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Major Structural Elements of the Cytoskeleton

Eukaryotes Have Three Basic Types of Cytoskeleton Elements

three major structural elements: microtubules, microfilaments, and intermediate filaments

Microtubules are composed of the protein tubulin and are about 25 nm

Microfilaments are polymers of the protein actin and are 7nm

Intermediate filaments differ depending on the cell type

Bacteria have cytoskeletal systems that are structurally similar to those in eukaryotes

rod shaped bacteria and the Archaea have polymer systems that function in a manner very similar to microfilaments, microtubules, and intermediate filaments

actin like MreB protein is involved in DNA segregation tubulin like FtsZ protein is involved in determining where bacteria cells divide and intermediate like crescentic protein is an important regulator of cell shape

FtsZ is produced by certain organelles in some eukaryotes (chloroplasts and mitochondria)

Bacterial proteins overall structure is the same as eukaryotes- equivalent proteins bind the same ph

The cytoskeleton is dynamically assem

microtubules and microfilaments are best known for their role in cell motility

microfilaments are essential components of muscle fibrils and microtubules are structural elements of cilia and flagella

specific drugs can be used to perturb cytoskeletal function

Microtubules

Two types of microtubules are responsible for many function in the cell

largest of the skeletal elements

two different groups

cytoplasmic microtubules: organization and maintenance of animal cell shape and polarity, chromosome movement, intracellular transport/trafficking and movement of organelles; form the mitotic and meiotic spindles that are essential for the movement of chromosomes. loosely organized dynamic network

axonemal microtubules: highly organized stable ground in sub cellular structures associated with cellular movement (cilia, flagella, basal bodies to which these appendages attach) Involved in cell motility

the axoneme of cilium of flagella consists of axonemal microtubules

Tubulin Heterodimers are the protein building blocks of microtubules

MT's are straight hollow cylinders with an outer diameter of 25nm and an inner diameter of 15nm

The MT wall consists of longitudinal arrays of linear polymers called protofilaments (usually 13 arranged side by side around the hollow center)

subunit of a protofilament is a heterodimer of the protein tubulin

the heterdimers that form the bulk of protofilametns are composed of one molecule of alpha tubilin and one molecule of beta tubulin

as soon as alpha and beta are synthesized they bind non-covalently to each other to produce an alphabeta-heterodimer that does not dissociate under normal conditions

alpha and beta each have a GTP-binding domain at the N-terminus, a domain in the middle to which colchicine can bind, and a third domain at the C-terminus that interacts with MT-associated proteins

all of tubulin dimers are oriented in the same direction such that all the alpha tubulin subunits face the same end - gives the protofilament and inherent polarity

tubulin isoforms- several closely related but non-identical genes for each of the alpa and beta tubulin subunits

tubulin can be chemically modified (acetylated tubulin tends to form more stable Mts than non-acetylated tubulin

Microtubules can form as singlets doublets or triplets

cytoplasmic MT's are simples tubes or singlet MT's built from 13 protofilaments

some axonemal MT's are more complex and can contain doublet or triplet MT's- these contain one complete 12 protfilament microtubule (A tubule) and one or two additional incomplete tubules called (b and C tubules) consisting of 10 or 11 protofilaments

doublets are found in cilia or flagella

triplets are found n basal bodies and centrioles

Microtubules form by the addition of tubulin dimers at their end

microtubules form by the reversible polymerization of tubulin dimers

critical step in the formation of MT's is the aggregation of tubulin dimers to into clusters called oligomers

oligomers serve as nuclei from which new microtubules can grow- process is called nucleation

once the MT has been nucleated it grows by addition of subunits at either end via elongation

initially slow (lag phase)- nucleation

elongation phase is relatively fast

eventually the mass increases to a point where the concentration of the free tubulin becomes limiting leading to the plateau phase- MT assembly is balanced by disassembly

microtubule growth in vitro depends on the concentration of tubule dimers

critical concentration is the plateau phase

MT's tend to grow when the tubulin concentration exceeds the critical concentration and depolymerize when the tubulin concentration falls below the critical concentration

