Cell Bio: Chapter 20- The Cytoskeleton and Vesicle Trafficking

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What is the cytoskeleton and what are its functions

• Network of protein filaments and tubules in cytosol that provide structural scaffolding or framework for cell

• Can be rigid but is constantly changing (dynamic)

Functions

• Defines cell shape

• Supports the structure of plasma membrane

• Vesicle movement and organizing organelle location and structure

• Cell movement

• Cell division

• Cell extracellular matrix interactions

What are the three structural fibers that make up the cytoskeleton?

Microtubules: Largest part of cytoskeleton (polymerize to be 25 nm in diameter), made from protein called tubulin

Microfilaments: Narrowest part of the cytoskeleton (polymerize to be 7nm in diameter); made from protein called actin

Intermediate FIlaments: Intermediate thickness (polymerize to be 8-12 nm in diameter); made from filamentous protein

• Filamentous protein: Not a single protein; made from alpha helices that wrap around each other

• Different proteins in different kinds of cells (epithelial, muscle) produce these

What is filamentous protein and what makes it?

• Subunit of intermediate filaments

• Made from mainly alpha helices

What are dimers and how do they affect microtubule polymerization in vitro

• Dimers are building block for MT

• Dimers are made from alpha tubulin and beta tubulin

• At high dimer concentration —> microtubules will polymerize (grow very fast)

• At low dimer concentration —> microtubules will depolymerize

• MT concentration will reach plateau phase which makes it polymerize and depolymerize continuously

What does it mean when the tubules are assymetrical?

• One side of the tubule has the alpha end exposed (negative end)

• One side of the tubule has the beta end exposed (positive end)

How do microtubules use GTP and how does a GTP cap form?

• Both the alpha tubulin and the beta tubulin of a dimer must be bound to GTP for the dimer to add to the growing microtubule

• Some time after addition, GTP bound to beta-tubulin is hydrolyzed

• When there is high dimer conc. GTP tubulin dimer is added to the end of the growing microtubule end which causes the GTP cap to form

  • Cap is a stable binding site for more tubulin to bind

uses GTP for polymerization

What is GDP tubulin?

Shortly after the GTP tubulin has been added, the GTP bound to beta-tubulin is hydrolyzed, which makes GDP tublin

What is microtubule catastrophe?

• Dimer conc. falls too low polymerization ceases

• Rate of GTP hydrolysis (GDP forming) > Rate of polymerization (GTP tubulin being added) then GTP cap is lost

• MT becomes unstable after losing cap and promotes rapid depolymerization = microtubule catastrophe

Dynamic instability

• Rapid growth and shrinkage of the MT

• Rescue of microtubules resumes growth after catastrophe

• More present in beta tubulin than alpha tubulin

How does microtubule nucleation happen in vivo (living cell)

• Microtubule nucleation = 1st step of putting dimers together; requires gamma tubulin

• Gamma tubulin ring complex: contains 13 copies of gamma tubulin and other accessory proteins

  • Stable formation for polymerizing microtubules

  • Serves as anchor for minus end (alpha end) of dimer

  • More dimers are added until it becomes a sufficient “seed”

  • Found in MTOC (centrosome)

    • Only 1 in animal cell

• Centrosome: MT are nucleated and spread out into cytosol

  • Centriole

Further explain the centrosome and centriole

• Centrosome: where MT are nucleated (aka the MTOC)

  • Contains a pair of centrioles

Centriole

• Contains 9 triplet structures of MT organized in cylinder

• Pericentriolar material = additional proteins that surround centriole

• Not required for nucleation

Where is the MTOC usually located in animal cells?

• Centrosome

• Microtubules stick out of the centrosome in different directions

• Positive end sticking out

Other MTOC locations

• Nerve Cell

  • Centrosome = MTOCs

  • Positive end of microtubules sticks out and goes along the axon

• Epithelial cell

  • Basal bodies = MTOCs

    • Structure is similar to centriole

      • Important for faciliatating formation of cillia

  • Cell has cilia (long finger-like projections) where the positive end of microtubules grow

• Red blood cell

  • No MTOC

  • Microtubules wrap around cell (marginal bundles of microtubules)

  • Mixed polarity = positive and negative ends point in different directions

Label this picture

Microtubules in yellow

Nucleus in magenta

MTOC on back side of nucleus (whiteish)

Microfilaments in blue (in plasma membrane)

• Can form cell cortex

Microtubule toxins

Inhibit polymerization: colchicine, colcemid (water soluble and can be easily washed out), nocodazole, vincristine

• Inhibits assembly of dimers

Inhibit depolymerization: paclitaxel (V and P used in chemo therapy)

• Stabilizes microtubules

Pulse/chase with colcemid

• Pulse in colcemid (inhibit polymerization)

• Wash away

• Chase with fresh media

• MT grow back

How are microtubules regulated?

