Cell Biology Test 3 Review
Chapter 12: Bioenergetics and metabolism:
Mitochondria, chloroplasts and peroxisomes. !
General Info!
Generation of metabolic energy is a major activity of alll cells. !
Mitochondria generate energy from the breakdown of lipids and carbohydrates. !
Chloroplasts use sunlight energy to generate ATP and reducing power to synthesize
carbohydrates from CO2 and H2O.!
Peroxisomes contain enzymes involved in a variety of metabolic pathways. Protein sorting to
these is distinct from vesicular transport. Proteins synthesized on free ribosomes and imported
into the organelles as completed polypeptides. !
Mitochondria!
Surrounded by a double membrane. These are the inner (with cristae that extend into the
interior —> matrix) and outer membranes which are separated by an intermembrane space. !
Matrix contains the genetic system (mitochondrial DNA) as well as the enzymes required for
oxidative metabolism. !
Pyruvate is transported to mitochondria where its complete oxidation to CO2 yields the bulk of
ATP (usable energy) obtained from glucose metabolism. !
Breakdown of fatty acids occurs in the mitochondrial matrix. !
Enzymes for the citric acid cycle are in the mitochondrial matrix. Oxidative phosphorylation
occurs in the inner mitochondrial membrane. Most energy is produced by this process. !
In the electron transport chain, high energy electrons from NADH and FADH2 are transferred
through a series of carries in the membrane to molecular oxygen. The energy from this process
is converted to potential energy that is stored in a proton gradient that drives ATP synthesis. !
Inner membrane is the principal site of ATP generation. Cristae increase the surface area.
The inner membrane of the mitochondria is impermeable to most ions and small molecules.
This is why it can maintain a proton gradient. !
ATP synthase is embedded in the inner membrane. The actual site of ATP synthesis is in the
matrix, because thats where reaction takes place. !
Outer membrane is highly permeable and it has porins, allowing the free di usion of small ff
molecules. pH in the intermembrane space = pH of cytoplasm. !
Mitochondria can move and fuse. They are generally positioned near locations of high energy
use like synapses in nerve cells. !
Mitochondrial genomes are usually circular DNA molecules, present in multiple copies. Most of
these genes encode only for a few proteins essential for oxidative phosphorylation, also
encode for the rRNAs and most of the tRNAs needed for translating the protein- coding
sequences. Human mitochondrial genome encodes 13 proteins involved in electron transport
chain and oxidative phosphorylation + 16S, 12S rRNAs and 22tRNAS required for translation. !
In the mitochondrial genome, the U in the tRNA anticodon can pair with any of the four bases
in the third codon position of mRNA. Four codons are recognized by a single tRNA. !
Many of the genes required for mitochondrial function and replication are in the nucleus. Most
proteins are synthesized on free ribosomes in the cytosol and the imported into the
mitochondria as complete peptides. !
Proteins are targeted into the matrix by amino-terminal signal sequences, presequences
(alpha-helices, hydrophobic and positively charged). These bind to receptors on the
mitochondria that are part of the Tom complex (translocase of the outer membrane). Proteins
are then transferred to another protein complex in the inner membrane, Tim complex
(translocase of the inner membrane). Some proteins cross the matrix via Tim23 and others exit
laterally, to be inserted/embedded into the inner membrane. !
Because the matrix is negatively charged, it attracts the presequence in, as it is being pulled
through by HSP70 proteins in an ATP dependent manner. Once in the mitochondrial matrix,
mitochondrial HSP70 allow the imported protein to fold into it’s 3D conformation. Import
happens in two steps: !
1. Insertion: it is the charge gradient driven step!
2. Translocation: it is the ATP driven step, so if ATP is removed protein cannot be
translocated. !
The proteins that make up Tom and Tim complexes, are coded for in the nucleus and must
therefore have a signal sequence that helps them embed into the membranes. !
Presequences are cleaved by matrix processing peptidase. Some proteins have multiple trans-
membrane domains and these must have internal import signals instead of presequences. For
this proteins, after being translocated across the outer membrane, they are bound by mobile
Tim9 and Tim10 chaperones, bringing them to Tim 22. Protein is then transferred laterally into
the inner membrane. !
Some inner membrane proteins are encoded by the mitochondrial genome. These are
synthesized in the mitochondrial matrix by mitochondrial ribosomes and then targeted to Oxa
translocase in the inner membrane. Proteins that are synthesized inside the matrix are
embedded by the Oxa translocase. !
Proteins destined for the outer membrane or the intermembrane space also pass through the
Tom complex. Proteins that have a-helical trans-membrane domains exit Tom laterally. !
B-barrel proteins pass through Tom and are then bound by Tim9 and Tim10, which carries the
to the mediates insertion into the SAM (sorting and assembly machinery - translocon). SAM
outer membrane. !
Phospholipid are imported from the cytosol. extract Phospholipid transfer proteins
phospholipids from the ER membrane and transport them through the cytosol, and the release
them at a new membrane (like the mitochondrial membrane). !
Mitochondria synthesize phosphatidylserine from phosphatidylethanolamine. They also
catalyze synthesis of a special type of phospholipid that improves the e ciency of cardiolipin, ffi
oxidative phosphorylation by restricting proton flow across the membrane. !
MECHANISM OF OXIDATIVE PHOSPHORYLATION . !
Electrons from NADH and FADH2 combine with oxygen. The energy released from these redox
reactions is then used to drive ATP synthesis. !
Transfer of electrons from NADH to O2 yields about -52.5kcal/mol for each pair of electrons
transferred. The electrons must pass through a series of carries. The electron transport
chain.
Electron carries are organized in 4 complexes in the inner mitochondrial membrane. !
Steps in the electron transport chain: !
1. Electrons from NADH enter the chain in complex I, these the pass to the mobile carrier
Coenzyme Q (ubiquinone) which carries them to complex III.!