Addition of tubulin dimers occurs more quickly at the plus ends of microtubules

two ends of microtubules differ chemically due to polarity

difference between two ends is that one end can inherently grow and shrink much faster than the other

the rapidly growing end of the microtubule is called the plus end and the other end is the minus end

minus ends are often located at the centrosome in this case MT dynamics are confined to plus ends

different growth rates of minus and plus ends reflect the different critical concentrations required for assembly

critical concentration for plus end is lower than that for the minus end

if the tubulin concentration is higher than the critical concentration for the plus end but lower than the critical concentration for the minus end assembly will occur at the plus end while disassembly takes place at the minus end- produces phenomenon known as tread milling

tread milling arises when a given tubulin molecule incorporated at the plus end is displaced progressively along the MT and eventually lost by depolymerization at the opposite end

Drugs can affect the assembly of microtubules

colchicine- an alkaloid which binds to tubulin monomers strongly inhibiting their assembly into MT's and fostering the disassembly of existing ones

tubulin-colchicine complex can still add to the growing end of the MT but it then prevents any further addition of tubulin molecules and destabilizes the structure promoting MT disassembly

Nocodazole is another compound that inhibits assembly- effects are more readily reversible when the drug is removed

Nocodazole compounds are called anti mitotic drugs because they disrupt the mitotic spindle of dividing cells blocking the further progress of mitosis

vinblastine and vincrstine (anti mitotic drugs) are useful when treating cancer cells since cancer cells divide rapidly so they are susceptible

taxol binds tightly to MT's and stabilizes them causing much of the free tubulin in the cell to assemble into Mts and arrests dividing cells in mitosis. - used in treatment of breast cancer

Both taxol and colchicine block cells in mitosis by do so by opposing effects on MT's

GTP hydrolysis contributes to the dynamic instability of microtubules

tubulin can assemble in vitro in presence of Mg2+ and GTP

GTP is required for MT assembly

each tubulin heterodimer binds to two GTP- the alpha tubulin binds to one GTP and the other GTP binds to beta tubulin and can be hydrolyzed to GDP sometime after the heterodimer is added to an MT

GTP is needed for MT assembly because the association of GDP bound tubulin heterodimers with each other is too weak to support polymerization

hydrolysis of GTP is not necessary for assembly since MT's polymerize from tubulin heterdomiers bound to non-hydorlyzable analogue of GTP

some microtubules can grow by polymerization at the same time others shrink by depolymerization- result: some Mt's enlarge at the expense of others

Dynamic instability model- explain how both polymerization and depolymerization could occur at the same time

growing MT's have GTP bound to their tubulin plus ends while shrinking Mt's have GDP instead

GTP-tubulin molecules are thought to protect MT's by preventing the peeling away of subunits from their plus ends (GTP cap provides stable MT tip to which further dimers can be added)

hydrolysis of GTP by Beta tubulin eventually results in an unstable tip at which point deploymerization may occur rapidly.

concentration of tubulin bound to GTP is crucial to dynamic instability model

when GTP-tubulin is readily available it is added to the microtubule quickly creating a large GTP tubulin gap

If concentration of GTP tubulin falls the rate of tubulin addition decreases

sufficiently low concentration of GTP tubulin the reate of hydrolysis of GTP on the beta tubulin near the tip of the MT exceeds the rate of addition of new GTP bound tubulin- result: shrinking of the GTP cap

when GTP cap disappears the MT becomes unstable and loss of GDP-bound subunits from its tip is favored

Microtubule catastrophe- when MT switched from growth to shrinkage- MT can disappear completely or it can abruptly switch back to growth phase (microtubule rescue)

frequency of catastrophe is inversely related to the free tubulin concentration

Microtubules Originated from Microtubule-organizing centers within the cell

MT formation in vivo is a more ordered and regulated process- sets of MT's in specific locations for specific cell functions

microtubules originate from microtubule-organizing center (MTOC)

an MTOC serves as a site at which MT assembly is initiated and acts as an anchor for one end of these MT's.

