composed of a single strand connected to an helix. The tetramericform of p53 assembles as a dimer of dimers. Two copies of p53 interactvia strands, forming a twostranded sheet. Two such dimers interactvia their helices to form the tetrameric assembly.



4.6 Disulfide Bonds

the center of a protease inhibitor. Most extracellular proteins containdisulfide bonds.



PDB ID number *: Cro repressor proteinfrom bacteriophage lambda (5CRO);Neuraminidase of influenza virus (1NN2);Deoxy human hemoglobin (1A3N); p53tetramerization domain (1C26)

PDB ID number *: 1BI6



2

4.7 Antibodies

that circulate in the blood stream. They bind to and inactivate foreign molecule consists of two light chains and two heavy chains. The heavychains have carbohydrates attached. The regions of the antibody thatbind to antigens are located at the very tips of the two arms.

the variable domains, contributed by the heavy and light chains, andhence called VH and VL . The variable domains are attached to twoconstant domains, again one each from the heavy and light chains, and H L .

Variable and constant domains share a similar structure, called the strands and one with five. A single covalent disulfide bridge holds thetwo sheets together, which results in a rigid and very stable domain.

As their name implies, the variable domains vary in amino acidsequence from one antibody molecule to another, thus providing thevast diversity in structure required by the immune system. The antigen

binding site in the variable domains is composed of hypervariable variations in the hypervariable loops are responsible for the specificityof antibodies to particular antigens.

Antigens bind to the tip of each antibody arm, generally two molecules antibody via a large contact surface, providing a tight and highly specificassociation.



PDB ID number *: Compilation ofimmunoglobin G1 & immunoglobin Fc andfragment B of Protein A complex (2IG2 &1FC2); FabD1.3-lysozyme complex (1FDL)

4.8 Lysozyme Reaction

The lysozyme enzyme cleaves polysaccharide chains. First, the enzymeand substrate associate, forming an enzyme-substrate complex. Theenzyme catalyzes a hydrolysis reaction that cleaves the substrate intoproducts, which are quickly released, allowing the enzyme to catalyzeanother reaction.

The cleft in the enzyme holds six sugar residues of a polysaccharide.The hydrolysis reaction occurs between residues.

Looking at the details of the reaction in solution, the sugar residuesadopt their most stable three-dimensional conformation. However,after the polysaccharide enters into the enzyme–substrate complex, theenzyme forces the sugar shown on the left into a strained conformationthat more closely resembles the transition state of the reaction.

Two amino acids within the enzyme facilitate the reaction. A glutamicacid donates a proton to sugar on the right and an aspartic acid attacks results in a transient covalent bond between the sugar and the aminoacid, and hydrolysis of the sugar-sugar bond



28

The deprotonated glutamic acid then polarizes a water molecule,drawing a proton away from it. This allows the water oxygen to attack

forming the enzyme-product complex. The enzyme and productsdissociate.

Storyboard and Animation: Sumanas, Inc., (www.sumanasinc.com)

4.9 Lysozyme Structure

Lysozyme is a small enzyme that binds to polysaccharide chains and domain is composed mostly of helices, while the other domain iscomposed mostly of strands. The interface between the two domainsforms a cleft in which the substrate binds. The structure shown herecontains one of the products of the hydrolysis reaction.

Lysozyme acts as a catalyst by adding a molecule of water to the bondbetween two sugars, breaking the bond. This reaction is catalyzed bytwo strategically positioned amino acid side chains in the enzyme’sactive site: glutamate 35 and aspartate 52. The highlighted group on thereaction product shown here would have formed the bond cleaved inthe reaction.



4.10 EF-Tu

each of its domains in a different color.

An important dynamic element in the structure of elongation factor Tu

is the switch helix. helix rearranges.

Tu.

Animation: Graham Johnson, Fivth Element (www.fivth.com)

PDB ID number *: 1LSZ



2

4.11 The ‘Safe Crackers’

assemblies,

in which their individual activities may be coordinated.

subunits,

such as the hydrolysis of ATP, lead to orderly movements throughout the

protein complex that accomplish a specific task.

4.12 Sickle Cell Anemia

oxygen transport molecule in the blood. The disease gets its name from blood cells become sickle-shaped and these elongated cells get stuck insmall blood vessels so that parts of the body don’t get the oxygen they This in turn alters one of the amino acids in the hemoglobin protein.Valine sits in a position where glutamic acid should be. The valinemakes the hemoglobin molecules stick together when oxygen tension islow, forming long fibers that distort the shape of the red blood cells, and

this brings on an attack.

Animation produced for DNA Interactive (www.dnai.org) © 2003 Howard Hughes

Medical Institute (www.hhmi.org) All rights reserved.

4.13 Anatomy of a PDB File

Three dimensional structures of macromolecules—that have been

the authors who solved the structure and experimental details of theanalysis. Next, the file lists the amino acid sequence of the protein.

a separate line. The first few columns define the atom as part of aparticular amino acid in the sequence. The later columns list a set ofx, y, and z coordinates that precisely locate the atom in the structure. files and use the coordinates to build three dimensional models on your

Original illustrations and storyboard:Nigel Orme and Christopher Thorpe

Graham JohnsonFivth Element (www.fivth.com)



30

PDB ID number *: MHC class I molecule(1A1M); Human T-cell receptor, viral peptideand Hla-A 0201 complex (1AO7)

4.14 MHC Class I: Protein Form and Function

peptides, or antigens, derived from normal cell proteins. Peptide- complex has two subunits. The smaller subunit, 2 microglobulin,resembles an immunoglobulin domain. The larger a subunit also hasan immunoglobulin-like domain which is linked to a head domain

containing the antigen-binding groove.

walls composed of long helices that rest on a floor composed of aneight stranded sheet. The peptide on display fits snugly between thehelices in the groove.

The peptide backbone is bound at both ends by highly conserved downwards into specific binding pockets in the groove, while other T cells.

a peptide foreign to the immune system, the T cell is activated by this complexed with a T-cell receptor reveal the exquisite precision withwhich the interacting surfaces fit together.





3

4.15 MHC Class II: Protein Form and Function

proteins are composed of two subunits that contribute to the structureof the head domain containing the antigen-binding groove.

proteins. As seen in this comparison, the different shape of the antigen

presenting cells and activate a different class of immune cells, calledhelper T cells.



PDB ID number *: Compilation of MHC classI molecule & MHC class II/superantigencomplex (1A1M & 2SEB)



The primary structure of a protein is its amino acid sequence. Aminoacids are held together in a chain by peptide bonds. This polypeptidechain can then fold into different shapes; these patterns constitute…



32


schematically in the figure below. Using the principles of feedbackinhibition, propose a regulatory strategy for this pathway that ensures


active site

molecule and catalyzes its chemical transformation.

allosteric


Science 181:223–230.

Genome Biol6:106–109.

active siteallosteric helixamino acid sequenceantibodyantigen sheet

KEY TERMS

Image courtesy of Robert Grant, StephanCrainic, and James M. Hogle



3

5.1 DNA Structure

backbone composed of phosphates and sugars to which the bases areattached. The bases form the core of the double helix, while the sugar–phosphate backbones are on the outside. The two grooves between the minor groove is too narrow.

polar.

A cyclic base is attached to each sugar. The bases are planar and extendout perpendicular to the path of the backbone. Pyrimidine bases are stacking contributes significantly to the stability of the double helix.

interactions, guanine always pairs with cytosine, and thymine withadenine.

always pairing with T—ensures that the two strands are complementary.



5.2 Chromosome Coiling

fit into the nucleus of every cell. The process starts with assembly ofa nucleosome, which is formed when eight separate histone protein these then stack on top of each other. The end result is a fiber of packed

nucleosomes known as chromatin.

Animation produced for DNA Interactive (www.dnai.org) © 2003 Howard HughesMedical Institute (www.hhmi.org) All rights reserved.

PDB ID number *: 132D



34


5.4 Quiz: Chapter 5



5.5 Media Assessment: DNA Structure

sugars.



3


nucleosomes. The nucleosomes are then pulled together into a compactchromatin fiber by interaction with the linker histone H1…


the figure below, along with their points of attachment to the sugar-phosphate backbones.

helix on these representations.


base pair

cell cycle


A structure for deoxyribose nucleic acids. Nature 171:737–738.

chromatin. Trends Biochem Sci 30:680–687.

base paircell cyclecentromerechromatinchromatin-remodeling complexchromosomecomplementary

KEY TERMS



36

6.1 DNA Polymerase

sequence of the template strand, colored yellow, to select each newnucleotide to be added to the 3 end of a growing strand, colored gray. differently.

end of the

growing strand, the blue finger domain of the polymerase moves inwardto correctly position the nucleoside triphosphate. A pyrophosphategroup is released when each nucleotide is added.

shown with the incoming nucleoside triphosphate and the templatenucleotide in light blue. The growing strand is green, and the templatestrand is red. When the finger domain moves inward, the nucleosidetriphosphate is tested for its ability to form a proper base pair with thetemplate nucleotide. When a base pair forms, the active site residuescatalyze the covalent addition of the new nucleotide to the 3 hydroxylgroup on the growing strand, and the entire process repeats at speedsup to 500 nucleotides per second.

On rare occasions, approximately once every 10,000 nucleotideadditions, the polymerase makes an error and incorporates a nucleotidethat does not form a proper base pair onto the end of the growingstrand. When this occurs, the polymerase changes conformation,and transfers the end of the growing strand to a second active site onthe polymerase, where the erroneous, added nucleotide is removed.The polymerase then flips back to its original conformation, allowing polymerase will make a mistake only about once every 107 to 108 nucleotide pairs.



6.2 DNA Helicase

Helicases separate nucleic acid duplexes into their component strandsusing energy from ATP hydrolysis.

T7, reveals an hexagonal arrangement of six identical subunits.

A model for the mechanism of how the enzyme might work explainsthis structural asymmetry. Of the six potential ATP binding sites, two phosphate, and two are empty. These three states may interconvert ina coordinate fashion as ATP is hydrolyzed, creating a ripple effect thatcontinuously runs around the ring.

and down, as seen in this cross section. The oscillating loops might pull in the process.

A frontal view shows the full dynamics of this fascinating proteinmachine.

Dale B. Wigley and Martin R. SingletonImperial Cancer Research Fund

Tom EllenbergerHarvard Medical School

Michael R. SawayaUniversity of California, Los Angeles

PDB ID number *: Human DNA polymerasebeta complexed with gapped DNA (1BPX);Compilation of human DNA polymerase betacomplexed with gapped DNA & human DNApolymerase beta complexed with gappedDNA and ddCTP (1BPX & 1BPY)



3

PBD ID number *: 1QE4

Original illustrations and storyboard:Nigel Orme and Christopher Thorpe

6.3 Sliding Clamp

relates to its function in a most intuitive way.



6.4 Replication I

Using computer animation based on molecular research, we are able assembly line of amazing miniature biochemical machines that are strands. One strand is copied continuously and can be seen spoolingoff to the right. Things are not so simple for the other strand because

Animation produced for DNA Interactive (www.dnai.org) © 2003 Howard HughesMedical Institute, (www.hhmi.org) All rights reserved.

6.5 Replication II

lagging strand.

the clamp.



38

Okazaki fragment, and the entire cycle repeats.

Music: Christopher Thorpe

6.6 Telomere Replication

hydroxyl group, the replication machinery builds the lagging strand by -hydroxyl groups atregular intervals along the lagging strand template.

Whereas the leading strand elongates continuously in the 5-to-3 direction all the way to the end of the template, the lagging strand stopsshort of the end.

chromosome, the lagging strand would not be complete.

The final primer would provide a 3 the primers would later need to be removed. The 3 -hydroxyl groups on -OH

progressively shorten during each replication cycle. This “end-replication” problem is solved by the enzyme telomerase. The ends Telomerase recognizes the tip of an existing repeat sequence. Using an

strand in the 5-to-3 direction, and adds additional repeats as it movesdown the parental strand.

information at the ends of linear chromosomes is completely copied in




3

Electron microscopy:

David DresslerUniversity of Oxford

Huntington PotterUniversity of South Florida

Molecular animation:

David A. WallerAstbury Centre for Structural Molecular

Biology, University of LeedsDavid Rice, Peter Artymiuk, John Rafferty,and David HargreavesKrebs Institute, University of Sheffield

6.7 Holliday Junction

of helicase proteins, seen here in the background, which use ATP

animation.

6.8 Quiz: Chapter 6

that were originally in the parent cell, while the double helix inthe other daughter cell consists of two newly made strands.

parental strand and one newly made strand.