2. In complex III, electrons transferred from cytochrome b to cytochrome c, which carries
electrons to complex IV (cytochrome oxidase), where they are finally transferred to O2. !
*In the matrix, pH is 8 because protons are constantly being pumped out. !
3. Complex II receives electrons from the citric acid cycle intermediate succinate. These
electrons are transferred to FADH2 rather than NADH and then to Coenzyme Q (ubiquinone).
Complex II does not yield free energy. !
*Coenzyme Q (ubiquinone) can receive electrons from either complex I or complex II. *!
Free energy from passage of electrons through complex I, III and IV is harvested by being
coupled to ATP synthesis. This is called coupling electron transport chemiosmotic coupling,
to ATP generation. First proposed by Peter Mitchell, 1961. !
Electron transport through complex I, III and IV is coupled to transport of protons from the
matrix to the intermembrane space. This stablishes a proton gradient across the inner
membrane.
Complex I and IV act as proton pumps that transfer protons as a result of conformational
changes induced by the electron transport. At complex III protons are carried by coenzyme Q
which accepts protons from complex I or II. !
Complex I and III each transfer 4 protons per pair of electrons. !
Complex IV 2 protons per electron pair are pumped across and another 2 protons combine
with O2 to form H2O in the matrix. !
4 protons per electron pair are transported at each complex. !
Potential energy in the proton gradient is electric and chemical in nature. There is a voltage
difference across the membrane enclosed since the matrix is negative and the intermembrane
space is positive. !
Both the pH gradient and electric potential drive protons back into the matrix. !
delta G = -5kcal/mol per proton. !
ATP synthase has two linked units: !
F0: spans inner membrane and forms a spring channel through which the protons move. !
F1: catalyzes synthesis of ATP. !
4 PROTONS NEEDED TO MAKE 1 ATP. !
1 NADH YIELDS 3 ATP !
1 FADH2 YIELDS 2 ATP!
The electrochemical gradient also drives the transport of small molecules in and out of the
mitochondria. !
ATP must be exported and ADP and Pi must be brought in. Adenine nucleotide translocator
transports one ADP into the mitochondrial in exchange for one ATP transferred from the
cytosol. !
This exchange is driven by the voltage component of the electrochemical gradient. ATP is -4
while ADP is -3. !
Pi is brought in as a phosphate (H2PO4 -) in exchange for hydroxyl ions (OH-). this exchange is
neutral but it is driven by the proton concentration gradient. Higher pH = higher OH
concentration, favoring OH translocation to outside. $
Chloroplasts !
These are the organelles responsible for photosynthesis and have some similarities to
mitochondria, such as: both generate metabolic energy contain their own genetic systems and
replicate by division.!
Chloroplasts:!
convert CO2 —> carbohydrates!
Synthesize amino acids, fatty acids and lipid components of their own membranes. !
NO2- reduction to NH3 !
Outer membrane contains porins and is permeable to small molecules. Inner membrane is
highly impermeable. Molecules must move through specific transporters. !
The stroma is functionally equivalent to the matrix. It contains the genetic system, metabolic
enzymes (needed for photosynthesis).!
Protons are pumped across thylakoid membrane from the stroma INTO the thylakoid lumen. !
pH of the stroma is 8. ATP is being made in the stroma !
Peptides in transit are recognized by a guidance complex, which recognizes them and directs
them to the translocase of the outer membrane Toc Complex. Hsp70 molecules keep the
polypeptide in an unfolded state and drive protein import by ATP hydrolysis.!
From the intermembrane space, proteins come into the on the inner membrane Tic Complex
and are then transported to the stroma by the action of Hsp93 chaperone. In the stroma, the
transit peptide is cleaved by stromal processing peptide (SPP). !
Proteins that have to cross the thylakoid membrane have a 2nd signal sequence, after the
transit peptide is cleaved. !
There are three ways that proteins can be translocated into the thylakoid lumen:!
1. Sec (get proteins into the lumen of the thylakoid)!
2. TAT (get proteins into the lumen of the thylakoid)!
3. SRP (deal with proteins that are embedded in the thylakoid membrane. !
Plastids!
These are a type of plant organelle with the same genome but di erent structure and function. ff !
For instance: chloroplasts are specialized in photosynthesis and the thylakoid membrane
makes them unique.!
Other plastids do have a double membrane envelope but no thylakoid. !
Classified according to the type of pigment they contain!
Chloroplasts - chlorophyll !
Chromoplasts - carotenoids (red, yellow, orange) !
Leucoplasts - nonpigmented but store a variety of energy sources in non photosynthetic
tissues!
Amyloplasts - store starch!
Elaioplasts - store lipids!
All of the plastids di erentiate from proplastids, which are small undi erentiated organelles in ff ff
rapidly dividing cells. !
In the photosynthetic cells of leaves, proplastids develop into chloroplasts, but only in the
presence of light. If kept in the dark development of pro plastids is arrested at an intermediate
stage (eitoplasts)
Photosynthesis!
Light reactions: sunlight energy drives synthesis of ATP and NADPH, coupled to
formation of O2 from H2O. Occur in the thylakoid membrane!
Dark reactions: the ATP and NADPH obtained in the light reactions drive glucose
synthesis. Occur in the stroma
-Chlorophylls are what absorb the sunlight, this absorption of light excites an electron
to a higher energy state, converting light energy into potential chemical energy.
Chlorophylls are collected in photo centers in the thylakoid membrane.!
Photon
Resonance
Reaction
center
Antenna
pigment
energy
transfer
chlorophyll
molecules
Photosynthetic
reaction
center
THE
CELL:
A
MOLECULAR
APPROACH
6e,
Figure
11.21
©
2013
Sinauer
Associates,
Inc.
Stroma
\
ms
Photon
“VA
Photon
=
eae
j
@
.
Thylakoid
lumen
THE
CELL:
A
MOLECULAR
APPROACH
6e,
Figure
11.24
©
2013
Sinauer
Associates,
Inc.
