Cell Biology Introduction

Cell Biology

Bio 108

I. Introduction A. The Origin and Evolution of Cells B. Cells as Experimental Models C. Tools of Cell Biology

Cell biology (formerly cytology, from the Greek kytos, "container") is an academic discipline that studies cells • their physiological properties

• their structure,• the organelles they contain• interactions with their environment,• their life cycle, division and death.

Cell – basic unit of life structurally and functionally.

History:a. Robert Hooke (1665)using his microscope

discovers cells in cork b. Schleiden ; Schwann and Virchow Cell theory:

1. All organisms are composed of one or more cells

2. The cell is the structural unit of life3. Cells can arise only by division from

preexisting cells

The Diversity and Commonality of Cells

Various cell types: shape, size, intracellular organizations,

polarization – Functions

Dynamic Nature of Cell

-it has the capacity to grow to reproduce become specialized ability to respond to stimuli adapt to changes in its environment

Fundamental properties shared by all cells: (conserved throughout evolution)

1. all cells employ DNA as their genetic material

2. surrounded by plasma membrane3. use the same basic mechanisms for

energy metabolism

Two Main Classes of Cells:

a.Prokaryotic cells – no nucleus - simpler structure (bacteria)

b.Eukaryotic cells - contain nucleus - more complex structure(protists,

fungi, plants & animals)

Fig.1.2.Average_prokaryote_cell-_en.svg (SVG file, nominally 494 × 402 pixels, file size: 135 KB)

A bacterium

The animal cell

A plant cell

The Main Functions of the Membrane-bounded Compartments of a Eukaryotic Cell

Compartment Main Function

Cytosol contains many metabolic pathwaysprotein synthesis

Nucleus contains main genomeDNA and RNA synthesis

Endoplasmic reticulum (ER)

synthesis of most lipidssynthesis of proteins for distribution to many organelles and plasma membrane

Golgi apparatus modification, sorting, and packaging of proteins and lipids for either secretion or delivery to another organelle

Lysosomes intracellular degradationEndosomes sorting of endocytosed material

Mitochondria ATP synthesis by oxidative phosphorylationChloroplasts (in plant cells)

ATP synthesis and carbon fixation by photosynthesis

Peroxisomes oxidation of toxic molecules

Organisms:1. Unicellular (eg. bacteria, amoebas &

yeasts) – capable of independent self-replication

2. Multicellular(eg. Humans)- composed of collection of cells w/c fxns in a coordinated manner w/ diff cells specialized to perform particular tasks.

All organisms: 1 or more cells

PROK

ARYO

TES

EUKA

RYOT

ES

  Prokaryotes EukaryotesTypical organisms bacteria, archaea protists, fungi, plants, animals

Typical size ~ 1-10 µm ~ 10-100 µm (sperm cells, apart from the tail, are smaller)

Type of nucleus nucleoid region; no real nucleus real nucleus with double membrane

DNA circular (usually) linear molecules (chromosomes) with histone proteins

RNA-/protein-synthesis coupled in cytoplasmRNA-synthesis inside the nucleusprotein synthesis in cytoplasm

Ribosomes 50S+30S 60S+40S

Cytoplasmic structure very few structureshighly structured by endomembranes and a cytoskeleton

Cell movement flagella made of flagellinflagella and cilia containing microtubules; lamellipodia and filopodia containing actin

Mitochondria none one to several thousand (though some lack mitochondria)

Chloroplasts none in algae and plants

Organization usually single cellssingle cells, colonies, higher multicellular organisms with specialized cells

Cell division Binary fission (simple division) Mitosis (fission or budding)Meiosis

DNA content (base pairs) 1 × 106 to 5 × 106 1.5 × 107 to 5 × 109

Table 1: Comparison of features of prokaryotic and eukaryotic cells

The Origin and Evolution of Cells

the First Cell:

-all present day cells (both prokaryotes & eukaryotes) descended from a single ancestor.

