Cytoplasm semi-fluid-like jelly within the cell division into
three subdivisions: cytosol, cytoskeleton & organelles
Slide 4
eukaryotic cells part of the cytoplasm about 55% of the cells
volume about 70-90% water PLUS ions dissolved nutrients e.g.
glucose soluble and insoluble proteins waste products
macromolecules and their components - amino acids, fatty acids ATP
unique composition with respect to extracellular fluids The Cytosol
Eukaryotic Cells Cytosol higher K+ lower Na+ higher concentration
of dissolved and suspended proteins (enzymes, organelles) lower
concentration of carbohydrates (due to catabolism) larger reserves
of amino acids (anabolism) ECF lower K+ higher Na+ lower
concentration of dissolved and suspended proteins higher
concentration of carbohydrates smaller reserves of amino acids
Slide 5
Cytoskeleton: internal framework of the cell gives the
cytoplasm flexibility and strength provides the cell with
mechanical support gives the cell its shape can be rapidly
disassembled in one area of the cell and reassembled in another
anchorage points for organelles and cytoplasmic enzymes also plays
a role in cell migration and movement by the cell
Slide 6
ATP Vesicle (a) Motor protein (ATP powered) Microtubule of
cytoskeleton Receptor for motor protein 0.25 m Vesicles Microtubule
(b) motility = changes in cell location and the limited movements
in parts of the cell the cytoskeleton is involved in many types of
motility requires the interaction of the cytoskeleton with motor
proteins some roles of motor proteins: 1. motor proteins interact
with microtubules (or microfilaments) and vesicles to walk the
vesicle along the cytoskeleton 2. motor protein, the cytoskeleton
and the plasma membrane interact to move the entire cell along the
ECM 3. motor proteins result in the bending of cilia and flagella
The Cytoskeleton and Cell motility
Slide 7
three major components 1. microfilaments 2. intermediate
filaments 3. microtubules Cytoskeleton:
Slide 8
Column of tubulin dimers Tubulin dimer 25 nm Actin subunit 7 nm
Keratin proteins 8 12 nm Fibrous subunit (keratins coiled together)
10 m 5 m
Slide 9
1. microfilaments = thin filaments made up of a protein called
actin -twisted double chain of actin subunits -forms a dense
network immediately under the PM (called the cortex) -also found
scattered throughout the cytoplasm
Slide 10
1.microfilaments = -function: 1. anchor integral proteins and
attaches them to the cytoplasm 2. interaction with myosin =
interacts with larger microfilaments made up of myosin - results in
active movements within a cell (e.g. muscle cell contraction) 3.
provide much of the mechanical strength of the cell resists pulling
forces within the cell 4. give the cell its shape 5. also provide
support for cellular extensions called microvilli (small
intestines)
Slide 11
Muscle cell Actin filament Myosin filament head (a) Myosin
motors in muscle cell contraction 0.5 m 100 m Cortex (outer
cytoplasm): gel with actin network Inner cytoplasm: sol with actin
subunits (b) Amoeboid movement Extending pseudopodium 30 m (c)
Cytoplasmic streaming in plant cells Chloroplast In muscle cells
motors within filaments made of myosin slide along filaments
containing actin = Muscle Contraction Examples of Actin/Myosin: In
amoeba interaction of actin with myosin causes cellular contraction
and pulls the cells trailing edge (left) forward -can also result
in the production of Pseudopodia (for locomotion, feeding) In plant
cells a layer of cytoplasm cycles around the cell -streaming over a
carpet of actin filaments may be the result of myosin motors
attached to organelles
Slide 12
2. intermediate filaments = more permanent part of the
cytoskeleton than other filaments -five types of IF filaments type
I to type V -made up of proteins such as vimentin, desmin, or
keratin -each cell type has a unique complement of IFs in their
cytoskeleton -all cells have lamin IFs but these are found in the
nucleus -some cells also have specific IFs -e.g neurons also posses
IFs made of neurofilaments type I IFs = acidic keratins type II IFs
= basic keratins type III IFs = desmin, vimentin type IV IFs =
neurofilaments type V IFs = nuclear lamins kidney cell -
vimentin
Slide 13
2. intermediate filaments = function: 1. impart mechanical
strength to the cytoskeleton specialized for bearing tension (like
microfilaments) 2. support cell shape e.g. forms the axons of
neurons 3. anchor & stabilize organelles e.g. anchors the
nucleus in place 4. transport materials e.g. movement of
neurotrasmitters into the axon terminals
Slide 14
3. microtubules = hollow rods or straws - made of repeating
units of proteins called tubulin - function: 1. cell shape &
strength 2. organelles: anchorage & movement 3. mitosis - form