Peroxisomes!
Single membrane-enclosed organelles that contain enzymes involved in metabolic reactions. !
They do not have their own genomes. Most peroxins are metabolic enzymes. Can replicate by
division but can also regenerate if entirely lost. !
Hydrogen peroxide is produced when substrates are broken down by oxidative reactions in
peroxisomes. Catalase converts hydrogen peroxide to water or uses it to oxidize another
organic compound.!
Involved in the synthesis of lipids and of amino acids. Specifically Lysine. In animal cells,
cholesterol and dolichol are synthesized in peroxisomes and in the ER. !
Peroxisomes have enzymes for synthesis of plasmalogens, which are important membrane
components of some tissues. !
Peroxisome assembly begins on the rough ER. Transmembrane protein Pex3 recruits PEx19. !
This interaction causes the vesicles containing both proteins to bud o the ER. ff $
Chapter 13: Cytoskeleton!
Basic Information
It is a network of protein filaments that extend throughout the cytoplasm of eukaryotic cells.!
It provides structural framework that helps determine cell shape, positions organelles and the
general organization of the cytoplasm. !
It is also responsible to movement of entire cells and internal transport of organelles and other
structures (like vesicles). It is a dynamic structure that is continually reorganized as cells move
and change in shape. !
Composition of the cytoskeleton:!
1. Actin filaments (microfilaments)!
2. Microtubules (largest)!
3. Intermediate filaments !
Actin !
It polymerizes to for actin filaments, also known as microfilaments. These are flexible fibers
7nm in dimeter and several μm in length. They can organize into 3-D structures like
bundles and networks. !
First isolated from muscle cells in 1942, very abundant in all eukaryotic cells (~5-10%
of total protein). !
Mammals have 6 actin genes: 4 expressed in muscle cells and 2 in non-muscle cells.!
The 3-D structure of actin molecules and filaments was determined in 1990.!
Each actin monomer (globular actin) has tight binding sites that [G] mediate head-to-
tail interactions 2 filamentous [F] actin. with other actin monomers. This forms
G actin —> dimerizes —> trimerizes —> forms filament with barbed and pointed ends.!
All actin monomers are oriented in the same direction. This makes them have polarity.!
This is important in their assembly and in establishing the direction of myosin
movement relative to actin. Directional movement.
Nucleation of actin is the which requires the correct alignment of the rate limiting step,
first three monomers. It is also the first step in actin polymerization. A trimer is formed
and then monomers are added to either end. Polymerization is reversible and filaments
can be broken down when necessary. !
The rate at which monomers are added is proportional to their concentration. !
Treadmilling: The barbed end of the filament grows 5 - 10 times faster than the pointed
end. !
Actin bound to ATP associated with the barbed ends and the ATP is hydrolyzed to ADP !
ADP-actin is less tightly bound than ATP-actin ad it dissociates at the pointed end. !
Treadmilling is critical in regulating actin filaments within the cell!
There are several drugs that a ect actin polymerization. ff !
Cytochalasin: bind to the barbed ends and block elongation (the end of major growth is
blocked and so actin can’t polymerize). This inhibits movements like cell division and
phagocytosis. !
Phalloidin: bind to actin filaments and prevents dissociation. This can drug can be
labeled with fluorescent dye to allow visualization of actin filaments. If added to cells, it
will also inhibit movement and phagocytosis. (This is because it doesn’t let the filament
re-arrange). !
The assembly and disassembly of actin filaments is regulated by actin-binding
proteins that act in diverse ways. !
Examples of these are:!
Some of the actin-binding proteins bind the length of actin filaments, stabilizing them
or cross-linking them to one another. !
Others stabilize actin filaments by capping the ends and preventing dissociation. !
Others promote dissociation and others regulate the exchange of ATP for ADP. !
- determine where filaments are formed by Formin and the Arp2/3 complex
facilitating nucleation. Formins are large dimers that mind to the initial monomers and
then move along the growing filament as monomers are added. Formins nucleate long
unbranched actin filaments, many of which are stabilized by tropomyosins that bind
lengthwise alonng the groove of actin filaments. !
- Where actin filaments need to move, actin is constantly turning over and branching
extensively, for this, Arp2/3 complex nucleates this filaments and helps bind actin-ATP
near the barbed ends. !
- ADF/cofilin (actin depolymerizing factor) modify actin filaments. They enhance the
rate of dissociation of actin/ADP monomers from the pointed end, and remain bound to
the monomers, preventing re-incorporation. ADF/cofilin can bind to and sever actin
filaments. !
Profilin reverses the e ect of ADF/cofilin by stimulating exchange of bound ADP for ff
ATP and dissociating the actin/ATP monomers from cofilin, so that they are available
for reassembly. !
Organization of actin filaments: !
Actin bundles: filaments are cross-linked into closely packed parallel arrays. !
Actin networks: filaments are cross-linked in arrays 3-D meshworks with the properties
of semisolid gels. !
This organization happens with the help of cross-linking proteins. !
Cross-linking proteins also known as actin-bundling proteins, are small rigid proteins
with at least two domains that bind actin and align the filaments closely. The proteins
that organize actin filaments into networks tend to be large flexible proteins that cross-
link perpendicular filaments. !
There are two types of actin bundles: !
Parallel bundles: closely spaced filaments are alligned in parallel, with the same
polarity, with barbed ends adjacent to the plasma membrane. Fibrin is a bundling
protein in parallel bundles, first isolated from intestinal microvilli. !
Contractile bundles: these are more widely spaced filaments and are cross-linked by -α
actinin. The increased spacing between filaments allows the motor protein myosin to
interact with the actin filaments. !
In actin networks, proteins like filamin form flexible cross-links. A filamin dimer is a
flexible V-shaped molecule with actin-binding domains at the end of each arm. !