40

6.9 Media Assessment: Replication Fork


reasoning.

replication at a replication origin; once a replication fork has been


next nucleotide, how is it that any mismatched nucleotides “escape” the



4


fragments.

collective term for the enzymatic processes that correct…


evolutionary rates and constraints in three mammalian genomes.Genome Res

spontaneous mutation. Nature Rev Genet

cancerDNA ligaseDNA polymeraseDNA repairDNA replicationhomologous recombination

lagging strand

KEY TERMS



42

7.1 RNA Structure

by the backbone.

The backbone is composed of repeating ribose sugars and phosphategroups.

Unlike the 2 attached to the 2 carbon. This ‘extra’ hydroxyl group influences the

adenine pairs with uracil instead of thymine. Uracil is never used in Th A-U pair like the A-T pair has two hydrogen bonds.

folds back onto itself.

single-stranded loop, in contrast, are either exposed or engaged innonstandard interactions.



7.2 Transcription

shown stretching away to the left. The assembled factors include an it copies one of the two strands. The yellow chain snaking out of the

replaced with the closely related base uracil, commonly abbreviated “U.”You are watching this process, called transcription, in real time.

Animation produced for DNA Interactive (www.dnai.org) © 2003 Howard HughesMedical Institute (www.hhmi.org) All rights reserved.

PDB ID number *: RNA duplex containinga purine-rich strand (1RRR); RNA tetraloop(1AFX)



4

PDB ID number *: 1I6H

PDB ID number *: TATA element ternarycomplex (1VOL); Compilation of TATAelement ternary complex & Gal4 complexwith DNA (1VOL & 1D66)

7.3 RNA Polymerase II

active site of the enzyme lies at the interface between the two largest was co-crystallized. New nucleotides would be continually added to the3

surface.

surface all the way to the active site. The nucleotide triphosphates used pore.



7.4 TATA-Binding Protein

promoter region of a gene. A crucial part of this initiation process isthe recognition and binding of the TATA sequence, a short stretch of

binding protein.

ninety degrees and is thought to provide a signal to assemble the rest ofthe transcription complex at the initiation site.





44

7.5 RNA Splicing

after transcription.

intron sequences. A few short nucleotide sequences provide the cellwith cues of what to remove. The elaborate molecular machine thatcarries out this task is called the spliceosome.

splice site by forming splice

Now in position, a conserved adenine nucleotide in the intron attacksthe 5 end of the intron covalently bonds to the adenine nucleotide forming alariat structure.

The spliceosome rearranges to bring together the exons, allowing the3 hydroxyl group of the first exon to react with the 5 end of the other. released and degraded.


7.6 tRNA

the 3 end at the tip of the shorter arm. The anticodon loop is positionedat the opposing end of the molecule and contains the anticodon base

triplet.The amino acid, a phenylalanine in this case, is covalently attached to terminus of all

The anticodon is comprised of three nucleotides complementary to

an extensive contact surface that includes recognition sites for the enzyme.



PDB ID number *: Phe-tRNA, elongationfactor Ef-Tu: Gdpnp ternary complex (1TTT);

Yeast aspartyl-tRNA synthetase (1ASY)



4

7.7 Translation I

the cytosol. Then in a dazzling display of choreography, all the to the ribosome. The amino acids are the small red tips attached to the

each of them carrying a three-letter nucleotide code that is matched to for each amino acid is read off, three letters at a time, and matched to the amino acid it carries is added to the growing protein chain. You arewatching the process in real time. After a few seconds the assembled bone marrow churn out a hundred trillion molecules of it per second!

And as a result, our muscles, brain and all the vital organs in our bodyreceive the oxygen they need.

Animation produced for DNA Interactive (www.dnai.org) © 2003 HowardHughesMedical Institute (www.hhmi.org) All rights reserved.

7.8 Translation II

To extend a growing polypeptide chain the ribosome must select the

is committed to be used in protein synthesis.

The ribosome catalyzes the formation of the new peptide bond and ribosome back to the state in which it can accept the next incoming



46

7.9 Translation: Atomic View

The crystal structure of the ribosome reveals many insights into themolecular mechanism of translation.

Zooming in on the large ribosomal subunit shows highly evolutionarily center, which catalyzes polypeptide bond formation. There are noribosomal proteins in the vicinity; peptide bond formation is catalyzed

site. This amino acid represents the carboxy-terminal amino acid of agrowing polypeptide chain on an actively translating ribosome with the position the amino acid precisely.

again held precisely by base pairing interactions between a conserved positions the reactive groups with the precise geometry required tocatalyze peptide bond formation.

now containing the growing polypeptide chain, moves from the A- tothe P-site, where it will be waiting for the next incoming amino acid torepeat the polymerization cycle.

The different states of the reaction cycle shown in this animation arebased on actual crystal structures, in which large ribosomal subunits mimicking the discrete steps in the reaction cycle.

T. Martin SchmeingThomas A. SteitzHoward Hughes Medical Institute, YaleUniversity

7.10 Polyribosome

to the cytoplasm, ribosomes begin to translate the sequence into amino

toward the 3 end. New ribosomes attach to the 5 end at the same rateas the previous ones move out of the way. These multiple initiationsallow the cell to make much more protein from a single message thanif one ribosome had to complete the task before another could begin.When a ribosome reaches a stop codon, the ribosome and the new micrograph depicts a membrane-bound polyribosome from a eucaryoticcell.


Electron Microscopy:

John HeuserWashington University in St. Louis



4

7.11 Ribosome Ratchet

the significant conformational changes that the ribosome is thought toundergo during each elongation cycle. The ratchet-like rearrangementsat the interface between the two ribosomal subunits may help move the

The models shown here are a computer reconstruction made frommany thousands of images of single ribosomes in vitreous ice that wereobserved with an electron microscope.

Animation: Amy Heagle Whiting, Howard Hughes Medical Institute, Health ResearchIncorporated at the Wadsworth Center, State University of New York at Albany

Final composition: Graham Johnson, Fivth Element (www.fivth.com)

Funded, in part, by NIGMS and NCRR, National Institutes of Health


7.13 Media Assessment: Translation

Joachim Frank and Rajendra K. AgrawalHoward Hughes Medical InstituteHealth Research Incorporated at theWadsworth Center, State University of New

York at Albany



48




alternative splicing


innovation. Cell 152(6): 1218-21.

Curr Opin Cell Biol 17:251–256.

Image courtesy of Ulrich Scheer

alternative splicingaminoacyl-tRNA synthetaseanticodoncodonexongenegene expression

KEY TERMS



4

8.1 Homeodomain

Homeodomains are found in many transcription regulatory proteins and overlapping helices packed together by hydrophobic forces. Helix 2

Amino acids in the recognition helix make important, sequence-specific

Three side chains from the recognition helix form hydrogen bonds with that forms when two neighboring electronegative atoms, like oxygenand nitrogen, share a single hydrogen.

protein contacts bases in the minor groove.



8.2 Zinc Finger Domain

binding proteins. They use centrally coordinated zinc atoms as crucialstructural elements. The zinc atom is coordinated by two cystine

residues from the sheet and two histidine residues from the a helix.A single zinc finger domain is only large enough to bind a few bases of

different zinc finger motifs allows precise control over the sequencespecificity of the protein.


Chime conversion and QuickTime production: Sumanas Inc. (www.sumanasinc.com

PBD ID number *: 1APL

PDB ID number *: Zinc finger DNA-bindingdomain (1ZNF); Zif268-DNA complex (1ZAA)



50

8.3 Leucine Zipper

A leucine zipper domain is comprised of two long, intertwined helices. Hydrophobic side chains extend out from each helix into thespace shared between them. Many of these hydrophobic side chainsare leucines, giving this domain its name. A spacefilling view revealsthe tight packing of side chains between the leucine zipper helices; thismakes the domain especially stable.

The specific interactions between side chains and bases are hydrogen guanine base.



8.4 Quiz: Chapter 8

1. Which of the following statements is not true about the differences

PDB ID number *: 1YSA


E. coli , how does the tryptophan repressor control the expression of

low…



5


devise a plausible explanation for how such proteins might…


cell memory

maintain their identity.

combinatorial control

work…


nuclear transfer from a cultured cell line. Nature

combinatorial controldifferentiationDNA methylationepigenetic inheritancegene expressionlong noncoding RNAmicroRNA (miRNA)

KEY TERMS



52

9.1 Conjugation

In this electron micrograph, one bacterium is seen contacting anotherthrough a protein lament known as a sex pilus. Such contact initiates atype of bacterial mating in which one bacterium—the donor with the sexpili—transfers DNA to a recipient, which lacks sex pili.

After the initial contact, the sex pilus retracts, reeling in the recipientcell. Thus brought into intimate contact, the two cells form a

cytoplasmic bridge to consummate the encounter.The donor cell contains a circular piece of DNA, called an F plasmid,that is transferred to the recipient. The recipient acquires the F plasmidthrough a process of DNA nicking and DNA synthesis.

Because the F plasmid contains all the genes required for makingsex pili and for transferring the DNA, both resulting bacteria are nowpotential DNA donors.

The F plasmid can also bring along other genes from the donor’schromosome, thereby allowing for a potentially extensive genetictransfer between the two cells.

9.2 Quiz: Chapter 9

1. Which of the following is not a mechanism of genetic variation?

• Mutation within the coding sequence of a gene

• Mutation within the regulatory DNA of a gene

• Purifying selection

• Gene duplication and divergence

• Exon shufing

9.3 Concept Questions

Analysis of a sample of skin cells reveals that the cells harbor mutationsthat may cause cancer. Should the owner of these skin cells be

concerned that the mutations will be passed along to his/her offspring?

In sexually reproducing multicellular organisms, such as humans, only…



5


the loss of purine bases. This defect causes the accumulation of about about 1%, it is only a matter of time until you turn into a chimp...


Alu sequence

of the human genome; this short, repetitive sequence is nolonger mobile on its own, but requires enzymes encoded byother elements to transpose.


understand evolution and disease. Genome Res 23(7): 1063-8.

diversity. Nature Rev Genet 3:370–379.

Image courtesy of R.C. Williams andH.W. Fisher

Alu sequenceconserved syntenycopy-number variationdivergenceexon shufflinggene duplication and divergencegene family

KEY TERMS



54

10.1 Polymerase Chain Reaction

fragment from a complex mixture.

specific oligonucleotide primers are added that are complementary toshort sequence stretches on either side of the desired fragment. After

they bind specifically to the ends of the desired target sequence. A heat polymerase extends the primers and synthesizes new complementary molecules are produced that contain the target sequence.

This cycle of events is repeated. The mixture is again heated to melt polymerase synthesizes new complementary strands.

synthesizes new complementary strands. At the end of the third cycle,

target sequence. Two of these molecules are precisely the length of the

of the correct length and 10 longer ones.

After 30 cycles there are over 1 billion fragments of the correct lengthbut only 60 longer ones. The product therefore consists of essentiallypure target sequence.


using:

Original illustrations, storyboard and music:Christopher Thorpe



5

10.3 Media Assessment: Polymerase Chain Reaction


represents only the coding sequences of a genome….


Q: The cells in an individual animal contain nearly identical genomes. To your surprise, the hybridization signal is much stronger in some cells



56


therefore…


Drosophila melanogaster. Science 287:2185–2195.

microdissection. Science

cDNAcDNA librarydideoxy (Sanger) DNA sequencingDNA cloningDNA libraryDNA ligase

DNA microarray

KEY TERMS



5

11.1 Fluidity of the Lipid Bilayer

To demonstrate the fluidity of the lipid bilayer, a piece of the plasmamembrane of this neuronal cell is pulled out with laser tweezers. not rupture the plasma membrane, which flows quickly to adapt to themechanical distortion.


11.2 Lipids and Lipid Bilayer

Phospholipids contain a head group, choline in this case, that isattached via a phosphate group to a 3-carbon glycerol backbone.Two fatty acid tails are attached to the remaining two carbons of theglycerol.

The head groups and the phosphate are polar, that is, they prefer to bein an aqueous environment.

from water. The fatty acid tails on phospholipids can be saturated, withno double bonds, or unsaturated, with one or more double bonds. Thedouble bonds are usually in the cis-configuration, which introducessharp kinks. When forming a bilayer, unsaturated fatty acid tails pack bonds, bilayers would solidify to a consistency resembling bacongrease.

hydroxyl group, a tiny polar head group so to speak, attached to a rigid thus stabilizes the bilayer.

are exposed to water and their hydrophobic tails are sandwiched in themiddle.


Source: Beckman Institute, The Theoretical Biophysics Group University of IllinoisUrbana-Champaign

H. Heller, M. Schaefer, K. Schulten. Molecular dynamics simulation of a bilayer of200 lipids in the gel and in the liquid-crystal phases. Journal of Physical Chemistry97:8343–8360, 1993.