-the 1st cell is thought to have arisen at least 3.8 B years ago as a result of enclosure of self-replicating RNA in a phospholipid membrane (RNA world hypothesis)

Present-Day Prokaryotes-divided into two groups: the archaebacteria and

the eubacteria which diverged early in evolution

Eukaryotic Cells-thought to have evolved from symbiotic

associations of prokaryotes (ENDOSYMBIONT THEORY)

ENDOSYMBIOSISA large anaerobic, heterotrophic prokaryote engulfs

a small aerobic prokaryoteThe aerobic endosymbiont has evolved into a mitochondrion

A portion of the plasma membrane has invaginated and evolved into a nuclear envelope and endoplasmic reticulum

(primitive eukaryote)

Nonphotosynthetic protist, fungal,animal cells

Engulfs a photosynthetic prokaryoteEvolve into a chloroplast

Algal & plant cells

Endosymbiont Theory

Fig.1.5. Time scale of evolution The scale indicates the approximate times at which some of the major events in the evolution of cells are thought to have occurred.

Figure 1.6. Generation of metabolic energy Glycolysis is the anaerobic breakdown of glucose to lactic acid. Photosynthesis utilizes energy from sunlight to drive the synthesis of glucose from CO2 and H2O, with the release of O2 as a by-product. The O2 released by photosynthesis is used in oxidative metabolism, in which glucose is broken down to CO2 and H2O, releasing much more energy than is obtained from glycolysis.

The Evolution of Metabolism:

Figure 1.6. Evolution of cells Present-day cells evolved from a common prokaryotic ancestor along three lines of descent, giving rise to archaebacteria, eubacteria, and eukaryotes. Mitochondria and chloroplasts originated from the endosymbiotic association of aerobic bacteria and cyanobacteria, respectively, with the ancestors of eukaryotes.

Cells as Experimental Models

E. coli S. cerevisiae Dictyostelium discoideum

Arabidopsis thaliana Caenorhabditis elegans Drosophila melanogaster

Xenopus laevis zebrafish

House mouse

Choosing the Right Experimental Organism for the Job

Organism Haploid DNA content (millions of base pairs)

BacteriaMycoplasma 0.6E. coli 4.6Unicellular eukaryotesSaccharomyces cerevisiae (yeast) 12Dictyostelium discoideum 70Euglena 3000PlantsArabidopsis thaliana 130Zea mays (corn) 5000AnimalsCaenorhabditis elegans (nematode) 97Drosophila melanogaster (fruit fly) 180Chicken 1200Zebrafish 1700Mouse 3000Human 3000

Table 1.2 DNA Content of Cells

C. Tools of Cell Biology

Tools of Cell Biology1. Light Microscopy

• Bright-field microscopy• Phase-contrast microscopy• Differential interference –contrast microscopy• Video-enhanced differential interference-contrast microscopy• Fluorescence microscopy• Confocal scanning microscopy

2. Electron microscopy• Transmission electron microscopy• Scanning electron microscopy

- To magnify an object, it uses a system of lenses to manipulate the path a light beam travels between the object being studied and the eye- Produce a maximum useful magnification of about1000 times the original size.- Has three lenses:

1. the condenser(focuses light on the specimen 2. the objective(s)

a. Low-power objectiveb. High-power objectivec. Oil-immersion objective

3. the eyepiece (ocular)

Light Microscope- earliest tool of cytologist- Limit of resolution is λ/2= 0.20-0.35 um

Objective Designation

Objective Magnification

Eyepiece Magnification

Total Magnification

Low Power 10 10 100

High Power (high dry)

40 10 400

Oil Immersion 100 10 1000

Table 2-1

Unit of length

Meter (m) Centimeter (cm)

Millimeter (mm)

Micrometer (um)

Nanometer (nm)

Micrometer (um)

0.000001 10-6

0.0001 10-4

0.001 10-3

1 1000 103

Nanometer (nm)