The -sheet spacer domain renders flexibility.β !
Actin filaments are concentrated at the cell periphery where they form a 3-D networ
beneath the plasma membrane. This network and associated proteins is called Cell
Cortex, which determines cell shape and is involved in activities such as movement. !
Red blood cells (erythrocytes) are useful for studies of the cell cortex. RBC have no
nucleus or organelles so the plasma membrane and associated proteins are easily
isolated. They also lack other cytoskeletal components, so the cell cortex (cortical
skeleton) is the main determinant of shape. !
Spectrin is a member of the calponin family of actin binding proteins. It is a tetramer of
two polypeptide chains: and . The ends of the spectrin tetramers associate with α β
short actin filaments, resulting in the spectrin-actin network. This network is strong and
flexible. !
Ankyrin links the spectrin-actin network and the plasma membrane by binding to
spectrin and a transmembrane protein (band 3). !
Protein 4.1 is another link that binds spectrin-actin junctions and the transmembrane
protein glycophorin. !
Dystrophin also a member of the calponin family of proteins, links actin filaments to
transmembrane proteins of muscle cell plasma membrane. The transmembrane
proteins link to the extra-cellular matrix to cytoskeleton, which helps maintain cell
stability during muscle contraction. (This protein was identified through investigations
on muscle dystrophy). !
Muscular dystrophy is an X-linked inherited disease and it results in progressive muscle
degeneration (skeletal). In Duchene’s (absent dystrophin) and in Becker’s (abnormal
dystrophin). !
Most cells have specialized plasma membrane regions that form contacts with
adjacent cells, the extracellular matrix of other substrata (like a Petri dish in cell
culture). These regions are also attachment sites for actin bundles, evident in
fibroblasts that are maintained in tissue culture (stress fibers). !
Cultured fibroblasts secrete extracellular matrix proteins that stick to the dish. The
fibroblasts attach to the matrix via the binding of transmembrane proteins called
integrins. The sites of attachment are called focal adhesions and they are also sites for
large actin bundles called stress fibers. !
Stress fibers are contractile bundles, cross-linked by -actinin and stabilized by α
tropomyosin. !
There are two other proteins, talin and vinculin which are involved in the binding of
stress fibers. !
In sheets of epithelial cells, cell-cell contacts (adherens junctions) form a continuous
adhesion belt around each cell. !
Contact is mediated by transmembrane proteins called cadherins. These proteins bind
to cytoplasmic catenins which anchor actin filaments to the plasma membrane. !
Cell surfaces have a variety of protrusions involved in cell movement, phagocytosis or
absorption of nutrients. These extensions are based on actin filaments, in relatively
permanent or rapidly rearranging bundles or networks. !
Microvilli are fingerlike extensions, which are abundant on cells involved in absorption. !
In epithelial cells lining the intestine form a layer on the apical surface (a brush border)
of about 1000 microvilli per cell. They increase the surface area for absorption by ten to
twentyfold. !
Intestinal microvilli contain closely packed parallel bundles of 20 to 30 actin filaments. !
These filaments are cross-linked by fimbrin and villin. The actin bundles are attached to
the plasma membrane by the Ca-binding protein calmodulin in association with myosin
I. !
!
Other surface protrusions are transient and form in response to environmental stimuli. !
Pseudopodia are extensions of moderate width, responsible for phagocytosis and the
movement of amoebas. !
Lamellipodia are broad, sheet-like extensions at the leading edge of fibroblasts. At the
end (two filopodia fused, this is how you get movement). !
Many cells also extend filopodia, thin projections of the plasma membrane supported
by actin bundles. !
Myosin is a molecular motor protein that converts chemical energy ATP to mechanical
energy, generating force and movement. Muscle contraction is the model for
understanding actin-myosin interactions and the motor activity of myosin. !
Skeletal muscles are bundles of muscle fibers, large cells formed by fusion of many
cells during development. Most of the cytoplasm consists of myofibrils, which are
bundles of thick myosin filaments and think actin filaments. !
Each myofibril is a chain of contractile units called sarcomeres, which give skeletal and
cardiac muscle their striated appearance. !
Sarcomeres have several regions discernible by electron microscopy. The bands
correspond to the presence or absence of myosin filaments. Actin filaments are
attached at the barbed ends to the Z-disc, which includes the cross linking protein -α
actinin. !
The sliding filament model of muscles contraction was proposed in 1954. During
contraction, each sarcomere shortens, bringing the Z discs together. !
There is no change in the width of the A band, but the I bands and H zone almost
disappear. !
Actin and myosin filaments slide past one another so that the actin filaments move into
the A band and H zone. The molecular basis for this is the binding of myosin to actin
filaments, allowing myosin to function as a motor function that drives filament sliding. !
Myosin II has two heavy chains and 2 pairs of light chains. The heavy chains have a
globular head region and a long -helical tail. The tails twist around each other in a α
coiled-coil. !
Thick filaments: several hundred myosin molecules in a parallel staggered array. !
The globular heads bind actin, forming cross-bridges between thick and thin filaments. !
The orientation of filaments reverses at the M-line. !
There are two other proteins in sarcomeres: !
1. Titin: it is extremely large, one titin molecule extends from the M line to the disc. !
Titin acts like a spring to keep myosin filaments centered in the sarcomere and
maintain the resting tension that allows a muscle to snap back if overextended. !
2. Nebulin: filaments that are associated with actin. These help organize and maintain
orientation and structure of the thin filaments. !
Myosin heads bind and hydrolyze ATP, providing energy to drive filament sliding. !
Swinging-cross-bridge model: ATP hydrolysis drives repeated cycles of interaction
between myosin heads and actin. !
The model of myosin function comes from in vitro studies and determination of the 3-D
structure of myosin: !
- The binding of ATP dissociates myosin from actin. !
- ATP hydrolysis induces a conformational change that displaces the myosin head group. !