Lipidat Database (www.lipidat.chemistry.ohio-state.edu/)

Steven M. BlockStanford University

PDB ID source: Compact lipid moleculestructure (Beckman Institute); Compilationof saturated and unsaturated fatty acids andcholesterol (Lipidat)



58

11.3 Membrane Disruption by Detergent

When detergent is added to this red blood cell, its membrane ruptures,and the cytosol spills out.

11.4 Bacteriorhodopsin

the membrane of Halobacter halobium, a purple archeon that lives in multipass membrane protein that traverses the plasma membrane ofthe cell with seven long helices. The helices surround a chromophore,retinal, that is covalently attached to the polypeptide chain and gives theprotein and cells their characteristic purple color.

attached to a lysine side chain of the protein. When retinal absorbsa photon of light, one of its double bonds isomerizes from a trans

to a cis configuration, thus changing the shape of the molecule. Thechange in retinal’s shape causes conformational rearrangements in thesurrounding protein.

The light-induced isomerization of retinal is the key event in protonpumping.

to an aspartate side chain, aspartate 85, that is positioned towardsthe extracellular side of the protein. Aspartate 85 quickly hands offthe proton to the extracellular space via a bucket brigade of watermolecules. The now negatively-charged retinal takes up a proton fromanother aspartate, aspartate 96; this one is positioned towards thecytosolic face of the protein. Upon re-protonation, the retinal returns

to the ground state. Aspartate 96 replenishes its lost proton from thecytosol, and the cycle can repeat. The net result: for each photonabsorbed, one proton is pumped out of the cell.

Molecular modelling by Tiago Barros, University of California at Berkeley




5

11.5 Membrane Effects in a Red Blood Cell

has an extremely tough cytoskeleton to which the plasma membraneis anchored. When the cell is placed in high-salt solution, however, pressure, water rushes out of the cell causing spike-like protrusions to

form as the cell collapses.


Henry Bourne and John SedatUniversity of California, San Francisco

Orion WeinerHarvard Medical School

11.6 FRAP

The lateral mobility of membrane proteins can be measured in photobleaching.

For this purpose, membrane proteins are often expressed as fusion fluorescence microscope.

A selected area of the cell is then bleached with a strong, computercontrolled beam of laser light.

Those membrane proteins that are not anchored and therefore can

diffuse in the plane of the membrane, quickly exchange places withtheir neighbors and fill back in the bleached area.

From the rate of this fluorescence recovery, the diffusion constant of theprotein can be calculated.

network of the endoplasmic reticulum.

After bleaching, we observe quick recovery of the fluorescence, showingthat the protein is very mobile in the plane of the membrane.

The same experiment can be repeated using a protein that is firmly a protein of the inner nuclear membrane that binds tightly to the

meshwork of the nuclear lamina.

After photobleaching, no fluorescence recovery can be seen over thesame time frame.


Video reproduced from: The Journal of Cell Biology 138:1193–1206, Figure 4B, 1997.© The Rockefeller University Press

Jennifer Lippincott-SchwartzNICHD, National Institutes of Health



60

11.7 Rolling Leucocytes

Leucocytes are white blood cells that help fight infection. At sites of

The endothelial cells then express surface proteins, called selectins. leucocytes, causing them to stick to the walls of the blood vessels. This

binding interaction is of sufficiently low affinity that the leucocytes canliterally roll along the vessel walls in search for points to exit the vessel.There, they adhere tightly, and squeeze between endothelial cells— without disrupting the vessel walls—then crawl out of the blood vessel

Here, leucocyte rolling is observed directly in an anaesthetized mouse.The up and down movement of the frame is due to the mouse’sbreathing. Two blood vessels are shown: the upper one is an artery— with blood flowing from right to left. The lower one is a vein—withblood flowing from left to right. Leucocytes only adhere to the surface ofveins; they do not crawl out of arteries.

through the vessel walls, whereas others have already left the vesseland are seen in the surrounding connective tissue.

When the blood flow is stopped temporarily by gently clamping thevessels, we can appreciate how densely both vessels are filled with red move so fast under normal flow that we cannot see them. When theblood flow is restored, some of the leucocytes continue rolling, whereasall noninteracting cells are immediately washed away by the shear.

Animation: Blink Studio Ltd. (www.blink.uk.com)

Marko Salmi and Sami TohkaMediCity Research Laboratory, University ofTurku, Finland.

11.8 Laser Tweezers

The light of a laser beam that is focused into a cone through a high refractive indices near the focal point. This experimental set-up is cells and organelles.

Christopher Thorpe



6


11.10 Media Assessment: Lipids and Lipid Bilayer

The image of a lipid bilayer shows:


Membrane lipids, such as phospholipids, have both hydrophilic and hydrophilic heads are attracted to water, while the hydrophobic tails…



62


suppose this process converts saturated fatty acids to unsaturated ones,

Vegetable oil is converted to margarine by reduction of double bonds...


amphipathic

phospholipid or a detergent molecule.

bacteriorhodopsin

membrane…


Nature

Cell Mol Life Sci 67(11): 1179-98.

amphipathicbacteriorhodopsincholesteroldetergentglycocalyxlipid bilayermembrane domain

KEY TERMS



6

12.1 Aquaporins

This image from a computer simulation shows a cross section of a lipid small and uncharged, they can diffuse directly across the bilayer. Thissimulation shows that water molecules occasionally and spontaneouslymove into the hydrophobic core of the bilayer, even in the absence ofany channel protein. The lipid bilayer, therefore, is semi-permeable towater.

The total concentration of solute particles inside the cell generallyexceeds the concentration of solute particles outside the cell, creatingan osmotic gradient that drives the diffusion of water molecules fromoutside the cell, across the membrane, and into the cell.

As the simulation runs, observe the water molecules being bouncedaround by thermal energy as they move randomly in the bilayer.Although the water molecules will ultimately travel from the area oflow solute concentration to high solute concentration, and thus into thecell, the likelihood of their entering the hydrophobic domain of the lipidbilayer is very small.

To allow water to move more readily across the membrane, many cell

types, such as the epithelial cells of the kidney, use specialized channelscalled aquaporins. Aquaporins selectively conduct water molecules, but simulation, we can see the helices of an aquaporin tetramer spanningthe plasma membrane; each monomer in the tetramer is a separatechannel through which water molecules can diffuse.

a narrow pore—one that is large enough for a single water moleculeto pass, but too small for hydrated ions to enter. One of the watermolecules is colored yellow to help track its path through the channel.The water molecules move through the channel single-file, oriented by

center of the pore are two strategically placed asparagines that serveas a selectivity filter that prevents protons from passing through thechannel.

and the egg on the right was used as a control. The eggs weretransferred from an isotonic solution (in which ions are at an equalconcentration inside and outside the cell) to a hypotonic salt solution(which has a low concentration of ions). This has no effect on thecontrol egg because its membrane is poorly permeable to water.However, the egg expressing aquaporins quickly begins to swell andeventually bursts as water rushes down the osmotic gradient. This

experiment demonstrates the role aquaporins play in channeling wateracross cell membranes.

Movie I

Jochen Hub & Bert de GrootMax Planck Institute for BiophysicalChemistry

Large Influence of Cholesterol on SolutePartitioning into Lipid MembranesChristian L. Wennberg, David van derSpoel, and Jochen S. HubJ. Am. Chem. Soc. 134, 5351–5361 (2012)

Movie IIJochen HubGeorg-August-Universität Göttingen

Movie III

Gregory Preston



64

12.2 Na+-K + Pump

Animal cells store energy in the form of ion gradients across the cell relative to the extracellular fluid, and conversely the potassium ionconcentration in the cytosol is kept high.

Like water behind a dam, these gradients harbor potential energy thatthe cell taps to fuel cellular work.

Animal cells use a membrane pump, called the sodium–potassiumpump, to maintain these ion gradients. To begin the pumping cycle,sodium ions enter binding sites on the cytosolic side of the pump.Although there are three sodium-binding sites on this pump, forsimplicity only one is illustrated here.

Pumping sodium against its concentration gradient requires energy,which is provided by cleaving ATP. ATP transfers a phosphate group tothe pump in a high-energy linkage.

Phosphorylation causes a dramatic change in the pump’s conformation,so that the sodium ions become exposed and released outside of thecell. This action also exposes binding sites for potassium ions in thepump. Although there are two potassium-binding sites, for simplicity

only one is shown here.

and the return of the pump to its initials conformation. The potassiumis then released inside the cell, and the cycle repeats. A complete cycletakes about 10 milliseconds.


12.3 Transport by Carrier Proteins

membrane. One type of transporter, called a uniport, carries only onetype of solute, selectively bringing it from one side of the membrane tothe other.

the transporter is called a symport.

its concentration gradient, from high concentration to low. The energy

released by the movement of this solute drives the movement of theother solute, represented by the square, against its concentrationgradient, from low to high concentration.

When the coupled transporter moves solutes in opposite directionsacross the membrane, it is called an antiport.

its concentration gradient, fueling the transport of the other solute(represented by the triangle) against its concentration gradient, that isfrom low to high concentration.




6

12.4 Glucose Uptake

One important task for cells lining the lumen of the gut is the uptake ofglucose produced by digestion of food. Yet glucose is typically higher inconcentration inside the cells than in the gut, and therefore transportingit into the cell requires energy. To this end, a glucose–sodium symportharvests the energy stored in the sodium gradient to pump glucose intothe cell.

According to one model, sodium and glucose can both bind to thepump, but the binding of one makes the binding of the other moreeffective. When the binding sites of the symport are open to the lumenof the gut, the high sodium concentration makes sodium very likely tobind, and thus glucose will bind more efficiently.

when both sodium and glucose binding sites are filled, both solutesare transported across the membrane in strict unison and are releasedtogether into the cell.

On the cytosolic side of the membrane, the solutes could, in principle,also bind and thus be exported again by the same route that broughtthem into the cell. However, while there is plenty of glucose inside the

cell, there is very little sodium. Therefore, the binding of both types ofsolutes only occurs very rarely, such that most of the glucose moleculesthat enter the cell will not leave by the same route. The import istherefore unidirectional.


12.5 Potassium Channel

The bacterial potassium channel is a multipass transmembrane protein are arranged symmetrically. A pore in the center of the protein allowsselective passage of potassium ions across the membrane.

Four rigid protein loops, one contributed by each subunit, form aselectivity filter at the narrowest part of the pore. This structure isresponsible for the channel’s high degree of selectivity for potassiumions over sodium ions.

carbonyl groups are spaced precisely to interact with an unsolvatedpotassium ion, balancing the energy required to remove its hydrationshell. Passage of a sodium ion through the channel is energeticallyunfavorable because the sodium is too small for optimal interactionwith the carbonyl groups.



PDB ID number *: 1BL8



66

12.6 Hair Cells I

a tuft of spiky extensions called stereocilia on its upper surface, andeach sends signals to auditory nerve fibers through its basal surface.

The hair cells are embedded in a layer of supporting cells and aresandwiched between two sheets of extracellular matrix—the tectorial

basilar membrane to vibrate, and this motion pushes the stereociliaagainst the tectorial membrane. The stereocilia tilt, triggering anelectrical response in the hair cell. The activated hair cell, in turn,activates the auditory nerve cells.

The hair cell membrane contains mechanically gated channels. Thesechannels are closed when the stereocilia are not tilted. However, whenthey tilt, a linking filament from one stereocilium to a channel on theneighboring stereocilium pulls at the channel, opening it. Positivelycharged ions flow into the cell, depolarizing the membrane.

12.7 Hair Cells II

waves.

pushed with laser tweezers to simulate this movement. Movementopens stressactivated ion channels in the plasma membrane, leadingto membrane depolarization. This is translated into the perception ofsound.

Moving an individual stereocilium demonstrates the flexible attachmentof these structures to the cell body.




6

12.8 Action Potentials

The fundamental task of a nerve cell is to receive, conduct, and transmitsignals. Neurons propagate signals in the form of action potentials,which can travel great distances along an axon without weakening.

To transmit an action potential over such a distance without weakeningrequires that the signal is continuously reamplified along the way.The central molecular players in this process are the voltage-gated

sodium channels, which undergo a cycle of finely choreographedconformational changes. When an action potential passes, sodium ions rush into the axon, further depolarizing its membrane. Within afraction of a thousandth of a second, however, the sodium channelsswitch to a new, inactivated state, in which they are closed but now can recover quickly after an action potential has passed. The sodiumchannels then reconvert to the closed state, ready to be opened againwhen the next action potential is encountered.

Let’s examine the changing state of the sodium channel during anaction potential. When no stimulus is present, the sodium channelsremain closed and the electrical potential measured across themembrane remains constant. However, if a depolarizing stimulus isapplied by a brief pulse of electric current, the membrane will start to of the sodium channels will open, permitting sodium ions to enter is sufficient, even more sodium channels open, and the membranepotential rapidly approaches the equilibrium potential for sodium (about conformation, where the channel is unable to open again even thoughthe membrane potential is still depolarized. The sodium channelswill remain in this inactivated state until a few milliseconds after themembrane potential returns to its initial negative value.