• Regulated by microtubule associated proteins (MAPS)

  • Motor MAPS: kinesin & dynein

  • Non-motor MAPS

Non-motor MAPs

• Numerous proteins bind to plus end of microtubules and promote polymerization, stabilization, and depolymerization

  • EB1- promote polymerization and stabilization

  • MCAK (catastrophin) - promote depolymerization

• Proteins bind laterally to microtubules and promote crosllinking: tau and MAP2

How do tau and MAP2 bind to microtubules?

• Tau/MAP2 bind laterally to one microtubule and cross link it to a neighboring one

• Tau forms a tighter crosslink than MAP2

• Entire structure of Tau/MAP2 and MT is called a bundle (MAP2 forms bundles with more width)

In image the light green is the microtubule and the dark green represents the tau/map2 and whole thing is the bundle

What are microfilaments (F-actin) made of?

G-actin

Explain how microfilaments polymerize and how the rate of polymerization is affected

• More G-actin; faster the rate of polymerization of microfilaments

• G-actin must be bound to ATP to be added to growing microfilament chain

• Shortly after G-actin is added, ATP is hydrolyzed

Explain how actin binding proteins bind to G-actin and regulate it

Polymerization (actin polymerizing proteins): Formin and profilin can help with polymerizing microfilaments

Depolymerization (monomer sequestering proteins): cofilin decrease polymerization of microfilaments

Explain how actin binding proteins bind to F-actin and regulate it

• Filament Length

  • Filament severing proteins: Cut and cap microfilaments into smaller pieces; this makes the cytosol more fluid

    • Ex: gelosin

  • Filament capping proteins: prevent new G-actin from being added

    • Ex: CapZ

• Filament Organization

  • Filament crosslinking proteins: link microfilaments; this makes the cytosol more gel-like

    • Ex: filamin

  • Branching: produces branching in microfilaments

    • Ex: Arp 2/3

  • Filament bundling proteins: help form bundles of microfilaments

    • Ex: fimbrin, alpha-actinin

What are microvilli?

• Small protrusions of the plasma membrane that increase the surface area

• Connected to actin microfilaments

• Digestive system has hundreds

What are the trailing and leading edge of the cell?

• Trailing edge: the rear end of the cell; if the cell is moving toward the right then the trailing edge is toward the left

• Leading edge: the front end of the cell; if the cell is moving toward the right then the trailing edge is toward the right

What are the 4 actin assemblies that help with cell movement

1. Stress Fibers (Contractile Bundles)

2. Cell Cortex (Gel)

3. Lamellipodium (Branched Network)

4. Filopodium (Parallel Bundle)

Explain what stress fibers are made of and how they help with cell motility

• Stress fibers are made of microfilaments that run antiparallel (mixed polarity because beta ends are pointing in different directions) to each other (the arrow head represents the beta end)

• All of them together are considered a contractile bundle

• Function: Pull the trailing end to keep up with the leading end

Explain what the cell cortex is made of and how they help with cell motility

• Made of microfilaments that are crosslinked

• Function: Help with supporting the cell and providing stability for lamellipodia and filopodia

Explain what the lamellipodium is made of and how they help with cell motility

• Made of microfilaments that form branched networks

• Function: help push the membrane out

Explain what the fliopodium is made of and how they help with cell motility

• Made of microfilaments that are parallel

• Same polarity

• All of them together are called a parallel bundle

• Function: push part of the membrane out

How does Arp2/3 help form actin networks?

• Arp2/3 is a actin related protein that helps form branches on microfilaments

How does branching work?

1. Actin binding protein (WASP) binds to and activates Arp2/3

2. Activated Arp2/3 binds to the side of an existing filament and a new filament grows at a 70 degree angle

3. Barbed end (+ ends) elongate and the membrane is pushed forward

4. Capping protein terminates elongation

What regulates actin assemblies?

• The Rho family (a G-protein) regulates the formation of different actin assemblies by regulating actin binding proteins (WASP)

What happens when G protein can’t hydrolyze its GTP ?

• Mutant G protein is unable to hydrolyze its GTP so it is always active and is called “dominant active”

• Thus there is an overexpression of actin assemblies

  • In Rho: stress fibers

  • In Rac: lamellipodium

  • In Cdc42: fliopodia