the spindle (chromosome movement) 4. form many of the
non-membranous organelles - cilia, flagella, centrioles -tubulin
-tubulin components of: 1.mitotic spindle 2.cilia and flagella
3.axons of neurons
Slide 15
3. microtubules -the basic microtubule is a hollow cylinder =
13 rows of tubulin called protofilaments -tubulin is a dimer two
slightly different protein subunits -called alpha and beta-tubulin
-alternate down the protofilament row -tubulin -tubulin
Slide 16
can be found as a single tube a doublet and a triplet -animal
cells microtubule assembly occurs in the MTOC (microtubule
organizing center or centrosome) -area of protein located near the
nucleus -within the MTOC/centrosome : 1. a pair of modified MTs
called centrioles 2. pericentriolar material made up of factors
that mediate microtubule assembly 3. - end of assembling
microtubules (MTs grow out from the centrosome) -other eukaryotes
there is no MTOC -have other centers for MT assembly
Slide 17
-MT disassembly is a mechanism of certain chemotherapy drugs
http://www.nature.com/nrc/journal/v4/n4/fig_tab/nrc1317_F4.html
Microtubule Assembly within the MTOC: -MTs are easy to assemble and
disassemble by adding or removing tubulin dimers -one end
accumulates or releases tubulin dimers much faster than the other
end called the plus end -the tubulin subunits bind and hydrolyze
GTP determines how they polymerize into the MT
Slide 18
A. Centrioles: short cylinders of tubulin - 9 microtubule
triplets -called a 9+0 array (9 peripheral triplets, 0 in the
center) -grouped together as pairs arranged perpendicular to one
another -make up part of the centrosome or MTOC -role in MT
assembly?? -also have a role in mitosis - spindle and chromosome
alignment Non-membranous Organelles
Slide 19
B. Cilia & Flagella cilia = projections off of the plasma
membrane of eukaryotic cells covered with PM BUT NOT MEMBRANOUS
ORGANELLES beat rhythmically to transport material power &
recovery strokes found in linings of several major organs covered
with mucus where they function in cleaning e.g. trachea, lungs
Trachea
Slide 20
B. Cilia & Flagella cytoskeletal framework of a cilia or
flagella = axoneme (built of microtubules) contain 9 groups of
microtubule doublets surrounding a central pair= called a 9+2 array
cilia is anchored to a basal body just beneath the cell surface
Microtubules Plasma membrane Basal body Longitudinal section of
motile cilium (a) 0.5 m 0.1 m (b) Cross section of motile cilium
Outer microtubule doublet Dynein proteins Central microtubule
Radial spoke Cross-linking proteins between outer doublets Plasma
membrane Triplet (c) Cross section of basal body
Slide 21
flagella = resemble cilia but much larger 9+2 array found
singly per cell functions to move a cell through the ECF -DO NOT
HAVE THE SAME STRUCTURE AS BACTERIAL FLAGELLA
Slide 22
Cilia, Flagella and Dynein motors Dynein protein Cross-linking
proteins between outer doublets Microtubule doublets in flagella
and motile cilia flexible cross-linked proteins are found evenly
spaced along the length blue in the figure these proteins connect
the outer doublets to each other and to the two central MTs of a
9+2 array each outer doublet also has pairs of proteins along its
length these stick out and reach toward its neighboring doublet
called dynein motors responsible for the bending of the
microtubules of cilia and flagella when they beat
Slide 23
dynein walking moves flagella and cilia dynein protein has two
feet that walk along the MT dyneins alternately grab, move, and
release the outer microtubules BUT: without any cross-linking
between adjacent MTs - one doublet would slide along the other
elongate the cilia or flagella rather than bend it so to bend the
MT must have proteins cross- linking between the MT doublets (blue
lines in figure) protein cross-links limit sliding forces exerted
by dynein walking causes doublets to curve = bending the cilium or
flagellum Cilia, Flagella and Dynein motors Microtubule doublets
Dynein protein ATP (a) Effect of unrestrained dynein movement
Cross-linking proteins between outer doublets ATP Anchorage in cell
(b) Effect of cross-linking proteins (c) Wavelike motion 1 2 3
Slide 24
Membranous Organelles completely surrounded by a phospholipid
bilayer similar to the PM surrounding the cell allows for isolation
of each individual organelle - so that the interior of each
organelle does not mix with the cytosol -known as
compartmentalization BUT - cellular compartments must talk to each
other therefore the cell requires a well-coordinated transport
system in order for the organelles to communicate and function
together -vesicular transport -active process requires ATP
Slide 25
Membranous Organelles major functions of the organelles 1.