The action potential is propagated along the length of the axon in only the sodium channels along a length of the axon, we can see why thisis so. As a depolarizing stimulus (represented in orange) reaches oursection of the membrane, sodium channels open and current flows into inactivation prevents the depolarization from spreading backward alongthe axon.




68

12.9 Synaptic Signaling

Neurons transmit chemical signals across synapses, like the onesshown in this electron micrograph. We can identify the dendriteof the receiving, or postsynaptic cell, as well as two presynapticnerve terminals loaded with synaptic vesicles. Note the narrow cleftseparating the pre- and postsynapitic cells.

The synapse converts the electrical signal of the action potential in

the presynaptic cell into a chemical signal. When an action potential 2+ channels in the 2+ ions to flow into the terminal. The 2+ in the nerve terminal stimulates synaptic vesicles to fusewith the plasma membrane, releasing their neurotransmitter cargo intothe synaptic cleft.

The released neurotransmitters diffuse across the synaptic cleft wherethey bind to and open the transmitter-gated ion channels in the plasmamembrane of the postsynaptic cell. The resulting ion flows depolarizethe plasma membrane of the postsynaptic cell, thereby converting theneurotransmitter’s chemical signal back into an electrical one thatcan be propagated as a new action potential. The neurotransmitter isquickly removed from the synaptic cleft—either by enzymes that destroyit, or by reuptake into the nerve terminals or neighboring cells.


12.10 Optogenetics

Photosynthetic green algae, like the Chlamydomonas reinhardtii seenhere, use light-gated channels in their plasma membranes to sensesunlight and navigate toward it.

Chlamydomonas —called channelrhodopsin —opens and allows Na+ to flow into the cell. This depolarizes the plasma membrane and,ultimately, modulates the beating of the flagella that algae use to swim.

into other, excitable cell types, such as neurons, which then becomeresponsive to light.

was introduced into a select subpoplation of neurons in a mousehypothalamus, a region of the brain involved in many functions,including the control of aggression.

The scientists then implanted a tiny fiber optic cable into the mouse’sbrain, in order to control the flow of blue light to the modified neurons.

This technique, called optogenetics, allows scientists to study neuronsand neural circuits in live animals.

with a fiber optic cable attached to its brain. On the right is a femalemouse.

When the light from the fiber optic cable is off, the male engages innormal mating behavior with the female. However, when the light isswitched on, the mouse immediately becomes aggressive, and violentlyattacks his female companion. When the light is turned back off, the

mouse returns to normal behavior.

Electron Microscopy:

Cedric S. RaineAlbert Einstein College of Medicine

Movie I

George WitmanUniversity of Massachusetts, Worcester

Anthony G. MossAuburn University

Gregory J. PazourUniversity of Massachusetts Medical School

Movies II & II

Dayu LinNew York University Langone MedicalCenter



6

an inflated rubber glove, is introduced to the cage.

The mouse largely ignores the glove when the light is off. However,when the light turns on, the mouse immediately attacks the glove. Oncethe light is switched back off, the mouse resumes its normal behavior.

Optogenetics may hold the potential to revolutionize the field ofneurobiology by allowing neuroscientists to analyze with remarkableprecision the neurons and circuits underlying complex behaviors.

12.11 Neurite Outgrowth

Neuronal precursor cells, taken from the hippocampus of an embryonic

rodent brain, differentiate in culture and send out long extensions,called neurites, that could later become dendrites or axons.

These neurites are pulled out of the cell body by growth cones that cancrawl independently over the substratum. Occasionally, a growth conedetatches from the substratum and the neurite retracts.

New growth cones can grow from the sides of existing neurites, formingbranches.


12.12 Neuronal Pathfinding

Xenopus, neurons extend axonsfrom the eye to connect to appropriate target cells in the midbrain. direction.

them on unerring courses toward their targets.

After entering the appropriate part of the midbrain, the optic tectum, theaxons slow down and send out branches, which can sample numeroustarget neurons and establish synaptic connections.

These two axons took six hours to grow to their targets less than a mil-limeter away.

Final Composition: Allison Bruce

Frank B. Gertler and Lorene LanierMassachusetts Institute of Technology

Sonia Witte and Christine E. HoltUniversity of Cambridge



70


1. Which of the following membrane transport proteins forms tinyhydrophilic pores in the membrane through which solutes can pass by

12.14 Media Assessment: Action Potential

What is the principle ion involved in membrane depolarization during

+

–

2+

+


The rigidity of plants ultimately stems from the movement of water.Water, like any molecule, tends to travel down its concentrationgradient when it diffuses across a cell membrane….



7


Q: Your boss is coming to dinner! All you have for a salad is somewilted, day-old lettuce. You vaguely recall that there is a trick to

you soak the lettuce in salt water, soak it in tap water, or soak it in


action potential

transient, self-propagating depolarization of the plasmamembrane in a neuron or other excitable cell; also calleda nerve impulse.


Al-Awqati Q (1999) One hundred years of membrane permeability: does Nature Cell Biol

in channels and pumps. Science

action potentialactive transportantiportaxonCa2+ pump (or Ca2+-ATPase)channelcoupled pumps

KEY TERMS

Image courtesy of Olaf Mundigl andPietro de Camilli



72

13.1 Glycolysis

molecule into two three-carbon molecules produces a net gain of energy breakdown product, pyruvate, is imported into mitochondria, where itultimately feeds into the citric acid cycle and the electron transport chain.

and fifth steps, this energy allows glucose to be split into two smallermolecules from which energy can be harnessed efficiently. And in the last chemistry that evolved to catalyze these reactions ensures that energy isreleased in small portions that can be efficiently captured. Less controlledcombustion reactions would release most of the energy as heat.

glucose. This investment of energy primes glucose for energy-releasingreactions later in glycolysis.

step of glycolysis is irreversible. catalyzes the opening of the ring form of glucose 6-phosphate to theopen chain form.

The same enzyme then performs a reversible reaction in which thecarbonyl group of glucose 6-phosphate changes position from the firstcarbon to the second carbon in the chain.

This reaction involves a water molecule, which donates a hydrogen ionto the carbonyl oxygen.

The hydrogen ion is then retrieved from the hydroxyl group on the

6-phosphate is formed.

The same enzyme, phosphoglucose isomerase, catalyzes the formationof fructose 6-phosphate into its ring form.

fructose 1,6-bisphosphate is formed.

This third step, in which the second phosphorylation event occurs, glycolysis.

The phosphorylations in steps 1 and 3 represent an investment of energythat will be paid back in the later stages of the pathway.

1,6- bisphosphate into its open chain form.

two molecules.

One molecule that is formed is the 3-carbon glyceraldehyde 3-phoshate.The enzyme performs additional reactions on the second 3-carbonmolecule. The second molecule is dihydroxyacetone phosphate.

the isomerization of dihyroxyacetone phosphate into glyceraldehyde3-phosphate.



7

The catalytic mechanism of this enzyme is very similar to that ofphosphoglucose isomerase, back in step 2. The result is two moleculesof glyceraldehyde 3-phosphate.

All of the subsequent steps of glycolysis will occur twice—once foreach molecule of glyceraldehyde 3-phosphate. These are the energygeneration steps of gylcolysis.

+ to oxidize glyceraldehyde 3-phosphate. The resulting molecule isconnected to the enzyme by a high-energy thioester bond.

A molecule of inorganic phosphate displaces the high-energy thioesterbond, forming a high-energy acyl-anhydride bond. The resultingmolecule is 1,3-bisphosphoglycerate.

dephosphorylates 1,3-bisphosphoglycerate. The high-energy phosphate 3-carbon molecule, a total of 2 ATPs are generated. At this point theenergy investment from the first three steps has been paid back.

energy of hydrolysis, is transformed by the enzyme phosphoglyceratemutase into 2- phosphoglycerate.

2-phosphoglycerate, creating phosphoenolpyruvate. The loss of waterredistributes energy within the molecule, creating a phosphate groupwith an extremely high free-energy of hydrolysis.

pyruvate.

in glycolysis from a single molecule of glucose is two molecules of ATP

The chemistry of glycolysis is conserved all the way from bacteria toanimal cells.

Chemistry Consultant: Patricia S. Caldera-Muñoz

Storyboard and Animation: Sumanas, Inc. (www.sumansinc.com)



74

13.2 Citric Acid Cycle

step pathways. For example, in the process of glycolysis, breakdownof glucose molecules releases energy that is captured by the energy pyruvate, is imported into mitochondria, where it is converted into generated by breakdown of fats or amino acids.

“burned”—that is, oxidized—and released one-by-one as the waste the electron transport chain in the inner mitochondrial membrane. Thisfuels the proton gradient that is then used for the production of ATP, thecell’s primary energy currency.

The molecule that enters the citric acid cycle is the 2-carbon compound the 6-carbon citrate.

carbon atoms from oxaloacetate marked in blue will be released duringthis cycle to form carbon dioxide.

that the hydroxyl group is in a different position in these two molecules.

converted to aketoglutarate. The hydroxyl-bound carbon is strippedof its hydrogen atoms, resulting in a carbonyl group. One of these + released as a proton. The carbon and 2 oxygen atoms are then released 2, creating the 5-carbon aketoglutarate.

2. The by the addition of the coenzyme A. The enzyme for this reaction adds ahigh-energy thioester bond to coenzyme A, releasing the carbon and 2 +

converted into succinate. The release of the coenzyme A group provides

Note that succinate is symmetrical molecule. The two end carbons arechemically identical, and the two carbons in the middle are chemicallyidentical. For convenience we will continue tracing only the 2 carbons

depicted in the upper half of the molecule. 2 2 is an energy carrier that feeds high-energy electrons to the electron 2.

resulting molecule is malate, with the water molecule added across thetwo central carbons.

malate is converted to oxaloacetate. The carbon carrying the hydroxyl



7

group is converted to a carbonyl group. This reaction releases hydrogen + the four-carbon oxaloacetate.

Oxaloacetate is thus replenished and can take part in another cycle,returning to step 1. Note the new position of the red carbon atoms, 2. Thegreen labels indicate the positions of the new carbons added during this

new cycle.

Chemistry Consultant: Patricia S. Caldera-Muñoz



1. The energy released by oxidizing glucose is saved in the high-energybonds of:

2 2.

13.4 Media Assessment: Glycolysis

The end product of glycolysis is:



76


reactions, starting with glycolysis and ending with oxidativephosphorylation...


When oxygen is added, glucose consumption drops precipitously and isthen maintained at the lower rate. Why is glucose consumed at a high


anabolic pathway

molecules are synthesized from smaller subunits; usuallyrequires an input of energy.

Image courtesy of Peter Tontonoz andRonald M. Evans

acetyl CoAADP, ATPanabolic pathwayscatabolismcell respirationcitric acid cycleelectron-transport chain

KEY TERMS



7


photosyntheis. Proc Nat Acad Sci USA 98:2170–2175.



78

14.1 Tomogram of Mitochondrion

A mitochondrion contained in a one-half micrometer thick sectionof chicken brain is viewed with a high voltage electron microscope.When the section is tilted in the microscope, it can be viewed frommany different angles, and a large amount of three-dimensional detail used to calculate a three-dimensional reconstruction, or tomogram, ofthe mitochondrion.

The tomogram of the same tissue slice is shown here as a series ofstacked images. The movie steps through the images one by one, fromthe bottom of the stack, to the top, and back. This allows us to traceindividual membranes in three-dimensions.

To create a three-dimensional model, membranes in an individual is traced in light blue, where it parallels the outer membrane, andtraced in yellow, where it folds into the cristae that protrude into themitochondrial interior. The tracings from all sections are then modeledas three-dimensional surfaces, and displayed as a three-dimensional any angle.

The cristae are colored differently and show the variety of shapes andconnections to the inner membrane in a single mitochondrion.

The model also shows the reconstitution of the outer mitochondrialmembrane, represented in dark blue, as well as two fragments of two organelles are quite frequently seen in cells. Note that there isno continuity between the mitochondrial and endoplasmic reticulummembranes. Lipids are thought to be shuttled between the twoorganelles by special carrier proteins that operate in this gap.

Final composition: Graham Johnson, Fivth Element (www.fivth.com)

14.2 Electron-Transport Chain

The mitochondrion is the site of most of the cell’s energy production.After food molecules are processed in the cytosol, they enter the cycle, the molecules are stripped of high-energy electrons, which are

The carrier molecules transfer the high-energy electrons to a chain ofproteins, called the electron transport chain, which is embedded in theinner mitochondrial membrane. The chain acts as a pump, using theenergy of the electrons to move protons from one side of the membraneto the other. The pumping creates a proton gradient across themembrane, which the mitochondrion can tap to make the fuel moleculeATP.

dehydrogenase complex. This complex has a higher affinity for electrons electrons are transferred from one protein to another in the complex,energy is released and used to pump protons across the membrane.