The myosin head binds to a new position of the actin filament and a is released. Pi !
The power stroke: myosin head returns to its original conformation which drives actin
filament sliding and ADP is released.
Contraction of skeletal muscle is triggered by nerve impulses which stimulate the release of
Ca2+ from the sarcoplasmic reticulum. !
The increased Ca2+ concentration in the cytosol a ects two actin filament binding proteins. ff
Tropomyosin and troponin.
Tropomyosin binds lengthwise along the actin filaments, and is also bound to troponin. !
When Ca2+ is absent, the tropomyosin-troponin complex binding of myosin to actin. blocks !
Binding of Ca2+ to troponin C shifts the complex, and allows contraction to proceed. !
In nonmuscle cells, contractile assemblies are similar to muscle fibers. !
They also produce contraction by sliding of actin filaments relative to one another. Examples of
contractile assemblies: stress fibers and adhesion belts. !
Cytokinesis: the division of a cell following mitosis. A contractile ring of actin and myosin II is
assembled by membrane-bound myosin just beneath the plasma membrane. Contraction of
this ring pinches the cell in two. !
In nonmuscle cells and smooth muscle, contraction is regulated primarily by phosphorylation
of a myosin light chain. !
It is catalyzed by myosin lightchain kinase, which is regulated by the Ca2+ binding protein
calmodulin. !
Unconventional myosins: nonmuscle myosins that dont form filaments and are not involved in
contraction. !
They function in a variety of cell movements, such as transport of vesicles and organelles. !
Myosin I: Have globular head groups that act as molecular motors. The short tails bind to
structures. Movement of myosin I along an actin filament can transport its attached cargo, like
a vesicle. !
Myosin V: Two headed dimer that transports vesicles and other cargo along filaments. Some
unconventional actins are involved in actin filament reorganization and anchor actin filaments
to the plasma membrane. !
Cell locomotion: !
The movement across a surface proceeds in 3 stages: !
Extension of the leading edge: involves branching and polymerization of actin filaments.
Inhibition of polymerization blocks formation of cell surface protrusion. !
Attachment of leading edge to the substratum!
Retraction of the rear of the cell into the cell body !
Cells move in response to signals from other cells or the environment. This happens in. Wound
healing. Signals that stimulate cell movement activate receptors in the cell membrane, leading
to recruitment of membrane proteins and specialized lipids. !
These signals in turn recruit actin binding proteins:!
Arp2/3 Complex!
WASP/Scar complex, the Arp2/3 activator !
Barbed-end tracking proteins that connect the growing actin filaments to the plasma
membrane !
As the barbed ends of actin filaments at the leading edge branch and grow, the pointed ends
are disassembled by ADF/cofilin. The ADP-actin monomers are carried to the growing barbed
ends by and deactivated through ADP/ATP exchange by profilin. twinfilin !
For slow-moving cells, attachment to the surface involves formation of focal adhesions. !
Cells moving more rapidly, such as amoebas or white blood cells, form more di use contacts ff
with the substratum, the molecules composition of which is not known. !
Retraction of the trailing edge involves small GTP-binding proteins. They regulate breakdown
of existing focal adhesions and stimulate endocytosis of the plasma membrane at the trailing
edge of the cell. !
Microtubules !
Rigid hollow rods. Dynamic structures that undergo continual assembly and disassembly. They
function in cell movements and in determining shape. !
Microtubules are made of the globular protein . tubulin !
Tubulin dimers consist of -tubulin and -tubulin which are encoded by related genes. α β !
γ-tubulin in the centrosome helps initiating microtubule assembly. !
Tubulin dimers polymerize to form microtubules. !
13 protofilamets around the hollow core. These protofilaments are head-to-tail arrays of tubulin
dimers arranged in parallel. They have a fast-growing plus end and a non-growing minus end.
This polarity determines the direction of movement. !
Microtubules can undergo rapid cycles of assembly and disassembly. Tubulin dimers with
GTP-bound to -tubulin associate with the growing end. GTP is hydrolyzed to GDP shortly β
after polymerization. This destabilizes the tubulin, causing rapid depolymerization and loss of
the tubulin bound to GDP from the minus end. !
In microtubules stabilized at the minus end, rapid GTP hydrolysis results in dynamic
instability. This means that there is an alternation between cycles of growth and shrinkage. !
As long as new GTP-bound tubulin dimers are added more rapidly than GTP is being
hydrolyzed, a GTP cap remains at the plus end and microtubule growth continues !
if GTP is hydrolyzed more rapidly than new subunits are added, GDP-bound tubulin at the plus
end of the microtubule leads to disassemble and shrinkage. !
Rapid turnover of the microtubules allows for remodeling of the cytoskeleton that occurs
during mitosis. There are drugs such as and that a ect microtubule colchicine colcemid ff
assembly. These are useful as experimental tools and in cancer treatment. !
Vincristine vinblastine and are used in cancer chemotherapy because they inhibit microtubule
polymerization and thus a ect rapidly dividing cells. ff !
Taxol stabilizes microtubules which also blocks cell division, since the microtubules are not
able to re-arrange. This drug is also used as chemotherapy!
In animal cells, most microtubules extend outward from the centrosome. !
During mitosis, they extend outward from duplicated centrosomes to form the mitosis spindle,
which is responsible for separation and distribution of chromosomes to daughter cells. !
Plant cells do not have an organized centrosome. Microtubules extend outward from the
nucleus. !
The centrosome is a microtubule organizing center in which the minus ends are anchored. If
cells are treated with colcemid, microtubules disassemble. When the drug is removed, new
microtubules can be seen growing outward from the centrosome. !
•- ends of the microtubules are anchored by the centrosome !
•+ ends are closer to the nucleus !
•+ ends are closer to the axons in the neurons !
The role of the centrosomes is to initiate microtubule growth. - tubular in the centrosome is γ
associated with other proteins in a ring shaped structure called This g-tubulin ring complex.
complex is thought to bypass the rate-limiting nucleation step. !