0.000000001 10-9

0.000000110-7

0.00000110-6

0.00110-3

1

Angstrom (Å)

0.0000000001 10-10

0.0000000110-8

0.000000110-7

0.000110-4

0.110-1

Table 2-2. Metric unit equivalents in expressing cell dimensions

Terms:1. Limit of Resolution-refers to how far apart

adjacent objects must be in order to be distinguished as separate entities. (eg. LOR of microscope is 400 nm)

2. Resolving Power-expressed in terms of λ(the wavelength of light used to illuminate the sample)

-the smaller is the limit of resolution the greater is the resolving power

A. Light Microscope

Resolving Power of any microscope-a measure of its ability to discriminate between two adjacentobjects.- is a function of the wavelength of light and the numerical aperture of the lens system- light microscopes (using visible light) have RP of approximately0.25 um which means that particles of a smaller size cannot be distinguished from one another).

Fig.1-9. Resolving PowerOf the Human Eye, theLight microscope and theElectron Microscope

1 um = 10-6 m (one-millionth of a meter)1 nm = 10-9 m (one-billionth of a meter)

1000 nm = 1 umAngstrom (Å) = 10-10 m or 0.1 nm

Type of Microscopy

Maximum useful magnification

Appearance of specimen

Useful Applications

Bright-field 1000-2000 Specimens stained or unstained; bacteria generally stained and appear color of stain

Gross morphological features of bacteria, yeasts, molds,algae and protozoa

Dark-field 1000-2000 Generally unstained; appears bright or “lighted” in an otherwise dark field

Microorganisms that exhibit some characteristic morphological feature in the living state and in fluid suspension e.g. spirochetes

Fluorescence 1000-2000 Bright and colored; color of the fluorescent dye

Diagnostic techniques where fluorescent dye fixed to organism reveals the organism’s identity

a. Differential-interference-contrast micrograph of a mitotic yeast cell.

b. Fluorescence microscopy

c. Phase-contrast micrograph of fibroblasts in culture.

Type of Microscopy

Maximum useful magnification

Appearance of specimen

Useful Applications

Phase-contrast 1000-2000 Varying degrees of “darkness”

Examination of cellular structures in living cells of the larger microorganisms, e.g yeasts, algae, protozoa and some bacteria

c) Dark-field photomi-crograph of Mysisd)Phase-contrast micrograph of a cheekcella) Flourescent microscope b) flourescent micrograph

of chromosomes and mitotic spindle

B. Electron Microscope-uses a beam of electrons controlled by a system of

magnetic fields -has high resolving power thus greater magnification

(can resolve objects separated by a distance of 0.003 umcompared to 0.25 um of light microscope).

-useful magnification is 200,000 to 400,000-use in the examination of viruses and the ultra-

stucture of microbial cells.- has two types:

a. Scanning electron microscopy (SEM)-employed to study the surface structure

of a specimen(eg. Attachment of bacterial cells toobjects)

b. Transmission electron microscopy (TEM)-used to view subcellular components

(even nucleic acid molecules)

Scanning electron micrograph of a flea

Transmission electron micrograph of Bacillus anthracis

Techniques and Methods of Studying Cells

Several different techniques exist to study cells:

1. Cell culture2. Cell Fractionation3. Immunostaining4. Computational Genomics5. DNA MICROARRAYS6. Gene knockdown7. In situ hybridization 8. Polymerase Chain Reaction

(PCR)

Figure 1-7. A procedure used to make a transgenic plant.

Figure 1.8. Using DNA microarrays to monitor the expression of thousands of genes simultaneously.

Figure 8-63. Using cluster analysis to identify sets of genes that are coordinately regulated.

Figure 8-47. Results of a BLAST search. Sequence databases can be searched to find similar amino acid or nucleic acid sequences. Here a search for proteins similar to the human cell-cycle regulatory protein cdc2 (Query) locates maize cdc2 (Subject), which is 68% identical (and 82% similar) to human cdc2 in its amino acid sequence.