Terrence G. FreySan Diego State University

Guy PerkinsUniversity of California, San Diego



7

shuttles them to the next way station, called the cytochrome b-c 1 each complex in the chain has a higher affinity for the electronsthan the previous one, the electrons keep moving through the chainunidirectionally.

Finally, cytochrome c delivers the electrons to the cytochrome oxidasecomplex, a third proton pump. The cycle repeats until the cytochrome

From there, they are handed over to molecular oxygen. Oxygen takesup the electrons as it combines with protons, forming water as product,thereby completing the step-wise path of the combustion of the foodmolecules.


14.3 ATP Synthase—A Molecular TurbineATP synthase is a molecular machine that works like a turbine toconvert the energy stored in a proton gradient into chemical energystored in the bond energy of ATP.

The flow of protons down their electrochemical gradient drives a rotor entry open to one side of the membrane and bind to rotor subunits.Only protonated subunits can then rotate into the membrane, awayfrom the static channel assembly. Once the protonated subunits havecompleted an almost full circle, and have returned to the static subunits,an exit channel allows them to leave to the other side of the membrane. mechanical, rotational energy.

The rotational energy is transmitted via a shaft attached to the rotor thatpenetrates deep into the center of the characteristic lollipop head, theF1 ATPase, which catalyzes the formation of ATP.

The F1 ATPase portion of ATP synthase has been crystallized.

influences the conformation and arrangement of the surrounding a temporal sequence as they would occur during rotation of the centralshaft.

concentration of ATP is high and the proton gradient low, ATP synthasewill run in reverse, hydrolyzing ATP as it pumps protons across themembrane.

To show the rotation of the central shaft, a short fluorescent actin single F1 ATPases can be visualized in the microscope.

When ATP is added, the filament starts spinning, directly demonstratingthe mechanical properties of this remarkable molecular machine.




80

14.4 ATP Synthase—Disco

Dance direction: Nagatsuta Bon-Odori

Camera work and production: Hiroyuki Noji

14.5 Bacterial Flagellum

Many species of bacteria propel themselves through their environmentby spinning helical motorized flagella. Rhodobacter cells have oneflagellum each, whereas E. coli cells have multiple flagella that rotate nanometers wide and up to 15 microns long and spins on the order of100 times per second. These animations show a series of schematizedand speculative models about how bacterial flagella might function andassemble.

Just outside of the cell wall, the filament is connected to a flexiblerotating hook.

The filament, the hook, and a structure called the basal body (locatedbelow the cell’s surface) make up the three parts of the flagellum. Thebasal body consists of a rod and a series of rings embedded in the innermembrane, the peptidoglycan layer, and the outer membrane.

layer and, as its name implies, remains stationary, and the rotor, whichrotates.

The motor derives its power from a proton gradient across the outside and a low concentration exists inside the cell.

The protons flow through the interface between two types of proteins,

proteins and therefore also contains two of these important aspartic

acids.Although the molecular mechanism of rotation is not known, onepossible model describes protons moving through the channels in the binding causes a conformational change in MotA proteins, resulting inthe first power stroke that moves the rotor incrementally.

At the end of the first power stroke, the two protons are released intothe cytoplasm. The proton loss causes a second conformational changethat drives the second power stroke, once again engaging the rotor.

Although the mechanism for motor function is not yet certain, manydetails of flagellar assembly have been determined.

Video: Howard C. Berg, Harvard University

3D Animation and Flagellar Structures:Keiichi Namba, Protonic NanoMachineProject, ERATO, JST & Osaka University



8

Flagella begin their assembly with structures in the inner membrane.26 subunits of an integral membrane protein called FliF come together make up the rotor.

Flagellar proteins destined to be part of the extracellular portion ofthe flagellum are exported from the cell by a flagellum-specific exportpathway and assembled at the center.

to the rigid peptidoglycan layer, keeping the stator proteins fixed inplace.

The subunits of the rod portion of the rotor move up through the hollowcylinder in the assembly and, assisted by cap proteins, build up the rodin a proximal to distal fashion.

Another set of rings, called L and P rings, are found in gram negativebacteria, such as E. coli. They penetrate the outer membrane forming abearing for the rod.

As the rod cap is exposed outside the L ring, it dissociates and is

replaced by a hook cap that guides the assembly of the hook proteins.

After the hook is assembled, the hook cap dissociates, and a series of

Finally, yet another cap is built and filament proteins assemble. Like therod and hook proteins, they travel through the hollow channel insidethe filament to reach the distal end. The cap rotates which causes thesubunits to build in a helical fashion. A complete filament can consist of20,000 to 30,000 subunits.

14.6 Photosynthetic Reaction Center

The bacterial photosynthetic reaction center is a large complex offour protein subunits. Three subunits, called the H, L, and M subunits,contain hydrophobic helices that span the membrane and anchor

the complex. The fourth subunit is a cytochrome that is peripherallyattached.

electrons generated after absorption of light move from centrallylocated chlorophylls to pheophytins. From there they move to aquinone, which is then released from the reaction center to feed the chlorophylls are replaced through a conduit of heme groups found inthe cytochrome subunit.


PDB ID number *: 1DXR



82

14.7 Light Harvesting

dedicated organelles contain a variety of membrane components ATP, which in turn fuel the production of sugars and other moleculesrequired by the cell.

envelope akin to that surrounding mitochondria, and the internalthylakoid membrane system. Within the thylakoid membranes largeantennae consisting of hundreds of light-absorbing chlorophyllmolecules capture light energy. When a chlorophyll molecule absorbslight, the energy bumps from one chlorophyll molecule to another, untilit passes to a special pair of chlorophyll molecules in the reaction center

electron transfers. First, a neighboring molecule accepts the high-energy donor molecule receives a low-energy electron from water. After thisseries of transfers occurs four times, two water molecules are split intoone molecule of oxygen and four protons.

The photosystem shares the thylakoid membranes with an electrontransport chain. When light bumps an electron out of the photosystem,the electron is removed by a small diffusible carrier molecule. As shownbefore, water replenishes lost electrons. The diffusible carrier moleculebrings the electrons to the cytochrome b6- f complex, which uses partof the electrons’ energy to pump protons across the membrane. From

antenna system and kicks electrons to an even higher energy level.

After two such high-energy electrons have been produced and delivered

To make the system work, each member of the electron transport chainhas to be finely tuned to have an appropriate tendency to receive or is excited by light, it has a high tendency to donate electrons. The nextcomponent is more likely to receive electrons. The loss of an electron receiving electrons from water. The next series of carriers in the chainmake better and better acceptors, drawing the electron through thechain.

The released energy is used to generate a proton gradient that fuels has to be bumped up to an even higher energy level than that absorbed




8


14.9 Media Assessment: Electron Transport Chain

The electron transport chain pumps protons:


electrons pass through a proton pump, which uses their movement togenerate an electrochemical proton gradient that ultimately producesATP…



84


The uncoupler dinitrophenol was once prescribed as a diet drug to aidin weight loss. How would an uncoupler of oxidative phosphorylation

An uncoupler promotes weight loss by decreasing the efficiency of…


antenna complex

membrane-bound photosystem that captures energy fromsunlight; contains an array of proteins that bind hundreds ofchlorophyll molecules and other photosensitive pigments.


resolution of F1-ATPase from bovine heart mitochondria. Nature370:621–628.

Annu Rev Biochem

antenna complexATP synthasecarbon fixationcell respirationchemiosmotic couplingchlorophyllchloroplast

KEY TERMS

Image courtesy of Chan B. Park



8

15.1 Nuclear Import

Nuclear import and export can be directly visualized in living cells that protein NF-AT.

NF-AT is normally localized in the cytosol, and excluded from the migrates to the nucleus.

This is done here experimentally by adding an ionophore that allowscalcium to enter the cells from the medium.

Upon removal of the ionophore, calcium levels return to normal andNF-AT is exported from the nucleus.

Final composition: Allison Bruce

15.2 Mitochondrial Protein Import

are encoded in the nucleus and translated into protein in the cytosol.

Proteins made in the cytosol must therefore be sorted and selectivelydelivered to their proper destinations.

Precursor proteins destined for a mitochondrion have a short segmentof amino acids, the signal sequence, that targets the proteins tothis organelle. The signal sequence has affinity for a receptor onthe mitochondrion’s surface and delivers the precursor protein to atranslocation apparatus for import.

At a contact site where the mitochondrion’s two membranes are closetogether, the precursor protein snakes in an unfolded state throughtwo sequential protein translocators, one in each of the mitochondrial

protein as it appears on the inside of the mitochondrion, and therebyprevent the protein chain from backsliding through the translocationtunnel.

Once inside, an enzyme, called a signal peptidase, cleaves thesignal sequence, which is no longer needed, from the precursor. Thechaperone proteins are released as the protein chain folds into its three-dimensional structure.


Frank McKeonHarvard Medical School

Futoshi ShibasakiThe Tokyo Metropolitan Institute of MedicalScience

Roydon PriceHarvard University

Annie Yang

Harvard Medical School

Electron Microscopy:Daniel S. Friend



86

15.3 ER Tubules

The endoplasmic reticulum is a highly dynamic network ofinterconnected tubules that spans the cytosol of a eukaryotic cell—like aspider’s web.

The network is continually reorganizing with some connections beingbroken while new ones are being formed.

Motor proteins moving along microtubules can pull out sections of

endoplasmic reticulum membranes to form extended tubules that thenfuse to form a network.

15.4 Protein Translocation

from the cytosol.

bind to it, forming a polyribosome. There are two separate populationsof polyribosomes in the cytosol that share the same pool of ribosomalsubunits.

Free ribosomes are unattached to any membrane. Membrane-bound

reticulum.

into the lumen, while transmembrane proteins only partially cross the

receptor function as molecular matchmakers, connecting ribosomes

to open the translocation channel. The signal peptide remains bound tothe channel while the rest of the protein chain is threaded through themembrane as a large loop.

Once the protein has passed through the membrane it is released into peptidase

Part I: Jennifer Lippincott-SchwartzNICHD, National Institutes of Health

Part II: Ron D. ValeHoward Hughes Medical InstituteUniversity of California, San Francisco



8

then released from the translocation channel into the membrane andrapidly degraded.

membrane as transmembrane proteins.

For clarity’s sake, the membrane-bound ribosome will be omitted to

a single membrane spanning segment, the N-terminal signal sequence process is halted by an additional sequence of hydrophobic amino acids,a stop-transfer sequence, further in the polypeptide chain. The stop-transfer sequence is released laterally from the translocation channeland drifts into the plane of the lipid bilayer, where it forms a membrane-spanning segment that anchors the protein in the membrane.

As a result, the translocated protein ends up as a transmembraneprotein inserted in the membrane with a defined orientation.

are thought to work in pairs: an internal start-transfer sequence servesto initiate translocation, which continues until a stop-transfer sequenceis reached; the two hydrophobic sequences are then released into thebilayer, where they remain anchored.

span the bilayer, additional pairs of stop and start sequences comeinto play: one sequence reinitiates translocation further down thepolypeptide chain, and the other stops translocation and causespolypeptide release... and so on for subsequent starts and stops.

Thus, multipass membrane proteins are stitched into the lipid bilayer

as they are being synthesized, by a mechanism resembling a sewingmachine.

Storyboard and Animation: Thomas Dallman, Bioveo (www.bioveo.com)

15.5 Clathrin

endocytosis, in which the plasma membrane invaginates and pinchesoff cargo-filled vesicles.

This movie shows a shows a series of electron micrographs that havebeen artificially morphed to show the process of endocytosis as it mayoccur.

The process involves a variety of molecules, including the cargomolecules that the cell takes in; the receptors that capture the cargomolecules; and molecules called adaptins that mediate contact betweenthe receptors and the clathrin molecules that act to shape the vesicleforming at the plasma membrane.



88

their leg domains, and ultimately form a closed cage.

Part I: Electron Micrograph, M.M. Perry and A.B. GilbertPart II: Animation and Storyboard, Sumanas, Inc. (www.sumanasinc.com)Part III: Electron Micrograph, Ernst Ungewickell, Hanover Medical SchoolPart IV : Tomás Kirchhausen, Harvard Medical School.

Animation: Alison Bruce

15.6 Cell Compartments

High voltage electron microscopy allows three-dimensionsional imaging slices of the cell are viewed in the microscope from different angles,which allows us to reconstruct a three-dimensional image.

structure.

and we can appreciate the size and shape of various compartments.

Using these outlines, a computer can construct a three-dimensional

apparatus, each traced in a different color. The cis are first delivered to the organelle, is light blue and the trans network, where they exit, is light blue.

packaged after leaving the trans

apparatus. They transport cargo between the cisternae or back to theendoplasmic reticulum.