during interphase have an MTOC called the centrosome positioned near the nucleus

centrosome is associated with centrioles surrounded by pericentriolar material

walls of centrioles are formed by nine pairs of triplet microtubules

centrioles are known to be involved in the formation of basal bodies which are important in the formation of cilia and flagella

when centrioles are missing microtubule-nucleating material disperses and the MTOC disappears

gamma tubulin are in large ring shaped protein complexes in the centrosome

gamma tubulin can be seen at the base of MT's that emerge from the centrosome - called gamma tubulin ring complexes (y-TURCs)- serve to nucleate the assembly of new MT's away from the centrosome - centrosomes will no longer nucleate in the absence

MTOC's organize and polarize the microtubules within cells

MTOC's ability to nucleate and anchor MT's. Because of this ability MT's extend out from an MTOC toward the periphery of the cell and grow out from an MTOC with a fixed polarity (their minus ends are anchored in the MTOC and their plus ends extend out toward the cell membrane)

Since the minus ends of many MT's are anchored at the centrosome dynamic growth and shrinkage of these MT's at the plus end tends to occur at the periphery of cells

MTOC can also influence the number of microtubules in a cell

Each MTOC has limited number of nucleation and anchorage sites that seem to control how many MT's can form

centrosomes associated with spindle poles in mitotic cells have the highest MT nucleating activity during prophase and metaphase

Microtubules stability is tightly regulated in cells by a variety of microtubule binding proteins

MT's have dynamic instability: meaning they grow out from the centrosome and then disassemble

process could account for randomly distributed and short-lived MT's.

Cells regulate MT's using variety of proteins- regulate structure assembly and function

some MT binding proteins use ATP to drive transport of vesicles and organelles or to generate sliding forces between MT's

Microtubule Stabilizing/Binding Proteins

MAPs account for 10-15% of mass of MT's isolated from cells

allow interaction with other filaments and cellular structures

increase MT stability

affect density of bundles of MT's

MT bundles are denser in axons than they are in dendrites

A MAP called Tau causes microtubules to form tight bundles in axons

MAP2 is present in dendrites and causes the formation of looser bundles of MT's

One portion of MAPs such as Tau or MAP2 binds along the length of an MT while the other portion extends at right angles to the microtubule where it can interact with other proteins

length of these controls the spacing of MT's in bundles MAP2 has a longer arm than Tau so MAP2 causes bundles to form that are less densely packed

Alzheimers disease- dense tangles of neurites (neurofibrilarry tangles) contain large amounts of hyperphosphorylated Tau protein which formed paired helical filaments

+-TIP Proteins

Mts are unstable to remain intact for long periods of time and will depolymerize unless they are stabilized in some way

Stabilize MT's- capture and protect their growing plus ends- -+TIP proteins associate with MT plus ends

Microtubule destabilizing/severing proteins

some proteins stabilize MT making them less likely to depolarize

other proteins promote destabilization

Microfilaments

smallest of cytoskeletal elements

known for role in contractile fibrils of muscle cells interact with myosin to causes muscle contractions

locomotion and structural functions

cell migration via lamellipodia and filopodia, amoeboid movement and cytoplasmic streaming- a regular pattern of cytoplasmic flow in some plant and animal cells

produce the cleavage furrows that divide the cytoplasm of animal cells during cytokinesis

found at sites of attachment of cells to one another and to the extracellular matrix

developing and maintaing cell shape

most animal cells have a dense network of MF's called the cell cortex just beneath the plasma membrane- provides structural rigidity on the cell surface and facilitates shape changes and cell movement

parallel bundles of MF's also make up the structural core of microvilli

Actin is the protein building block of microfilaments

synthesized as a single polypeptide consisting of 375 amino acids- once synthesized it folds into a U shape with central cavity that binds to ATP or ADP

Individual actin molecules are referred to as G-actin

G-actin polymerize to form microfilaments and in this form it is referred to as F-actin

Actin in G or F form bind to a wide variety of other proteins known as actin-binding proteins

