Cytoskeleton

Exam 3

Define Cytoskeleton and Identify its elements

The cytoskeleton is a dynamic system of proteins and fibers of various sizes that help organize the cell and move components within it.

Elements: microfilaments, microtubules, and intermediate filaments

What are actin (microfilament) monomers?

G actin

• globular protein

• 43 kDa protein

• 375 amino acids

• has nucleotide binding site for ATP or ADP

• has polarity (plus/barbed and minus/pointed end)

• highly conserved (sequence for it the same in many organisms)

• very abundant (5-10% of proteins in a eukaryotic cell are G actin)

How do actin monomers polymerize?

• Head to tail: all pointed ends face one way, and all barbed ends face the other

1. Nucleation: G actin —> dimer —> trimer (nucleus of actin)

   1. This is the rate limiting step (takes the longest b/c requires precise collisions)

2. ATP bound G actin added to plus end (high affinity)

3. ADP bound G actin leave the minus end (low affinity)

Technically ATP can be added or removed from either end, but it’s physically harder considering its affinity

Types of actin binding proteins (APBs)

1. monomer binding proteins

2. filament severing proteins - cut and expose ends for polymerization/depolymerization

3. filament capping proteins - keep fixed length

4. filament crosslinking proteins - allow for actin network formation

5. filament bundling proteins - allow for actin bundle formation

6. filament anchoring proteins - allow acting to bind to other things (ex. plasma membrane)

Ways microfilaments can be assembled. (What are they used for)

1. bundles

2. networks

3. contractile elements

Example of actin bundle

Microvilli

stabilized finger-like structure by actin filaments (rigid)

Examples of actin networks

Terminal web beneath microvilli, Branched actin at leading edge of cell

provide structural support, but more flexible than bundles

Microfilament Movement requires what?

• motor proteins (myosin)

• ATP

• Actin

Myosin Movement Steps

1. rigor conformation - myosin head attached to actin because high affinity (ATP binding site empty)

2. ATP put into myosin binding site, so myosin lets go because it loses its affinity for actin

3. ATP is hydrolyzed (now ADP) which cocks head group back medium affinity for acting

4. Power stroke triggered when phosphate group is forcibly ejected

5. At the end of power stoke ADP is released and myosin returns to original conformation

How are microfilaments related to cytokinesis?

contractile ring of actin formed around cell with myosin motors. actin “belt” is pulled through “buckle” by myosin, pinching the cells and creating the cleavage furrow, eventually dividing the cell in two

Cell Crawling Steps

1. Cell attached to surface (substrate)

2. actin filaments branch and polymerize at front of cell, pushing the leading edge forward (lamellipodium)

3. leading edge attached to substrate via focal adhesion points (stabilized and connected to actin filaments within cell)

4. cell body (where most of the organelles happen to be) brought forward

5. trailing edge snaps up into area cell just moved to

Vesicle Transport

the outside of the vesicle is covered in unconventional myosin which roll vesicle in appropriate direction. the unconventional myosin moves quickly but in same contractile motion at regular

Muscle Parts

the muscle is a collection of bundles

bundles are a collection of muscle fibers (muscle cells)

muscle fibers are a collection of myofibrils

Sarcomere Components

Components:

z-disc/line - dark lines between sarcomeres

I-band - region that spans z-disc with actin but no myosin

A-band - total width of all myosin

M-line - middle of sarcomere

H-zone - region in A-band with only myosin

During contraction:

• z-discs get closer

• h-zone gets smaller (b/c of increased overlap of actin and myosin)

• i-band gets smaller (b/c of increased overlap of actin and myosin)

• a-band stays same (myosin don’t change length)

Conventional myosin

• those in muscle fibers

• tails long and wrapped around each other

• moving toward plus end of actin filament

What are microtubules used for?

• guide intracellular transport via microtubules array

• separating chromosomes during mitosis

• propulsion or sweeping of fluid over membrance

What are microtubules made out of?

Rigid, hollow, tubes made of tubulin. Tubulin itself is a dimer of alpha tubulin and beta tubulin. Each subunit has a nucleotide binding site which can hold GTP and GDP which determines its affinity.

How are microtubules made?

• 13 linear protofilament surrounding hollow core

• Protofilaments are tubulin dimers stacked vertically in a head-to-tail arrangement, so the microtubule has polarity (directionality).

• the alpha tubulin is the minus end

• the beta tubulin is the plus end

• Polymerization/Depolymerization is possible at both ends (one did not favor over another), but most disassembly occurs at the plus end because the minus end is typically occupied.