protein synthesis ER and Golgi 2. energy production mitochondria 3.
waste management lysosomes and peroxisomes
Slide 26
Membranous Organelles the organelles of a eukaryotic cell are
not constructed de novo they require information in the organelle
itself when a cell divides it must duplicate its organelles also in
general the cell enlargens existing organelles by incorporating new
phospholipids and proteins into them the bigger organelle then
divides when the daughter cell divides during cytokinesis
Slide 27
The Endomembrane System: A Review endomembrane system is a
complex and dynamic player in the cells compartmental organization
divides the cell into compartments includes the: Nucleus
Endoplasmic Reticulum Golgi apparatus lysosomes, endosomes vacuoles
and vesicles
Slide 28
The Endomembrane System: A Review proteins travelling through
the ER and Golgi are destined for 1. Secretion outside the cell 2.
Plasma membrane 3. Lysosome
Slide 29
1.Endoplasmic reticulum (ER) = series of membrane-bound,
flattened sacs in communication with the nucleus and the PM -each
sac or layer = cisternae -inside or each sac = lumen (10% of total
cell volume) -distinct regions of the ER are functionally
specialized Rough ER vs. Smooth ER
Slide 30
-three functions: 1. synthesis phospholipids, lipids and
proteins proteins phospholipids & lipids 2. storage
intracellular calcium 3. transport site of transport vesicle
production
Slide 31
1.Endoplasmic reticulum (ER) -two types: Rough ER - outside
studded with ribosomes -continuous with the nuclear membrane
-protein synthesis, phospholipid synthesis -also the initial site
of processing and sorting of proteins
Slide 32
1.Endoplasmic reticulum (ER) -the import of proteins into the
RER is a co-translational process -import of proteins into an
organelle = translocation -proteins are imported as they are being
translated by ribosomes -in contrast to the import of proteins into
other organelles (e.g. chloroplasts, mitochondria, peroxisomes) and
the nucleus = post-translational process
Slide 33
Co-translational Protein Synthesis two kinds of proteins enter
the ER: 1.ER proteins transmembrane proteins that stay stuck in the
ER membrane PLUS ER lumen proteins that remain in the ER 2.