Assembled microtubules can be released from the microtubule organizing center to organize
elsewhere in the cell. This is specially evident in polarized epithelial cells, nerve cells, and plant
root cells during growth of root hairs. !
Most animal cell centrosomes have a pair of oriented perpendicular to each other centricles,
and surrounded by Centrioles are cylindrical, based on nine triplets of pericentriolar material.
microtubules. !
Centrioles also form basal bodies of cilia and flagella. But they are not found in plant cells,
many unicellular eukaryotes, and most meiotic animal cells. In these cells the pericentriolar
material initiates microtubule assembly. !
Microtubule stability is also regulated by post-translational modification of tubulin by
phosphorylation, acetylation, etc. These modifications a ect microtubule behavior by ff
providing sites for binding of specific microtubule-associated proteins (MAPs).!
Interactions of microtubules with MAPs allows cells to stabilize microtubules in particular
locations and help determine cell shape and polarity. !
Many MAPs are cell-type specific. The tau protein is a MAP and it is characteristic of lesions
found in the brains of Alzheimer’s patients.!
Nerve cells have two types of processes supported by stable microtubules: !
-Axons: microtubules have + ends towards the tips, associated with tau.!
-Dendrites: microtubules are oriented in both directions, associated with MAP2.!
There are two families of motor proteins responsible for powering movements in which
microtubules participate: !
Kinesins: most of these move along microtubules towards the + end. Towards the synapse. !
Dyneins: these move toward the - end, back towards the nucleus. !
Organelles and vesicles are moved along microtubules with motor proteins. !
Axonemal dynein: this was the first dynein to be identified, it is very abundant in cilia. Other
motor proteins are present in lower amounts and isolation is required. Development of in vitro
assays by video enhanced microscopy. !
Cytoplasmic dynein moves along microtubules towards the - end. This protein was previously
identified as axonemal dynein from cilia. Is extremely large with 2-3 heavy chains, and a
variable number of light and intermediate chains. often acts with cytoplasmic dynein Dynactin
to move cargoes over long distances.!
Kinesin I: moves along microtubules in one direction, towards the + end. !
In axons, however, vesicles were also observed moving back towards cells.!
Kinesin I has two heavy chains and two light chains. The heavy chains have α-helical regions
that form coiled-coils. X-ray christallography shows that kinesin and myosin evolved
from a common ancestor and are structurallly similar. !
Many kinesins have been identified. Some move towards the microtubule + ends and
some don’t move at all. !
The direction of movement depends on the position of the motor domain:!
•+ end directed: N-terminal
•- end directed: C-terminal
•No movement: the is in the motor domain middle of the heavy chain.
A major role of microtubules is to transport vesicles and organelles through the
cytoplasm. Di erent members of the kinesin and dynein families are thought to ff
transport cargo in opposite directions. !
Cargo selection can be very specific. Several types of molecular motors associate with
a given cargo at the same time, allowing for precise positioning. A future challenge is to
determine how transport is controlled by switching among the di erent motors. ff !
Microtubules and motor proteins also position organelles within the cell. Example: !
The ER extends to the periphery of the cell in association with microtubules, which
involves Kinesin I.!
Drugs that depolymerize microtubules cause the ER to retract toward the cell center.
Cilia and flagella are microtubule-based projections of the plasma membrane. They
are responsible for movement of a variety of eukaryotic cells.!
Some bacteria have flagella but they are protein filaments projecting from the cell
surface. !
Cilia beat in a coordinnated back-and-forth motion, which either moves the cell
through fluid or moves fluid over the surface of the cell. !
Flagella are longer, and have a wave-like pattern of beating. !
Structure of cilia and flagella is similar:
The axoneme consists of microtubules in a 9 + 2 pattern: a central pair surrounded by
9 outer doublets. !
Each of. The doublets is a complete fused to an incomplete A tubule B tubule.
Nexin links the tubules and 2 arms of dynein are attached to each A tubule. !
The microtubule - ends are anchored in a basal body, similar in structure to a centriole.
This has 9 triplets of microtubules. Basal bodies initiate growth of axonemal
microtubules and anchor cilia and flagella to the surface of the cell. !
Movement of cilia and flagella results from sliding of outer microtubule doublets relative
to one another. Powered by motor activity of axonemal dyneins. !
Dynein bases bind to A tubules, while the head groups bind to B tubules of adjacent
doublets. !
Microtubules completely reorganize during mitosis. Interphase microtubule array
disassembles and free tubulin subunits are reassembled into the mitotic spindle. !
The centrosome is duplicated to form two microtubule organizing centers at opposite
poles of the mitotic spindle. !
As the cell enters mitosis, the rate of microtubule disassembly increases, resulting in
shrinkage of microtubules. The number of microtubules emanating from the two
centrosomes increases. !
There are four types of microtubules that make up the mitotic spindle: !
1. Kinetochore microtubules: attach to the condensed chromosomes at the
centromeres, stabilizing them.
2. Chromosomal microtubules: connect to chromosome ends via chromokinesin.
3. Polar microtubules: are not attached to chromosomes but are stabilized by
overlapping with each other in the center of the cell.
4. Astral microtubules: extend outward from the centrosomes and have exposed +
ends.!
After the centrosomes move to opposite sides of the cell, the duplicated chromosomes
attach to kinetochore and chromosomal microtubules, and align on the metaphase
plate. Then the links between the sister chromatids are severed and anaphase begins.!
Chromosome movement occurs by two mechanisms: !
1. Anaphase A: chromosomes move towards spindle poles along kinetochore
microtubules, which shorten as the chromosomes move. Movement towards the
centrosomes is driven by kinetochore-associated minus-end directed motor
proteins. This is coupled to disassembly and shortening of kinetochore and
chromosomal microtubules, mediated by kinesins that act as microtubule
depolymerizing enzymes.!