• GTP hydrolysis weakens the affinity for other tubulin

How are microtubules made in vitro?

1. individual dimers form oligomers (rate determining step)

2. oligomers form protofilaments

3. protofilaments are elongated and arranged to form hollow tube (fast)

4. tubulin dimers are equally added to plus and minus end until no more available

5. microtubule reaches a state of equilibrium where it polymerizes and depolymerizes at the same rate on both sides of the structure

What does dynamic instability mean in terms of microtubules?

Microtubules in real live get longer, then shorter, then longer, then shorter, and continue repeating this process all based on the microenvironment levels of GTP bound tubulin.

What is the microtubule GTP cap?

Unlike caps for microfilaments, microtubule caps are not physical structures. These are simply areas at the plus end of a microtubule that have a lot of GTP bound tubulin, preventing the subunits from peeling away. In the absence of a GTP cap, rapid depolymerization occurs.

What ways to drugs affect microtubule behavior?

1. binding tubulin

   1. induce depolymerization by hiding available tubulin

   2. some nonspecifically affect all microtubules (ex. colchicine, colcemid)

   3. some target rapidly dividing cells (ex. vincristine, vinblasts)

2. binding microtubules

   1. drug rests on available end of microtubule making it stable

   2. stabilization sounds good, but it’s not always because the cytoskeleton is supposed to be dynamic, especially in M phase (chromatids can’t pull apart so cell can’t divide)

   3. some are good because they can stop cancer cells from dividing

   4. some versions specifically target rapidly dividing cells (ex. taxol)

Where do new microtubules come from?

Microtubule Organizing Centers (MTOC)

In animal cells the primary MTOC is the centrosome, which lies adjacent to nucleus

more specifically: new microtubules come from gamma tubulin in the pericentriolar material (PCM)

Centrioles are not needed to make new microtubules

What is the centrosome made out of?

• two centrioles (in t shape) - can only send microtubules out in 3 directions

• each centriole is arranged in a 9-triplet microtubule structure

• each complete microtubule has 13 protofilaments, anything fewer is incomplete

• a triplet has one complete microtubule (alpha tubule) and two incomplete (beta and gamma tubules) (10-11 protofilaments)

What is the pericentriolar material (PCM)?

an amorphous (without defined shape) collection of protein around the centrioles from which microtubules come from. Gamma tubulin in the PCM is the origin of all new microtubules (start off preformed rings). All microtubules originate in the centrosome, but they don’t all stay.

How does gamma tubulin lead to the formation of new microtubules?

Gamma tubulins have pre-formed platforms for easy polymerization by tubulin dimers. The minus end is always what is anchored to the gamma tubulin. One type of microtubule associated protein (MAP) is responsible for cutting off the microtubules. The end must be capped to prevent depolymerization.

What does polarity of a cell type in terms of microtubules?

• make cells look different

• stable microtubules can make one side look different than another (ex. neuron dendrites)

What are microtubule associated motor proteins?

1. Dyenin - move towards minus end of microtubules

2. Kinesisns - get closer to plus end of microtubules

What are the functions of microtubules and motor proteins?

1. intracellular vesicle transport

2. organelle movement and positioning

3. color change (movement of pigment vesicles)

4. bending of cilia and flagella

5. separation of sister chromatids and chromosomes during M phase

What is the axoneme?

The fundamental unit of movement for cilia or flagella

9 + 2 arrangement (9 doublets, pair of microtubules in middle)

each doublet has a complete and incomplete microtubule

all ends (minus side) are anchored into a basal body

What are the different types of microtubule configurations?

9-triplet: found in centrioles and basal body (root of axoneme made by copying centriole), alpha (complete), beta and gamma (incomlete)

9 + 2: found in axoneme, 9-doublets (alpha complete, beta incomplete) and pair of microtubules in middle. The doublets have dynein motor proteins on them

What’s the difference between axoneme in flagella vs. cilia

• flagella have longer axoneme

• flagella axoneme bend faster

• flagella have 1 or 2 axoneme

• cilia have dozens to hundreds of axonemes

How do axonemes bend?

The rate of movement depends on the type of axoneme

• flagella move fast and in segments

• cilia move more gently

Dynein arms connect alpha to beta so when it tries to move to the negative end, both flops over, and since the doublet are connected, they all also fall over.

ATP is what causes dynein to walk in the first place

All doublets are connected.