When all the other organelles are combined into a single image, wecan see the incredible crowding of organelles in the cytosol. Here,

reticulum and ribosomes are shown in yellow. The purple organelles areprobably endosomes.

components work in synchrony to allow the cell to achieve its tasks.



8

15.7 Constitutive Exocytosis Pathway

plasma membrane after synthesis in the endoplasmic reticulum.

They are first dispersed throughout the extensive membrane network ofthe endoplasmic reticulum from where they move to exit sites that formin random locations in the membrane network. At each of these sites,the membrane proteins are concentrated and packaged into transport

intermediates.

At the next stage, transport intermediates move along microtubule are now pulled outward on microtubules, which deliver them to theplasma membrane.

content proteins disperse.

Video reproduced from: The Journal of Cell Biology 143:1485–1503, Figure 1A, 1998.© The Rockefeller University Press.

15.8 Exocytotic Transport

surface are often packaged into tubular transport vesicles of significantsize.

the plasma membrane.

The transport vesicles move along microtubules which are stainedhere with a red fluorescent dye.

The green cell in the corner does not contain fluorescent microtubules.




Patrick Keller and Kai SimonsEuropean Molecular Biology Laboratory



90

15.9 Phagocytosis

Phagocytosis allows cells to take up large particles, such as these yeastcells that are being engulfed by the slime mold Dictostelium.


Video reproduced from: M. Maniak, R. Rauchenberger, R. Albrecht, J. Murphy, andG. Gerisch. Coronin involved in phagocytosis. Cell 83:91–924. © 1995, withpermission from Elsevier Science.

15.10 Receptor-Mediated Endocytosis

freely, cholesterol molecules are derivatized and packed inside low surrounds the cholesterol molecules. The protein portion is recognized

receptor that protrudes into the cytosol. Adaptin recruits clathrinmolecules, which start coating the membrane. Assembly of the clathrincoat causes the membrane to bend and invaginate, forming a vesicle particles bound to them.

Once inside the cell, the vesicle uncoats and fuses with the endosome,the intracellular compartment that first receives all endocytosed receptors to release their cargo.

trip from the plasma membrane to the endosome and back every 10minutes.

content is delivered to a lysosome, which contains hydrolytic enzymesthat can digest the particles. Free cholesterol is liberated together with The cholesterol is then released into the cytosol to be used in thesynthesis of new membranes.


Markus ManiakUniversity of Kassel, Germany



9

15.11 Endosome Fusion

binds to endosomes and promotes their fusion with one another,thereby increasing the steady-state size of individual endosomalcompartments.

magnification.


15.12 Freeze Fracture of Yeast Cell

15.13 Pancreas: View 1

Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet. Springer-Verlag. New York,1975.

Philip D. Stahl, Alejandro Barbieri andRichard RobertsWashington University School of Medicinein St. Louis



Lelio Orci and Alain Perrelet



92

15.14 Pancreas: View 2

Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet. Springer-Verlag. New York,1975

15.15 Pancreatic Secretory Cell

Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet. Springer-Verlag. New York,1975.


1. The outer membrane of the nucleus is continuous with the membrane





9


Molecules enter and exit the nucleus through a structure called the the tangled meshwork of disordered polypeptides that fill the centralchannel of the…


lysosomes, and the plasma membrane. One measure of the difficulty of

the sorting problem is the degree of “purification” that must be…


autophagy

and organelles that are damaged or obsolete.

clathrin

that…


Proc Natl Acad Sci USA

Nature RevGenet

autophagychaperone proteinclathrincoated vesicleendocytosisendomembrane systemendoplasmic reticulum (ER)

KEY TERMS



94

16.1 Calcium Signaling

culture.

small amounts of a neurotransmitter, individual cells light up randomlyas ion channels open up and allow calcium ions to enter the cell.

16.2 G-Protein Signaling

binding domain.

When an appropriate protein ligand binds to this domain, the receptorundergoes a conformational change that is transmitted to its cytosolic protein for short).

protein in the plasma membrane.

with the inactive receptor, while, in other cases, as shown here, it only to dissociate.

protein, activating both the alpha subunit and beta-gamma complex. from the activated beta–gamma complex, whereas in other cases thetwo activated components stay together.

activity of target proteins in the plasma membrane, as shown here for a

Ann H. Cornell-Bell Viatech Imaging

Steven FinkbeinerGladstone Institute of Neurological Diseaseat the University of California, San Francisco

Mark S. CooperUniversity of Washington

Stephen J. Smith

Stanford University School of Medicine



9

signal to other components in the signaling cascade.

inactivates the subunit. This step is often accelerated by the binding events.

As long as the signaling receptor remains stimulated, it can continue receptors eventually inactivate, even if their activating ligands remainbound.

the activated receptor. Once a receptor has been phosphorylated in thisway, it binds with high affinity to an arrestin protein, which inactivates

Arrestins also act as adaptor proteins, and recruit the phosphorylatedreceptors to clathrin-coated pits, from where the receptors areendocytosed, and afterwards they can either be degraded in lysosomes

or activate new signaling pathways.

Animation: Thomas Dallman, Bioveo (www.bioveo.com)

16.3 cAMP Signaling

Adenylyl cyclase is a membrane-bound enzyme whose catalytic domain

Activated adenylyl cyclase converts ATP to cyclic AMP which then coupled receptor to other components in the cell.

of two catalytic subunits and two regulatory subunits. The binding ofcyclic AMP to the regulatory subunits alters their conformation andliberates the catalytic subunits which are now active and phosphorylatespecific target proteins.

subunits enter the nucleus, where they phosphorylate a transcription protein. This complex activates transcription after binding to specific

regulatory regions that are present in the promoters of appropriatetarget genes.

Animation: Thomas Dallman, Bioveo (www.bioveo.com)

Original illustrations:Nigel Orme



96

16.4 Calcium Wave During Fertilization

When a sperm cell fuses with this sea urchin egg cell, calcium ionsbegin rushing into the cell at the site of fusion.

measured with a fluorescent dye that becomes increasingly brighter themore calcium is present.

dimensional display, into peak heights, where red and high peaksrepresent the highest calcium concentrations.

A second rise of the calcium concentration can be observed after pronuclei to meet and fuse near the center of the egg.

Final composition: Allison Bruce

Part I:Carolyn A. LarabellLawrence Berkeley National Laboratory

Jeff HardinUniversity of Wisconsin, Madison

Part II:Michael WhitakerUniversity of Newcastle Upon Tyne

Isabelle GillotUniversity of Nice-Sophia Antipolis

16.5 Calmodulin

affinity calcium-binding sites.

calmodulin.

Most notably, the two globular domains rotate relative to each other.These conformational changes enable calmodulin to bind to targetproteins and regulate their activity.

captures helical peptides on target proteins by wrapping tightly aroundthem. To make this possible, the central helix of calmodulin breaks intotwo helices now connected by a flexible loop. Although the calciumions remain tightly bound during this remarkable reaction, they are notshown in the animated part of this movie.



Source: Intermediate structures provided by Eric Martz (www.umass.edu/microbio/rasmol/) and calculated by the Yale University Morph Server, Mark Gerstein andWerner Krebs (bioinfo.mbb.yale.edu/)

PDB ID number *: Calcium-free calmodulin(1CFD); Compilation of calcium-freecalmodulin & calcium-bound calmodulin(1CFD & 1OSA); Compilation of calciumbound calmodulin & calcium-boundcalmodulin complexed with rabbit skeletalmyosin light chain kinase (1OSA & 2BBM)



9

16.6 Ras

switch 1 and switch 2, change conformation dramatically. The changein conformational state allows other proteins to distinguish active downstream target proteins in the cell signaling pathways.

A space-filling model shows that the conformational changes between the protein. The two switch regions move the most.

arginine side chain directly into the active site. The arginine, together


PDB ID number *: Compilation of c-H-Rasp21 protein catalytic domain complex withGDP & structure of p21-Ras complexedwith GTP at 100K (4Q21 & 1QRA);Ras-Rasgap complex (1WQ1)

Henry Bourne and John SedatUniversity of California, San Francisco

Orion WeinerHarvard Medical School

16.7 Chemotaxis of Neutrophils

These human neutrophils, taken from the blood of a graduate student, infection. They are attracted there by chemical signals that are releasedby other cells of the immune system or by invading microbes.

micropipette. When neutrophils sense these compounds they polarizeand move towards the source. When the source of the chemoattractantis moved, the neutrophil immediately sends out a new protrusion, andits cell body reorients towards the new location.



98

16.8 Lymphocyte Homing

was anaesthetized and its fin pierced with a needle to introduce a smallwound.

A vein is seen at the bottom of the frame.

lymphocytes crawl out of the blood vessel and migrate towards the

wound.

They are attracted there by chemicals released from damaged cells,invading bacteria, and other lymphocytes.

restricted to the wounded area.

The static cells that are dispersed in the connective tissue arefibroblasts.

seconds.



1. When the hormone insulin is released into the bloodstream, what

16.10 Media Assessment: G-Protein Signaling



9


The three main classes of cell-surface receptors are ion-channel- plasma…

16.12 Challenge Questions: Chapter 16

How is it that different cells can respond in different ways to exactly the

molecule because of differences in the internal machinery...


adaptation

a cell or organism to register small changes in a signal despitea high background level of stimulation.


genomic and evolutionary perspective of plasma membrane receptorsinvolved in signal transduction. Sci STKE

Philos Trans R Soc Lond B Biol Sci

Image courtesy of Michael Snyder

adaptationadenylyl cyclaseCa2+/calmodulin-dependent protein kinase (CaM-kinase)calmodulincell signalingcyclic AMP

KEY TERMS



100

17.1 Intermediate Filaments

filaments, microtubules, and actin filaments—that provide the cells withstrength, structure, and movement. Although all eucaryotic cells containmicrotubules and actin filaments, intermediate filaments are found onlyin vertebrates and a number of other soft-bodied animals.

desmosomes.

These cables of intermediate filaments have a high tensile strength.Without these filaments, stretching or pressure on the epithelial sheetwould cause it to rupture.

precise hierarchical arrangement of protein subunits. At the lowestlevel, two monomers associate with each other to create a twisteddimer.

Two dimers then line up to form a staggered tetramer. Note that the twodimers are arranged in opposite orientations, with their amino terminal

ends away from each other, so that the two ends of the tetramer areindistinguishable.

Tetramers then link end-to-end, thus building up one strand of anintermediate filament.

A total of eight strands stack together and twist around each otherto create the intermediate filament. This stacking provides theextensive lateral contacts between the strands that give the filament itsremarkable mechanical strength. An electron micrograph shows theappearance of intermediate filaments that have been assembled in atest tube.


17.2 Dynamic Instability of Microtubules

Microtubules continually grow from this centrosome added to a cellextract. Quite suddenly however, some microtubules stop growing andthen shrink back rapidly, a behavior called dynamic instability.


Electron Microscopy:D.E. Kelly

Timothy MitchisonHarvard Medical School



10

17.3 Microtubule Dynamics in vivo

of microtubules.

dynamics of the microtubule cytoskeleton.

Note that many but not all microtubules in this cell grow from thecentrosome.

Only the ends of growing microtubules are visible in this experiment;

extent of the microtubule cytoskeleton emerges.


Yuko Mimori-KiyosueKAN Research Institute

Shoichiro TsukitaFaculty of Medicine, Kyoto University

17.4 Microtubule and ER Dynamics

constantly extend into the leading edge of a migrating cell and retractagain.

red), the membrane network of the endoplasmic reticulum (shown herein green) exhibits its own dynamic behavior as tubes are extended bymotor proteins on the microtubule tracks.

Video reproduced from: C.M. Waterman-Storer and E.D. Salmon. Endoplasmicreticulum tubes are distributed in living cells by three distinct microtubule dependentmechanisms. Current Biology 8:798–806. © 1998, with permission from ElsevierScience. Clare M. Waterman-Storer

The Scripps Research Institute

Edward D. (Ted) SalmonUniversity of North Carolina at Chapel Hill



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17.5 Organelle Movement on Microtubules

organelles is added to microtubules.

Motor proteins are normally attached to the organelles. When ATPis added as a fuel for the motor proteins, some organelles bindmicrotubules, and are moved along the tracks by their motors.

Most kinesin motors move towards the plus end of microtubules.

are used to transport organelles, and occasionally a single organelle,which must have both types of motor attached, can be seen to switchdirections.

The bi-directional traffic observed here is reminiscent of that in an intactcell.

Nira PollackUniversity of California, San Francisco

Ron D. ValeHoward Hughes Medical InstituteUniversity of California, San Francisco

17.6 Kinesin

The motor protein kinesin is a dimer with two identical motor heads. kinesins pull organelles along microtubule tracks. The organelleattaches to the other end of the long coiled-coil that holds the twomotor heads together. The organelle is not shown here.

heads encounters a microtubule, it binds tightly. Microtubule binding enters the empty nucleotide binding site.