Different types of Actin are found in cells

actin from diverse organisms will copolymerize into filaments

actin can be divided into two major groups

muscle-specific actions (alpha actins)

nonmuscle actins (beta and gamma actins)

beta and gamma actin localize to different regions of the cell and appear to have different interactions with actin binding proteins

G actin monomers polymerize into F-actin microfilaments

G actin monomers can polymerize reversibly into filaments with a lag phase corresponding to filament nucleation followed by more rapid polymer elongation phase

within a microfilament all the actin monomers orient in the same direction so that an MF has inherent polarity

polarity is demonstrated by incubating MF's with myosin sub fragment 1 (S1)- proteolytic fragment of myosin

S1 fragments bind to the actin MF's to give a distinctive arrowhead pattern with all S1 pointing in same direction

minus end is the pointed end

plus end is the barbed end

polarity is reflect in more rapid addition of loss of G actin at the plus end and slower addition or loss of G actin at the minus end

as G actin monomers assemble onto a MF the ATP bound to them is slowly hydrolyzed to ADP

the ends of growing MF tend to had ATP F-actin whereas the bulk of MF's is composed of ADP F actin

ATP hydrolysis is not a requirement for MF elongation since they can also assemble from ADP G actin or from nonhydrolyzable analogues of ATP G actin

Specific drugs affect polymerization of microfilaments

cytochalasins- prevent the addition of new monomers to existing polymerized MF's. As subunits are gradually lost from the minus ends of MF's they eventually depolymerize

latrunculin A acts by sequestering actin monomers preventing their addition to the plus ends of growing MF's

either drug- net result is loss of MF

Cells can Dynamically assemble actin into a variety of structures

cells that crawl have specialized structures called laellipodia and filopodia

in cells that adhere tightly to underlying substratum and do not move well organized bundles of actin called stress fibers stretch from tail to front

rapidly moving cells usually don't have such striking actin bundles- cell cortex is cross linked into a gel or very loosely organized lattice of microfilaments

MF's form highly oriented polarized cables with their plus ends oriented toward the tip of protrusion

Actin Binding proteins regulate the polymerization length and organization of microfilaments

actin binding proteins- control where actin assembles and the structure of the resulting actin network

control of the process of MF polymerization occurs at nucleation of new MF's, the elongation and severing of preexisting MF's, and the association of MF's into networks

Proteins that regulate polymerization

growth of MF's depend on the concentrations of ATP bound G actin

if concentration is high MF's will assemble until the G actin is limiting

in the cell a large amount of G actin is not available for assembly because it is bound by the protein thymosin B4

profilin competes with thymosin B4 for binding to G-actin monomers

when profillin concentration is high polymerization is favored- only if free filament ends available

ADF/cofilin is known to bind to ADP-G-actin and F-actin- thought to increase the rate of turnover of ADP- actin at the minus ends of MF's- ADP on these G actin monomers can then be exchanged for a new ATP and the ATP G actin can be recoiled for addition to growing plus ends

ADF/cofilin also severs filaments creating new plus ends as it does

Proteins that Cap actin Filaments

capping protein binds the end of a filament and prevents further addition or loss of subunits thereby stabilizing it

CapZ- when bound to end of filament further addition of subunits at plus end is prevented

tropomodulins- bind to minus end of actin filaments preventing loss of subunits from the pointed ends of F-actin

Proteins that Crosslink Actin Filaments

actin networks form loose crisscrossing, cross linked MF's

filamin is important for crisscrossing- consists of two identical polypeptides joined head to head with actin binding site at each tail

Proteins that sever Actin Filaments

other proteins play opposite role breaking up MF's and causing the cortical actin gelt to soften and liquefy

sever and or cap MF's

Proteins that bundle actin filaments

actin can be highly ordered

proteins mediated this highly ordered bundling

alpha actinin- required to make adhesive connections to the extracellular matrix as they migrate

Microvilli- ordered actin arrays

in microvilli the plus ends point toward the tip where they are attached to the membrane- the MF's in the bundle are also connected to plasma membrane by lateral cross links consisting of proteins myosin I and calmodulin that extend outward

at base of microvillus the MF bundle extends into a network of filaments called the terminal web - composed mainly of myosin and spectrin which connect the MF's to each other, to proteins within the plasma membrane, and to the network of intermediate filaments

terminal web gives rigidity to microvilli by anchoring their MF bundles securely so that they project straight out from the cell surface