proteins destined for the Golgi, PM or lysosome or secretion
Slide 34
Co-translational Protein Synthesis transport from the ribosome
across the ER membrane requires the presence of an ER signal
sequence (red in the figure) 16-30 amino acids at the beginning of
the peptide sequence (N-terminal)
Slide 35
a complex of proteins will bind this signal in the cytoplasm =
signal recognition particle/SRP the ER membrane has receptor for
the SRP and ribosome SRP receptor (yellow protein in figure) the
ribosome is docked next to a hole in the ER membrane (blue protein
in figure) = translocon translocon recognizes the signal sequence
and binds it guides the rest of the translating polypeptide into
the ER lumen once the polypeptide is fed into the ER lumen a
peptidase (located in the SRP receptor complex) cleaves the signal
sequence off Co-translational Protein Synthesis
Slide 36
Translocation try this animation it might be a bit complicated
but give it a try anyway
http://www.rockefeller.edu/pubinfo/proteintarget.html heres a
figure from a molecular biology text that summarizes the
process
Slide 37
once the polypeptide is fed into the ER lumen a peptidase
cleaves the signal sequence off = PRODUCES A SOLUBLE PROTEIN
localizes to the ER lumen the presence of another sequence of amino
acids within the polypeptide stop-transfer sequence the
translocator stops translocating and transfers the polypeptide into
the ER membrane = PRODUCES A TRANSMEMBRANE PROTEIN
Slide 38
Modifications in the RER 1. folding of the peptide chain
actually a spontaneous process due to the side chains on the amino
acids only properly folded proteins get transported to the Golgi
for additional processing and transport many proteins located in
the ER which supervise this folding 2. formation of disulfide bonds
help stabilize the tertiary and quaternary structure of
proteins
Slide 39
Modifications in the RER 3. breaking of specific peptide bonds
proteolytic cleavage or proteolysis 4. assembly into multimeric
proteins (more than one chain) for an animation go to
http://sumanasinc.com/webcontent/animatio
ns/content/proteinsecretion_mb.html
Slide 40
Modifications in the RER 5. addition and processing of
carbohydrates = glycosylation N-linked glycosylation = attachment
of 14 sugar residues as a group to an asparagine amino acid within
the protein the sugar is actually built and then transferred as one
unit to the nearby translating protein by a transferase protein
needs to be trimmed down in order to allow protein folding most
proteins made in the ER undergo N-linked glycosylation
Slide 41
Smooth ER extends from the RER -free of ribosomes main function
is transport vesicle synthesis area where this happens can be
called transitional ER 1.Endoplasmic reticulum (ER)
Slide 42
-but other cell types have SER with enzymes embedded in it for
additional functions: 1.lipid and steroid biosynthesis for
membranes 2. detoxification of toxins and drugs 3. cleaves glucose
so it can be released into the bloodstream 4. uptake and storage of
calcium
Slide 43
2. Ribosomes = can be considered a nonmembranous organelle made
in the nucleolus 2 protein subunits in combination with rRNA -large
subunit = 28S rRNA, 5.8S rRNA, 5 rRNA + 50 proteins -small subunit
= 18S rRNA + 33 proteins proteins are translating in the cytoplasm
and imported into the nucleus rRNA is transcribed in the nucleolus
ribosomes found in association with the ER = where the peptide
strand is fed into from the ribosome also float freely within the
cytoplasm as groups = polyribosomes
Slide 44
3. Golgi Apparatus = stacks of membranes called cisternae
(cisterna, singular) -the first sac in the stack = cis-face (faces
the ER) -the last sac in the stack = trans-face -the ones in the
middle = medial cisterna or cisternae Named after Camillo Golgi in
1897
Slide 45
3. Golgi Apparatus -associated with the cis and trans faces are
additional networks of interconnected cisternal structures -called
the cis Golgi network (CGN) and trans Golgi network (TGN) -the TGN
has a critical role in protein sorting
Slide 46
3. Golgi Apparatus site of final protein modification and
packaging of the finished protein functions: 1. protein
modification A. glycosylation - creation of glycoproteins and
proteoglycans B. site for phosphate addition to proteins =
phosphorylation C. protein trimming 2. production of sugars Golgi
makes many kinds of polysaccharides 3. formation of the lysosome 4.
packaging of proteins and transport to their final destination TGN
acts as a sorting station for transport vesicles
Slide 47
Modifications in the Golgi glycosylation = produces a
glycoprotein or a proteoglycan most plasma membrane and secreted
proteins have one or more carbohydrate chains sugars help target
proteins to their correct location; are important in cell-cell and
cell- matrix interactions two kinds: N-linked and O linked O-linked
sugars are added one at a time in the Golgi to the amino acids
serine, threonine or lysine (usually one to four saccharide
subunits total) N-linked sugars are added as a group (about 14
sugars!) in the ER Proteoglycan
Slide 48
glycosylation: glycosylation starts in the ER N-linked
glycosylation addition of N- linked oligosaccharides many of these
N-linked sugar residues are trimmed off within the ER important for
folding of the protein glycosylation continues in the cisternae of
the Golgi addition of O-linked oligosaccharides to proteins PLUS
modification of the N-linked oligosaccharides - either addition or
removal of sugar residues
Slide 49
Why Glycosylation? the vast abundance of glycoproteins suggests
that glycosylation has an important function N-linked is found in
all eukaryotes including single-celled yeasts a type of N-linked
can even be found in archaea in their cell walls WHY GLYCOSYLATION?