This nucleotide exchange triggers the neck linker to zipper onto thecatalytic core. This action throws the second head forward, and brings itnear the next binding site on the microtubule.

The attached trailing head hydrolyzes the ATP, and releases phosphate.As the neck linker unzippers from the trailing head, the leading headexchanges its nucleotide, and zippers its neck linker onto the catalytic

core, and the cycle repeats. microtubule.


Animation reproduced with permission from Vale & Milligan, Science 288:88–95,Supplemental Movie 1. © 2000 American Association for the Advancement ofScience.

Ron D. ValeHoward Hughes Medical InstituteUniversity of California, San Francisco

Ron MilliganThe Scripps Research Institute



10

17.7 Neutrophil Chase

this spread a neutrophil is seen in the midst of red blood cells. Staphylococcus aureus bacteria have been added. The small clump ofbacteria releases a chemoattractant that is sensed by the neutrophil.The neutrophil becomes polarized, and starts chasing the bacteria. Thebacteria, bounced around by thermal energy, move in a random path, with the bacteria and engulfs them by phagocytosis.

Digital capture: Tom Stossel, Brigham and Women’s Hospital, Harvard MedicalSchool

Music: Freudenhaus Audio Productions (www.fapsf.com)

David Roger Vanderbilt University

17.8 Crawling Actin

Myosin motors can be attached to the surface of a glass slide.Fluorescent actin filaments will bind to the motor domains of theattached myosins. When ATP is added, the myosin motors move theactin filaments.

This rapid movement can be observed in a fluorescence microscope asthe actin filaments appear to crawl across the slide.

James SpudichStanford University School of Medicine



104

17.9 Muscle Contraction

When a neuron stimulates a muscle cell, an action potential sweepsover the plasma membrane of the muscle cell. The action potentialreleases internal stores of calcium that flow through the muscle cell andtrigger a contraction.

Muscle cells have an elaborate architecture that allows them to

invaginations of the plasma membrane, called T-tubules, criss-cross thecell. When the cell is stimulated, a wave of depolarization—that is anaction potential—spreads from the synapse over the plasma membraneand via the T tubules deep into the cell. A voltage-sensitive protein cells, thereby releasing a burst of calcium ions all throughout the cytosolof the cell.

Within a contractile bundle of a muscle cell, called a myofibril, the contracting unit, or sarcomere, thin actin and thick myosin filaments because myosin-binding sites on the actin filaments are all covered bya rodshaped protein called tropomyosin. A calcium-sensitive complex,called troponin, is attached to the end of each tropomyosin molecule.When calcium floods the cell, troponin binds to it, moving tropomyosinoff the myosin-binding sites. Opening the myosin-binding site on theactin filaments allows the myosin motors to crawl along the actin, returned to the sarcoplasmic reticulum by the action of a calcium pump.Without calcium, myosin releases actin, and the filaments slide back totheir original positions.




10

17.10 Myosin

Muscle myosin is a dimer with two identical motor heads that act lever arm. A coiled-coil rod ties the two heads together, and tethersthem to the thick filament seen on top. The helical actin filament isshown at the bottom.

and phosphate, and have weak affinity for actin.Once one of the heads docks properly onto an actin subunit, phosphateis released. Phosphate release strengthens the binding of the myosinhead to actin, and also triggers the force-generating power stroke that empty nucleotide binding site, causing the myosin head to detach fromthe actin filament.

On the detached head, ATP is hydrolyzed, which re-cocks the leverarm back to its pre-stroke state. Thus, like a spring, the arm stores theenergy released by ATP hydrolysis, and the cycle can repeat.

The actin filament does not slide back after being released by the motorhead, because there are many other myosin molecules also attached to

it, holding it under tension.

The swing of the lever arm can be directly observed on single myosinmolecules, here visualized by high-speed atomic force microscopy.


Animation reproduced with permission from Vale & Milligan, Science 288:88–95,Supplemental Movie 1. © 2000 American Association for the Advancement ofScience.

Part I:Ron D. ValeHoward Hughes Medical InstituteUniversity of California, San Francisco

Ron MilliganThe Scripps Research Institute

Part II:Toshio AndoKanazawa University, Japan

17.11 Listeria ParasitesThis mammalian cell has been infected with pathogenic Listeria monocytogenes. These bacteria move throughout the cytosol byrecruiting host cell actin which polymerizes and pushes them forward,producing a comet’s tail in their wake.

Whenever a bacterium is pushed into the plasma membrane, it creates atemporary protrusion and is then bounced back to continue its randompath.

actin tails and start moving about.

These bacteria can also form actin comet tails and move in cell extracts.Here, the bacteria are expressing the green fluorescent protein, andactin is labeled red with a fluorescent dye.

The dynamics of the actin tails, that propel the bacteria through thecytosol, can be modeled, based on known biochemical and physicalproperties of actin and actin filaments.


Video reproduced by permission from Nature Reviews Molecular Cell Biology 1:110–119. © 2000 Macmillan Magazines Ltd.

Part I:Julie A. TheriotStanford University School of Medicine

Daniel A. PortnoyUniversity of California, Berkeley

Part II:Julie A. TheriotStanford University School of Medicine

Frederick S. SooStanford University

Part III:Jonathan B. AlbertsUniversity of Washington, Seattle



106

17.12 Heart Tissue

17.13 Heart Muscle Cell

17.14 Gut Epithelium: View 1

Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet, Springer-Verlag. New York,1975








10


Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet, Springer-Verlag.New York, 1975.


Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet, Springer-Verlag.New York, 1975

17.17 Tracheal Epithelium

Originally published in Freeze-Etch Histology: A Comparison between Thin Sectionsand Freeze-Etch Replicas by Lelio Orci and Alain Perrelet, Springer-Verlag.New York, 1975.






108


when cells are stretched

17.19 Media Assessment: Muscle Contraction


shrinkage shown by microtubules. As free tubulin dimers are added to a



10


Q: Polymerization of tubulin subunits into microtubules occurs with anincrease in the orderliness of the subunits (see figure below). Yet tubulinpolymerization occurs with an increase in entropy (decrease in order).


actin filament

this cytoskeletal element is essential for cell movement and forthe contraction of muscle cells.


relation for growing microtubules. Science 278:856–860.

Science 306:1021–1025.

Image courtesy of Albert Tousson

actin-binding proteinactin filamentcell cortexcentriolecentrosomeciliumcytoskeleton

KEY TERMS



110

18.1 Cdk2

proteins in the cell cycle. When activated, these kinases transferphosphate groups from ATP to serine and threonine side chains on occluded by a loop, often referred to as the T loop.

As their name suggests, cyclin-dependent kinases are activated by

and exposes the bound ATP, allowing it access to target proteins. Thus complex.

phosphorylate its target proteins.

the target serine or threonine side chains are precisely positioned withrespect to the phosphate of the bound ATP.

the kinase active site and block kinase activity by interfering with ATPbinding. Other inhibitors bind near the active site and interfere withsubstrate binding.



18.2 Early Embryonic Cell Division

This video shows the remarkable synchrony of the early embryonic celldivisions for the clawed frog Xenopus laevis. About 25 frog eggs werefertilized simultaneously in this petri dish, and then filmed over the first 25 minutes in real time. Not only do all the cells in each embryo dividesimultaneously, but the entire dish of embryos maintains essentially thesame clock time over several hours after fertilization.

PDB ID number *: Human Cyclindependentkinase 2 (1HCK); Cyclin A/Cyclin-dependentkinase 2 complex (1FIN); Phosphorylatedcyclin-dependent kinase 2 bound to cyclinA (1JST); Phosphorylated Cdk2–cyclin Asubstrate peptide complex (1QMZ); p27/cyclin A/Cdk2 complex (1JSU)



11

18.3 p53-DNA Complex

p53 is a tumor suppressor protein that prevents cells from dividinginappropriately. Loss of p53 function is associated with many forms of

through water molecules, with bases in the minor groove. Mutations

from both loop 2 and loop 3 cooperate to sequester a zinc ion, formingthe rigid heart of a zinc finger motif. Mutations that disrupt interactionsin this motif are also common in tumor cells.



18.4 Plant Cell Division

As this plant nucleus, taken from a blood lily, prepares to divide, thechromosomes first condense during prophase. Next, they gradually lineup across the center of the mitotic spindle.

At the metaphase to anaphase transition, the sister chromatids ofevery chromosome pair separate suddenly, in striking synchrony. Thechromosomes are pulled along the microtubules of the spindle toopposite ends.

After chromosome separation, small vesicles line up in the center of thespindle and fuse with each other to form a new cell wall between thetwo daughter nuclei.

At telophase, the chromosomes decondense in the newly formed nuclei. Andrew S. BajerJadwiga A. Molè-BajerUniversity of Oregon



112

18.5 Animal Cell Division

mitotic events in a lung cell grown in tissue culture.

starts to condense.

The two chromatids in each chromosome remain paired as thechromosomes become aligned on the metaphase plate.

The chromatids then separate and get pulled by the mitotic spindle intothe two nascent daughter cells.

The chromatin decondenses as the two new nuclei form and cytokinesiscontinues to constrict the remaining cytoplasmic bridge until the twodaughter cells become separated.


Video reproduced from: The Journal of Cell Biology 122:859–875, 1993. © TheRockefeller University Press

Edward D. (Ted) Salmon and Victoria SkeenUniversity of North Carolina at Chapel Hill

Robert SkibbensLehigh University

18.6 Mitotic Spindle

The mitotic spindle of a dividing human cell is reconstructed here inits full beauty from multiple optical sections that were recorded with stained in blue, and the kinetochores—where microtubules attach to the


18.7 Mitotic Spindles in a Fly Embryo

Drosophila embryo, nuclei divide rapidly and in perfect different fluorescent dyes.

After the mitotic spindle has assembled, the microtubules—shown in

green—start pulling the blue chromosomes to either pole.

The chromosomes decondense and fill the newly formed round nuclei.

and migrate to opposite poles of each nucleus where they form newmitotic spindles and the process repeats.

The whole embryo rhythmically contracts with each division cycle.

Kevin F. SullivanThe Scripps Research Institute

William SullivanUniversity of California, Santa Cruz

Claudio E. Sunkel, Tatiana Moutinho-Santos, Paula Sampaio, Isabel Amorim andMadalena CostaInstitute of Biologia Molecular and CellBiology, University of Porto, Portugal



11

Shigekazu Nagata,Kyoto university

Sakura Motion Picture Company, © 2007Sakura Motion Picture Company

18.8 Mitosis

microscopy, we observe a HeLa cell in late prophase. The membranes ofthe endoplasmic reticulum and nuclear envelope are tagged green, andthe chromosomes, which have already replicated and condensed, arered.

As the nuclear envelope breaks down during prometaphase, the

chromosomes attach to microtubules via their kinetochores and beginto move. Note how the endoplasmic reticulum absorbs the nuclearenvelope, with which it is continuous, and maintains its integrity.

cell.

At anaphase, the sister chromatids synchronously separate and arepulled towards opposite poles of the dividing cell. Note that the spaceoccupied by the mitotic spindle between the separating chromosomes

reassembles around each set of chromosomes, resealing the nuclearcompartment.

And finally, during cytokinesis, the cytoplasm is divided in two bya contractile ring of actin and myosin filaments, (which cannot beobserved here), that constrict the plasma membrane and create twodaughter cells.

After mitosis, the chromosomes in each daughter cell lose their distinctshape as they de-condense.

When we play the movie again at a faster speed, we can betterappreciate the dynamics and beauty of the endoplasmic reticulum andchromosomes during mitosis.

Tom KirchhausenHarvard Medical School

18.9 Apoptosis

Apoptosis, a form of programmed cell death, has been induced in these attachment to the substratum that they have been growing on andshrivel up without lysing.



114

18.10 Interpretive Mitosis

Chromosomes: Mari Nishino, Han Li, Lisa Watson, Manisha Ray, Beatrice Wang,Sarah Foss

Cleavage Furrow: Ryan Joseph, Ahnika Kline, Chris Cain, Arthur Millius

Centrosomes: Ben Engel, Andrew Houk

Camera Work: Will Ludington

Directed & Edited: Ben Engel

18.11 Mitotic Chromosomes


1. Which two processes together constitute the M phase of the cell





11


Why are the kinases of the cell-cycle control system known as cyclin-

Kinases of the cell-cycle control system are called cyclin-dependent…


One important role of Fas and Fas ligand is to mediate elimination and colon tumors, half the tumors were found to have amplified andoverexpressed a gene for a secreted protein that binds to Fas ligand.