Basic Local Alignment Search Tool (BLAST)

-a technique that can be used to better visualize cells and cell components under a microscope.

-by using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell.

- Most stains can be used on fixed, or non-living cells, while only some can be used on living cells; some stains can be used on either living or non-living cells.

Cell staining

Why Stain Cells?

-most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope.

-Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

- to determine biomass in an environment of interest.

How Are Cells Stained and Slides Prepared?Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:

•Permeabilization - treatment of cells, generally with a mild surfactant, which dissolves cell membranes in order to allow larger dye molecules to enter inside the cell.

•Fixation - serves to "fix" or preserve cell or tissue morphology through the preparation process. This process may involve several steps, but most fixation procedures involve adding a chemical fixative that creates chemical bonds between proteins to increase their rigidity. Common fixatives include formaldehyde, ethanol, methanol, and/or picric acid.

•Mounting - involves attaching samples to a glass microscope slide for observation and analysis. Cells may either be grown directly to the slide or loose cells can be applied to a slide using a sterile technique. Thin sections (slices) of material such as tissue may also be applied to a microscope slide for observation.

•Staining - application of stain to a sample to color cells, tissues, components, or metabolic processes. This process may involve immersing the sample (before or after fixation or mounting) in a dye solution and then rinsing and observing the sample under a microscope. Some dyes require the use of a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate. The mordanted stain will remain on/in the sample when excess dye solution is washed away.

A. Dye-cellular interactions

____________________________________Fig. 1. Schematic representation of dye-protein interactions. At pI of protein (center), negligible binding of charged dyes occurs. Below pI (left), protein binds acid(=anionic) dyes; above pI (right), protein binds basic (=cationic) dyes. At physiologic pH, specific proteins may exhibit net (+) or net (-) charges and are therefore characterized as acidophilic (=affinity for acid dyes) or basophilic (=affinity for basic dyes), respectively.

Important stains and reactions1. Cationic ("basic") Dyes: tissue components stained with these dyes are basophilic.

a. Examples of basic dyesi. Hematoxylins (behave as cationic

dyes)ii. Azuresiii. Methylene blueiv. Toluidine blue

b. Examples of basophilic tissue componentsi. Nuclei and nucleoliii. Cytoplasmic RNA

(e.g . ,ergastoplasm, Nissl)

2. Anionic ("acid") Dyes: tissue components stained with these dyes are acidophilic.

a. Examples of acid dyesi. Aniline blue: blueii. Eosin: pink-rediii. Fast green: greeniv. Orange G: orangev. Picric acid: yellow

b. Examples of acidophilic tissue components

i. Most cytoplasmii. Hemoglobiniii. Keratiniv. Collagens

3. Special Stains and Reactionsa. Alcian blue (=basic dye) for

polyanions and acidic glycoproteins (alcianophilia)b. Elastic stains (e.g., orcein, resorcin

fuchsin, Verhoeff)c. Feulgen reaction for DNAd. Lipid colorants/stains (e.g., Sudan black, oil red O, osmium tetroxide)e. Masson trichrome for differential staining of several tissuesf. Metachromasia (e.g., toluidine blue for polyanions)g. PAS reaction for vic-glycolsh. Silver stains for Golgi, basement membranes, reti- cular fibers, neurofibrils (argyrophilia)i. Vital stains (e.g., trypan blue, India ink)

j. Oxidation-reduction reactions andstainingk. Romanowsky dyes: mixtures of acid and basic dyes for staining blood smears

i. Mechanism• Orthochromasia -cellular componentsstained either pink/red due to eosin or blue due to basic dye component

• Polychromasia –layered staining with both dyes

• Metachromasia -color shift of basic dye from blue to violet-purple due to high concentrations of polyanions

ii. Examples• Wright stain• Giemsa stain

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