2. Anaphase B: Spindle poles separate, accompanied by elongation of polar
microtubules. The overlapping polar microtubules slide against one another to push
the spindle poles apart. Plus-end directed kinesins cross-link polar microtubules
and move them towards the plus end. !
Spindle poles are pulled apart by the astral microtubules. Cytoplasmic dynein
anchored to the cell cortex moves along astral microtubules in the - end direction. !
Simultaneous depolymerization of astral microtubules by middle motor kinesins leads
to separation of the spindle poles. !
Intermediate Filaments!
The diameters of intermediate filaments are between actin filaments and microtubules. These
are not directly involved in cell movements, but provide mechanical strength and sca old for ff
localization of cell processes. These are not found in yeast, plants and some insects. !
Intermediate filaments are composed of many types of proteins that are expressed in di erent ff
types of cells. Type I and II are in epithelial cells. keratins, !
Vimentin forms a network extending out from the nucleus towards the cell periphery. !
Desmin is expressed in muscle cells, where it connects the Z discs of individual
contractile elements. !
Neurofilament (NF) proteins (with -interxin) are the major intermediate filaments of α
many neurons. These provide support for long axons. !
Type V (nuclear lamins) form a meshwork underlying the nuclear membrane. !
Nestins (type VI) are expressed during embryonic development in some stem cells. !
Intermediate filaments have a central -helical rod domain which plays a central role in α
filament assembly. The head and tail domains determine the specific functions. !
Filament assembly: the central rod domains of 2 polypeptides form a coiled coil. The
diners associate in a staggered antiparallel fashion to form tetramers, which assemble
end-to-end to form protofilaments. !
Eight protofilaments wind together to form a filament. !
Intermediate filaments do not have distinct ends. They are more stable and do not
exhibit the dynamic behavior of actin filaments or microtubules. But phosphorylation
can regulate assembly and disassembly (like nuclear lamins are disassemble during
mitosis). !
Intermediate filaments form a cytoplasmic network in most cells, extending from a ring
around the nucleus to the plasma membrane. They can also associate with other
cytoskeleton elements, providing a sca old that organizes the internal structure of the ff
cell.!
Epithelial cells have specialized contacts: !
1. Desmosomes: junctions between adjacent cells. Keratin filaments attach to dense
protein plaques on the cytoplasmic side. Attachment is mediated by desmoplakin
( a plakin family protein). !
2. Hemidesmosomes: junctions between epithelial cells and underlying connective
tissue. Keratin filaments are attached to di erent plakins (plectin). Transmembrane ff
integrins link to the extracellular matrix. !
Some plakins link intermediate filaments to other elements of the cytoskeleton. Plectin
binds actin filaments and microtubules, forming bridges between them and
intermediate filaments. This increases the mechanical stability of the cell. !
Direct evidence for the function of intermediate filaments is recent. Some cells in
culture dont make intermediate filaments. Injection of cultured cells with antibody
against vimentin disrupts intermediate filament networks without a ecting cell growth ff
or movement. !
Cells in organisms however, are subjected to mechanical stresses that trigger
development of intermediate filaments. Their role was shown in studies using
transgenic mice with a keratin mutation. The mutation disrupted the formation of a
normal keratin cytoskeleton, resulting in severe skin abnormalities. !
These experiments also pointed to the molecular basis of some human diseases.
Epidermolysis bullosa simplex (EBS) patients develop skin blisters from cell lysis after
minor trauma. They also have keratin gene mutations. !
Abnormalities of neurofilamets result in diseases such as amyotrophic lateral sclerosis
(ALS), involving progressive loss of motor neurons, leading to muscle atrophy and
paralysis. $
Chapter 14: Plasma Membrane !
All cells are surrounded by a plasma membrane. It defines cell boundary and separates it from
the environment. It is a selective barrier and determines the composition of the cytoplasm. It
also mediates interactions between the cell and its environment. !
The fundamental structure of the plasma membrane is the phospholipid bilayer. Proteins
embedded in the bilayer carry out specific functions including: selective transport of molecules
and cell-cell recognition. !
The bilayer structure can be seen in electron micrographs. The polar head groups appear as
dark lines because they bind the electron-dense metal stains. The hydrophobic fatty acid
chains in the center are lightly stained. !
Animal cell plasma membranes have types of phospholipids:5 !
•In the outer leaflet: phosphatidylcholine and sphingomyelin PCS
•In the inner leaflet: phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol !
Animal cell plasma membranes also contain: with the glycolipids only in the outer leaflet,
carbohydrate portions exposed on the cell surface. These are built on a sphingosine backbone. !
Cholesterol is present in about the same molar amounts as phospholipids. !
Phospholipid structure is responsible for the basic structure of membranes: separating
aqueous compartments. !
The bilayer interiorly consists of hydrophobic fatty acid chains, so it is impermeable to water
soluble molecules, ions and most biological molecules. !
Bilayers are viscous fluids, not solid. The fatty acids have one or more double bonds, which
make kinks in the chain and keep them from packing together. Phospholipids and proteins are
free to di use laterally within the membrane. ff !
Cholesterol a ects membrane fluidity, depending on temperature. ff !
•At , it interferes with the movement of fatty acid chains, high temperatures making the outer
leaflet less fluid and reducing its permeability to small molecules.!
•At , it interferes with the interactions between fatty acid chains, cholesterol low temperatures
prevents membranes from freezing and maintains fluidity.
Cholesterol and the sphingolipids (sphingomyelin and glycolipids) tend to cluster in small
patches or lipid rafts. !
Lipid rafts are more highly ordered than most of the phospholipid bilayer. The sphingolipids
have di erent melting temperatures than phospholipids derived from glycerol. ff !
Most plasma membranes are about 50% lipid and 50% protein by weight. Since proteins are
much larger than lipids, this means that 1 protein per 50-100 lipids. !