N-linked in the ER is important for proper protein folding N-linked
also limits the flexibility of the protein the sugar residues can
prevent the binding of pathogens sugar residues also function as
signaling chemicals sugar residues function in cell
interactions
Slide 50
Why Glycosylation? O-linked glycosylation O-linked are added
one at a time in the Golgi to the amino acids serine, threonine or
lysine (one to four saccharide subunits total) added on by enzymes
called glycosyltransferases human A, B and O antigens are sugars
added onto proteins and lipids in the plasma membrane of the RBC
everyone has the glycosyltransferase needed to produce the O
antigen those with blood type A have an additional Golgi
glycosyltransferase enzyme which modifies the O antigen to make the
A antigen a different glycosyltransferase is required to make the B
antigen both glycosyltransferases are required for the creation of
the AB antigen coded for by specific gene alleles on chromosome 9
(ABO locus)
Slide 51
some PM proteins and most secretory proteins are synthesized as
larger, inactive pro-proteins that will require additional
processing to become active this processing occurs very late in
maturation processing is catalyzed by protein-specific enzymes
called proteases some proteases are unique to the specific
secretory protein trimming occurs in secretory vesicles that bud
from the trans-Golgi face processing could be at one site (albumin)
other proteins may require more than one peptide bond (insulin)
Modifications in the Golgi: Protein Trimming
Slide 52
The Golgi: Protein transport within the cytoplasm protein
transport within the cell is tightly regulated most proteins
usually contain tag or signals that tell them where to go in the
Golgi - specific sequences within a protein will cause: 1.
retention in the Golgi 2. will target it to lysosomes 3. send it to
the PM for fusion 4. send it to the PM for secretion a lack of a
signal means you will automatically be secreted = constitutive
secretion
Slide 53
targets: 1.secretory vesicles for exocytosis 2.membrane
vesicles for incorporation into PM 3.transport vesicles for the
lysosome SO - WHERE DO PROTEINS GO AFTER THE GOLGI??? -proteins
budding off the Golgi have three targets:
Slide 54
ER proteins stay in the ER never traffic to the Golgi these ER
proteins will have a retention signal Ribosomal proteins
translation of ribosomal proteins are done in the cytoplasm by
polyribosomes assembled into the large and small protein subunits
in the cytoplasm imported into the nucleus rRNAs are transcribed in
the nucleolus - no translation!!! protein subunits and rRNAs are
assembled in the nucleolus to form the small and large ribosomal
subunits exported from nucleus mitochondrial proteins the
mitochondria has its own DNA, transcribes its own mRNA and has its
own ribosomes for translation WHAT IF YOU ARENT ONE OF THESE
PROTEINS??