Proteins that link Actin to Membranes

for MF's to exert force on plasma membrane during cytokinesis and cell movement they must be connected to the plasma membrane.

the connection is indirect and requires one or more linker proteins that anchor MF's to transmembrane proteins embedded within the plasma membrane

band 4.1, ezrin, radaxin, moesin family of actin-binding proteins function in linking MF's to membrane

Proteins that promote actin branching and growth

cells can assemble actin into branched networks

Arp2/3 complex helps branches to form by nucleating new branches on the side of existing filaments

Arp2/3 branching is activated by a family of proteins that includes the Wiskott-Aldrich syndrome protein (WASP) and WAVE/Scar

people who can't produce wasp have defects in their ability for their platelets to undergo changes in shape and so have difficulties forming blood clots

long actin filaments can be mores useful than branched ones- actin polymerization can be regulated independently of Arp2/3 through proteins known as formins

forming are required to assemble certain unbranched F-actin structures (actin cables and contractile ring)

Cell signaling regulates where and when actin-based structures assemble

actin-binding proteins regulate the types of actin based structures that cells assemble- cell signaling regulates the activity of these proteins

plasma membrane lipids and several small G proteins related to Ras regulate the formation stability and breakdown of MF

Inositol Phospholipids

type of membrane phospholipid that regulates actin assembly

PIP2 can bind to profiling CapZ and proteins such as ezrin recruiting them to the plasma membrane and regulating the ability of these proteins to interact with actin

CapZ binds tightly to PIP2 resulting in its removal from the end of a microfilament thereby permitting the filament to be disassembled

Rho Family GTPases

regulation of actin cytoskeleton is the dramatic change in the cytoskeleton of cells exposed to certain growth factors.

In response to stimulation by platelet derived growth factor (PDGF) fibroblasts will begin to grow divide and form actin rich membrane extensions that resemble lamellipodia

LPA induce cells to form stress fibers

many of these signals result in the changes in the actin cytoskeleton through their action on a family of monomeric G proteins known as Rho GTPasses

key members of Rho GTPases are: Rho, Rac, and Cdc42 - regulators of actin cytoskelelton

Rho activation-formation of stress fibers

Rac activation- extension of lamellipodia

Cdc42 activation-formation of filopodia

Rho GTPases are stimulated by guanine nucleotide exchange factors which foster an exchange of a bound GDP to GTP

GAP's stimulate Rho GTPases to hydrolyze their bound GTP thereby inactivating them

GDI's can sequester inactive Rho

Intermediate Filaments

keratin- important component of structures that grow form skin

most stable and least soluble

don't appear to be polarized

Intermediate Filament Proteins are Tissue specific

differ in amino acid composition from tissue to tissue

grouped into 6 classes

classes I and II- keratins. class I are acidic and class II are basic

Class III- vimentin, desmin, glial fibrilary acidic proteins

Class IV- neurofilament proteins

Class V- nuclear lamina A B C- inner surface of the nuclear membrane

Class VI- nestin

these genes are encoded by a single family of related genes and can be classified according to amino acid sequence

Intermediate Filaments assemble from fibrous subunits

all are fibrous rather than globular proteins

homologous central rodlike domain of 310-318 amino acids

central domain consists of 4 segments of coiled helices interspersed with three short linker segments

N and C terminal domains differ greatly in size sequence and function

two IF polypeptides intertwined into a coiled coil - central domains parallel- two dimers then align laterally to form tetrameric protofilament

Intermediate filaments confer mechanical strength on tissues

tension bearing role

mutations of keratin give rise to blistering kin disease

also think to blame for ALS

The cytoskeleton is mechanically integrated structure

mechanical integration of all three cytoskeleton elements is made possible by specific linker proteins that connect them known as plakins

Chapter 15

Chapter 15 part 2

Chapter 15 Part 3