How do you suppose that overexpression of this protein might…


anaphase

separate and are pulled toward opposite ends of the dividingcell.

chromatids…


rereplication via multiple mechanisms in eukaryotic cells. Genes Dev

division cycle in yeast. Science

Image courtesy of Andrew Bajer

anaphaseanaphase-promoting complex

(APC)apoptosisasterBcl2 familybi-orientation

KEY TERMS



116

19.1 Meiosis

number of sperm binding to its surface. Note the large difference in sizebetween these male and female gametes.

Although the sperm are much smaller than the egg, a single sperm hasthe same number of chromosomes as the egg. When a single sperm

and egg fuse during fertilization, each contribute a set of chromosomesto the resulting fertilized egg, called a zygote. The zygote will have thesame number of chromosomes as the other cells in the body, since eachparental gamete supplies a half-set of chromosomes.

four resulting gametes will have half the number of chromosomes asthe germ-cell precursor, and each of the gametes will be geneticallydifferent from the other gametes.

dissimilar gametes, we need to look more closely at the key molecular

events that occur during the meiotic cycle.The germ-cell precursor begins with two complete sets of replication, a complete copy of each set is made. The copies align withthe original set of chromosomes, and then link tightly, forming twin setsof chromosomes, called sister chromatids.

The maternal and paternal sister chromatids then align on themetaphase plate, where they form a set of four paired chromatids, chromatids, mixing chromosomal information at sites called chiasmata. of genetic variation.

After recombination, the reshuffled chromatids separate, and eventually

will have one half the number of chromosomes as the parent cells and,due to recombination, each gamete will be genetically different from theothers.

Animation: Graphic Pulse, Inc. (www.graphicpulse.com)



11

Mark TerasakiUniversity of Connecticut Health Center

19.2 Sea Urchin Fertilization

A sea urchin egg during fertilization is visualized here simultaneouslyby phase contrast microscopy and by fluorescence microscopy. Theegg contains a fluorescent dye that becomes brighter in the presence ofcalcium ions.

When a sperm cell fuses with the egg, the fluorescence image showsa wave of calcium ions that sweeps through the cytosol, starting from

the initial point of sperm–egg fusion. Following the path of the calciumwave, we see a membrane, called the fertilization envelope, risingfrom the cell surface. The fertilization envelope protects the fertilizedegg from the outside environment, and prevents the entry of additionalsperm. The rise in cytosolic calcium triggers an elevation of thefertilization envelope through the process of exocytosis.

released hydrolases causes a swelling of material surrounding the cell,which in turn elevates the fertilization envelope.

the plasma membrane is labeled with a fluorescent dye, seen on the

membrane which, in the optical sections shown, appears as a ring ofincreased fluorescent staining.

On the left, differential interference contrast microscopy is used todirectly view the exocytic vesicles that underlie the plasma membrane.The vesicles are visible here, because they are densely packed withprotein and consequently have a different refractive index from the contents and disappears from the image. This effect is best seen when




118


during meiosis, the maternal and paternal homologs come together and


allele

may exist in the gene pool of the species.

asexual reproduction

parent…


functional interactions among basic chromosome organizational

features govern early steps of meiotic chiasma formation. Cell111:791–802.

Nature

alleleasexual reproductionbivalentchiasma (plural chiasmata)classical genetic approachcomplementation testcrossing-over

KEY TERMS



11

Sheryl Denker and Diane BarberUniversity of California, San Francisco

Stephen J. SmithStanford University School of Medicine

Cynthia AdamsFinch University of Health Sciences andChicago Medical School

Yih-Tai ChenCellomics, Inc.

W. James NelsonStanford University School of Medicine

20.1 Wound Healing

Fibroblasts grown in vitro in a culture dish form a confluent monolayer inhibits their migration.

with a needle.

of the wound become migratory and quickly move to repair the gap.

Animation: Blink Studio Ltd. (www.blink.uk.com)

20.2 Adhesion Junctions Between Cells

These epithelial cells express green fluorescent cadherin. They are labeled cadherin is diffusely distributed over the whole cell surface.

As cells crawl around and touch each other, cadherin becomes

completely surrounded by neighbors and form a tightly packed sheet ofepithelial cells.




120

20.3 Drosophila Development

Drosophila embryo undergoes many complexmorphological changes. We first see migration of pole cells from theposterior end. These cells are destined to become the germ cells of thefly. A crest develops which separates a region that will develop into thehead, mouth parts, and fore gut. At this stage, the future tail end of the defined.

The first three segments will give rise to the head and mouth parts,the next three to the thorax, and the remaining ones to the abdomen. about 10 hours.

We can appreciate the complexity of these events by morphing a seriesof individual scanning electron micrographs into a continuous temporalsequence: migration of pole cells; development of various surfaceindentations, including openings to the air ducts, or tracheal tubes;segmentation, and tail retraction.

A similar sequence viewed from the top—or the dorsal side. Pole cells

migrate and then move into the interior as the hind gut invaginates. Therear end is temporarily folded over onto the dorsal side and eventuallystarts retracting to straighten out the embryo.

deep groove forms during gastrulation, as mesodermal cells migrateinward, where they become the precursor cells for many internalorgans. The groove then seals off as the cells that remain exterior zipperup.


20.4 Early Zebrafish Development

The first divisions of a zebrafish egg occur synchronously about every 30minutes and create a mass of cells sitting on top of a enormous yolk.

This blastoderm then begins to spread as a continuous sheath over the yolk.

interior of the embryo. They will eventually form the lining of the gut, aswell as the musculature, skeleton, and other internal tissues.

The first body segments, the head process and tail bud become visible.

The tail bud continues to extend, and we clearly see the eye develop.

17 hours into development, we can already see a recognizablevertebrate emerging, wrapped around the ball of yolk that will nourish itfor the first few days of its existence.


Video reproduced from: R.O. Karlstrom and D.A. Kane, Development 123:461. ©1996 The Company of Biologists Ltd.

Thomas C. KaufmanHoward Hughes Medical InstituteIndiana University, Bloomington

SEM:

Rudi TurnerIndiana University, Bloomington Morphing:

Michael Kaufmann, Jeffrey Giacoletti andChris Macri

Indiana University, Bloomington

Rolf O. KarlstromUniversity of Massachusetts at Amherst

Donald A. KaneUniversity of Rochester, New York



12

20.5 Megakaryocyte

Megakaryocytes are the precursor cells from which blood plateletsderive. These gigantic cells undergo an elaborate fragmentation processthat pinches off portions of the cell’s cytoplasm. These fragments arethe platelets, which are then swept away in the blood stream. Platelets


Video reproduced from: The Journal of Cell Biology 147:1299–1312, 1999. © TheRockefeller University Press.

20.6 Embryonic Stem Cells

such as red blood cells, neurons or muscle cells.

When grown in culture and exposed to an appropriate cocktail of signalmolecules, previously homogeneous, undifferentiated embryonic stem cells in these groups start contracting rhythmically and in synchrony,indicating that they have formed a fully functional contractile apparatus

appropriate gene expression programs are selectively activated in thebeating cells

20.7 Breast Cancer Cells

Normal human breast epithelial cells can be grown in cell culture. Theyform structures that resemble the little sacs of cells from which the

mammary gland, this space would be connected to ducts, and the cellswould secrete milk into it.

conditions, divide aggressively and in an uncontrolled fashion. They arealso more migratory and grow into disorganized clumps which wouldform tumors in the body.


Joseph E. Italiano, Jr.Brigham and Women's Hospital andHarvard Medical School

Ramesh A. ShivdasaniDana-Farbwe Cancer Institute and HarvardMedical School

Bruce R. ConklinGladstone Institute of CardiovascularDisease, University of California, SanFrancisco

Mina J. Bissell, Karen Schmeichel, Hong Liuand Tony HansenLawrence Berkeley Laboratories



122

20.8 The Intestinal Crypt and APC Loss

The lining of the small intestine, like the lining of most of the gut, is epithelial cells, usually called enterocytes, help absorb nutrients from the small intestine, the surface of the lining of the gut is increasedenormously by thousands of villi that protrude into the lumen. Herewe see a single villus and its internal architecture. The surface of the

villus is covered by a single layer of enterocytes, which extend down between paneth cells, divide and make copies of themselves, and alsomake transit-amplifying cells. The transit amplifying cells proliferaterapidly and move up the walls of the crypt. As the cells migrate upwardsthey begin to differentiate into goblet cells and enterocytes. While thecells are moving up the sides of the villus, they carry out the essentialfunctions of the small intestine, notably absorption of nutrients. Whenthe differentiated cells reach the tip of the villus, they undergo apoptosisand are shed into the lumen of the small intestine. The entire process The process of out-migration and rapid cell replacement is a defense

mechanism against the development of colon cancer, since almostall epithelial cells, including those that have accidentally sustainedmutations, are shed within days of their formation. Therefore, the onlymutations that can lead to the development of a cancer are those thatare retained in the crypt. This dictates that such mutations must blockthe outmigration of mutant cells from the crypt. The outmigrationof transit amplifying cells from the bottom of the crypt depends on leading to the accumulation of transit amplifying cells in the crypt. an induced gene inactivation, which appears to mimic the mutation

inactivate the intracellular protein called -catenin; the inactivation of-catenin permits the differentiation of the transit amplifying cells and -catenin accumulates within the transit amplifyingcells, which blocks both their outmigration and differentiation.The accumulated transit amplifying cells do not themselves form acarcinoma. However, they and their descendants can now accumulateadditional mutations that will drive such cells progressively to becomefull-fledged carcinoma cells.

Animation by Digizyme, Inc (www.digizyme.com)Models, Animations, Surfacing, Composite: Eric KellerStoryboard and Art Directions: Gael McGill© 2009 by Hans Clevers

Hans CleversHubrecht Institute



12

20.10 Junction Between Two Muscle Cells



20.9 Angiogenesis

As a normal part of growth and development, the body must generate see endothelial cells sprouting to form new branches from the aorta endothelial cells. The process begins when an endothelial cell of asmall vessel is activated by an angiogenic stimulus, such as vascular

endothelial cell becomes motile and extends filopodia that guide thedevelopment of a capillary sprout. The leading or “tip cell” continues tomove away from the capillary as cells behind it migrate in and divide, process, pinocytic vesicles fuse with one another. The large vacuolesformed in this way then fuse with one another, creating a lumen that example shown here, the individual cells contain either a red or a greenfluorophore. Note that the areas of green and red are distinct—even

though cells share a lumen, they do not share cytoplasm and remainseparate cells after the fusion events. Angiogenesis is critical not only innormal development and wound healing, but also in the developmentof tumors. A tumor must stimulate blood vessel formation to grow more in both normal cells and tumors. When cells within a tumor become the tissues, activating endothelial cells on nearby vessels. This results

Movie I:

Brant M. WeinsteinNational Institutes of Health

Movie II:

Georgina E. DavisUniversity of Missouri School of Medicine



124


20.11 Endothelial Cell in Liver

20.12 Liver Cells: Sinusoid Space







12


What would happen if fibroblasts produced mature collagen rather

unstructured…


Q: Mortality due to lung cancer was followed in groups of males in the dying from lung cancer as a function of age and smoking habits for four

groups of males: those who never smoked, those who stopped at age30, those who stopped at age 50, and those who continued...


of epithelium; actin filaments inside the cell attach to itscytoplasmic face.


Nature

microscopic imaging of connexin trafficking. Science 296:503–507.

adherens junctionapicalbasalbasal laminacadherincancercell junction

KEY TERMS



126

GENERAL CREDITS

Artistic and scientific direction:Peter Walter, Howard Hughes Medical Institute, University of California, San Francisco

Narrated by:Julie Theriot, Stanford University School of Medicine

Produced by:

Michael Morales, Garland Science

Animation and video production:Sumanas, Inc. (www.sumanasinc.com)Blink Studio Ltd. (www.blink.uk.biz)Graham Johnson, University of California, San FranciscoAllison BruceThomas DallmanAmy Heagle Whiting, Health Research Incorporated at the Wadsworth Center, StateUniversity of New York at AlbanyMichael Kusie (www.mkmstudio.com)Graphic Pulse Inc. (www.graphicpulse.com)Michael Morales and Lamia Harik, Garland SciencePeter Walter, Howard Hughes Medical Institute, University of California, San Francisco

High-resolution electron micrographs:Doug Bray, The University of Lethbridge, CanadaLelio Orci and Alain PerreletBrian Oates and Cyprien Lomas, The University of British Columbia

Audio recording and engineering:Adam Rossi (www.ar-audio.com)Johannes Luley, mysonictemple (www.mysonictemple.com)Freudenhaus Audio Productions

Original music:Freudenhaus Audio ProductionsChristopher ThorpeAdam Rossi

Licensed music and sound effects:CSS Music (www.cssmusic.com)Sounddogs (www.sounddogs.com)

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