Slide 55
4. Lysosomes = garbage disposals -dismantle debris, eat foreign
invaders/viruses taken in by endocytosis or phagocytosis -also
destroy worn cellular parts from the cell itself and recycles the
usable components = autophagy -form by budding off the trans-Golgi
network?? -cell biologists not really sure exactly how the lysosome
forms
Slide 56
4. Lysosomes -contains powerful enzymes to breakdown substances
into their component parts -over 40 kinds of hydrolytic enzymes
-these enzymes are collectively known as acid hydrolases -acidic
interior - critical for function of these enzymes -the hydrolytic
enzymes of the lysosome need to be cleaved first in order to become
enzymatically active -done by the acidity of the lysosomes interior
- acidic interior created and maintained by a hydrogen pump (H+
ATPase) that pumps H+ into the interior - Active transport
-chloride ions that diffuse in passively through a chloride channel
- forms hydrochloric acid (HCl)
Slide 57
4. Lysosomes several different kinds of lysosomes diverse in
shape and size types: lysosome form from the budding and fusion of
vesicles from the TGN these vesicles contain lysosomal enzymes
early endosome forms through receptor-mediated endocytosis from the
plasma membrane late endosome forms by fusion of early endosomes
with vesicles containing lysosomal enzymes endolysosome fusion of a
late endosome with a pre-existing lysosome transforms it into a
lysosome an endolysosome may be considered an immature
lysosome
Slide 58
Diseases at the Organelle Level Tay Sachs and lysosomes: also
known a Hexosaminidase A deficiency -named after Waren Tay and
Bernard Sachs -key identifying mark = cherry red spot in the retina
-lack one of the 40 lysosomal enzymes hexosaminidase -results in
the accumulation of gangliosides (phospholipid) in the cell
membrane of neurons -death of the neuron results failure of nervous
system communication -infantile form of the disease = death by 4
yrs -juvenile form = death from 5 to 15 yrs -adult onset not fatal;
progressive loss of nervous function -most common in Ashkenazi
Jews, French Canadians and Cajun populations in Lousiana (same
mutation as Jews)
Slide 59
5. Mitochondria -surrounded by a dual phospholipid bilayer an
outer mitochondrial membrane an inner mitochondrial membrane a
fluid-filled space = mitochondrial matrix (contains ribosomes!)
-the inner membrane is folded into folds called cristae -these
increase the membrane surface area for the enzymes of Oxidative
Phosphorylation
Slide 60
outer membrane - 50% phospholipid & 50% protein -very
permeable - contains pores for the import and export of critical
materials inner membrane - 20% phospholipid & 80% protein -less
permeable vs. the outer membrane -folded extensively to form
partitions = cristae -contains proteins that work to create an
electrochemical gradient -contains enzymes that use this gradient
for the synthesis of ATP -also contains pumps to move ATP into the
cytosol matrix - lumen of the mitochondria -breakdown of glucose
into water and CO 2 ends here (enzymes of the Transition phase
Krebs Cycle)
6. Peroxisomes: found in all cells but abundant in liver and
kidney cells -only identified in 1954 -may arise from pre-existing
peroxisomes or may bud from the ER -major function is oxidation
(breakdown) of long chain fatty acids (beta-oxidation) -results in
the conversion of the fatty acid into acetyl coA Krebs cycle -in
plant cells beta-oxidation is only done by the peroxisome -in
animal cells the mitochondria can also perform this reaction
-oxidation is done by oxidases = enzymes that use oxygen to oxidize
substances -remove hydrogen atoms from the fatty acid -this
reaction generates hydrogen peroxide (H 2 O 2 )
Slide 63
6. Peroxisomes: found in all cells but abundant in liver and
kidney cells PROBLEM #1: H2O2 is very corrosive -therefore
peroxisomes also contain an enzyme called catalase to break this
peroxide down into water and oxygen PROBLEM #2: the electron
transport chain in mitochondria produces superoxide radicals (O2 -
) as a normal consequence of electron leaking (from complex I)
-peroxisomes also contain anti-oxidant enzymes to break down other
dangerous oxidative chemicals made by the cell during metabolism
e.g. SOD breaks down O2 - to make H2O2 -other functions of
peroxisomes: 1. synthesis of bile acids 2. breakdown of alcohol by
liver cells
Slide 64
Adrenoleukodystrophy and peroxisomes: -X linked disorder in the
gene ABCD1 (transporter protein) -1:20,000 to 1:50,000 births -in
ALD - peroxisomes lack an essential enzyme -leads to a build up of
a long-chain saturated fatty acids on cells of throughout the body
-can results in the loss of the myelin sheath not known why
-lethargy, skin darkens, blood sugar drops, altered heart rhythm
imbalanced electrolytes, paralysis, death *** slowed by a certain
triglyceride found in rapeseed oil Lorenzo Odone = Lorenzos Oil
(mixture of unsaturated fatty acids that slows the development of
these saturated FAs) F-actin and peroxisomes