The fluid mosaic model was proposed by Singer and Nicolson in 1972. They distinguished
two classes of membrane proteins. Peripheral and integral.!
Peripheral membrane proteins: these are associated with the membrane through protein-
protein interactions, often ionic bonds. These bonds can be disrupted by polar reagents, salts
or extreme pHs. These are part of the cortical skeleton: !
*- Spectrin !
*- actin!
*- band 4.1 !
Integral membrane proteins: these are inserted into the lipid bilayer, they can be dissociated
only by reagents that disrupt hydrophobic interactions. Examples of these reagents include
detergents (amphipathic molecules that ca solubilize these proteins)!
Transmembrane proteins: these are a type of integral proteins that completely span the lipid
bilayer with portions exposed on both sides. They can be seen in electron micrographs of
plasma membranes prepared by freeze-fracture technique. !
Membrane spanning portions are usually a-helices of hydrophobic amino acids; they are
inserted into the ER membrane during synthesis (co-translational). !
Carbohydrate groups are added (asymetrically) in the ER and Golgi. Most are glycoproteins
with oligosaccharides exposed on the cell surface. !
•Glycophorin: has a single trans-membrane a helix. !
•Band 3: is the pH regulator and the transporter of HCO3- and Cl- ions with 14
transmembrane a-helices. !
Porins: are special transmembrane proteins in the outer membrane of mitochondria,
chloroplasts and some bacteria. Porins cross the membrane as B-barrels. They make the outer
membrane highly permeable to ions and small polar molecules. !
Some proteins are anchored in the plasma membrane by covalently attached lipids or
glycolipids. are added to the C terminus of some Glycophosphatidylinositol (GPI) anchors
proteins in the ER. These proteins are glycosylated and then exposed on the cell surface. !
Lateral movement of proteins and lipids in the membrane was first demonstrated in 1970.
Human and mouse cells were fused in culture, then analyzed for membrane proteins using
fluorescent antibodies. Some membrane proteins have restricted movement because of
association with the cytoskeleton, or association with other membrane proteins on adjacent
cells or with the extracellular matrix. !
Many epithelial cells are polarized, plasma membranes are divided into apical and basolateral
domains.!
In the small intestine, the apical surface is covered by microvilli that increase the surface area
for absorption. The basolateral surface is specialized to mediate transfer of absorbed nutrients
in the blood.!
Cell surfaces have a glycocalyx, carbohydrate coat made of glycolipids and glycosylated
proteins on the outer membrane. It protects the cell from ionic and mechanical stress and is a
barrier to invading microorganisms.!
Plasma membranes are selectively permeable to small molecules. Specific transport proteins
carrier proteins and channel proteins, mediate passage of small molecules allowing the cell to
control the composition of its cytoplasm.!
In passive di usion: ffmolecules dissolve in the phospholipid bilayer and di use across it. The ff
direction of the transport is determined by the concentrations of the molecules inside and
outside of the cell. Net flow is ALWAYS down the concentration gradient. !
Only small, relatively hydrophobic molecules are able to passively di use across a ff
phospholipid bilayer at significant rates: !
*Gases (O2 and CO2)!
*Hydrophobic molecules (steroid hormones) !
*Small polar molecules (H2O and ethanol) !
Facilitated di usion: ffdirection of movement is determined by concentration gradients.
Transport is mediated by proteins which allow polar and charged molecules to cross the
plasma membrane (carbohydrates, amino acids, ions and nucleosides).!
•Carrier proteins: bind molecules on one side of the membrane, then undergo conformational
changes that allow the molecule to pass through and be released on the other side.!
•Channel proteins: form open pores through the membrane, allowing free di usion of any ff
molecule of the appropriate size and charge. !
Carrier proteins allow facilitated di usion of sugars, amino acids and nucleosides. The glucose ff
transporter has 12 a-helical transmembrane segments (typical of many carrier proteins).!
14 glucose transporters have overlapping specificities for glucose, fructose, and related
molecules. 3-D structure of GLUT1 and GLUT2 have recently been determined. The
transporters function by alternating between two conformational states.!
Once glucose is taken up, it is rapidly metabolized, so intracellular glucose concentrations
remain low and glucose continues to be transported into the cell. Glucose transport can also
be reversed, like in liver cells when glucose is synthesized and needs to be released into
circulation. !
Channel proteins such as porins, form open pores in the membrane that allow molecules to
pass freely. allow water molecules to cross the membrane rapidly. Aquaporins !
They are impermeable to charged ions, allowing the passage of water without a ecting the ff
electrochemical gradient. !
•Ion channels: well studied in nerve and muscle cells, where their opening and closing is
responsible for transmission of electric signals. The transport through ion channels is
extremely rapid, more than a million ions per second. !
Ion channels are highly selective. Specific channel proteins allow passage of Na+, K+, Ca2+
and Cl-. Most of these channels have gates that only open to certain stimuli. !
Ligand gated channels: open in response to the binding of neurotransmitters or other
signaling molecules. !
Voltage-gated channels: open in response to changes in the electric potential across the
plasma membrane. !
Patch clamp technique: developed by Neher and Sakmann in 1976 allows the activity of
individual ion channels to be studied. A micropipette is used to isolate a small patch of
membrane, allowing the flow of ions through a single channel to be analyzed. !
Ion pumps use energy from ATP hydrolysis to actively transport ions across the plasma
membrane to maintain concentration gradients. Thus, the ionic composition of the cytoplasm
is substantially di erent from that of extracellular fluids. ff !
Because ions are electrically charged, pumping results in electric gradients across the
membrane. In resting squid axons, there is an electric potential of about 60mV. The inside of
the cell is negative with respect to the outside. !
Na+ is pumped out of the cell, while K+ is pumped into the cell. The plasma membrane
contains open K+ channels, so the flow of K+ makes the largest contribution to resting
membrane potential. !
Nernst Equation
