Cell 201: Lecture 12 - Cytoskeleton

What is the cytoskeleton?

Network of interconnected filaments + tubules extending through the cytosol

What Role do cytoskeletons play?

Cell movement + division

Moving vesicles around

structure + shaping

True or False: The cytoskeleton is a fixed shape and doesn’t change often

False: it is dynamic + Changable

What are the 3 Main families of cytoskeleton components? List from largest to smallest

1. Microtubules

2. Intermediate filaments

3. Microfilaments

What are each of the 3 Families of cytoskeleton components made up of?

1. Microtubules = tubulin

2. Intermediate filaments = Actin

3. Microfilaments = various proteins

How are each of the 3 cytoplasmic components located/arranged?

Intermediate = around nucleus + Provide strain relief off plasma membrane. Involved in cell to cell connections

Microtubules = emanate off the same area

Actin = Periphery = maintain shape of PM

What is the structure of Intermediate filaments?

8 protofilaments joined end to end with staggered overlaps

Is there polarity in intermediate filaments?

No

• no chemical distinction between either end

What are some functions of intermediate filaments?

• structural support

• Maintenance of cell shape

• Formation of nuclear lamina + scaffolding

• Strengthening of nerve cell axons

• Keeping muscles fivers in register

What is the main distinction between the intermediate filaments + the other 2?

No polarity

• the other 2 have polarity

Why are microtubules + microfilaments polar?

their monomers assemble in a specific repeating way

What is the structure of Microtubules?

Hollow tube with a wall consisting of 13 protofilaments

• protofilaments are made up of alpha beta heterodimers

What is the polarity of the microtubules

They have a + and - end

• One end is all Alpha 

• One end is all beta

What are some functions of microtubules?

• Cytoplasmic

• Organization + maintenance of cell shape + polarity

• Chromosome movement

• Intracellular transport/trafficking + movement of organelles

• Cell motility

What is the structure of Microfilaments?

2 "intertwined” chains of F actin

• made up of G-actin monomers subunits are pointing the same way

What is the polarity of the microfilaments

They have a + and - end

• G actin subunits have a polarity + are assembled head to tail

What is the difference between F vs. G actin?

F actin = the polymer of the monomer ==> FILAMENT

G actin = GLOBULAR single monomer

• same protein diff assembly

What are some functions of Microfilaments?

• Muscle contraction

• cell locomotion

• Cytoplasmic streaming

• Cytokinesis

• Maintenance of cell shape

• Intracellular transport + trafficking

What are some tool (4) used to study the cytoskeleton?

1. Fluorescence microscopy (fixed specimens)

2. Live cell fluorescence microscopy (tag the subunits)

3. Computer enhanced digital microscopy (subunits are too small so need computers)

4. Electron microscopy

True or false: there are naturally produced substances that affect cytoskeleton

True

• Drugs affecting Microtubules microfilaments

What is an example of a drug that affects the cytoskeleton?

Cytochalasin D

• prevent new monomers from adding to + end of Microfilaments

True or false: Bacteria + archaea have cytoskeletal systems that are structurally similar to those in eukaryotes

TRUE

• Bacteria + archaea have polymer systems that function similarly to eukaryotic cytoskeleton elements

In bacteria the actin-like _____ protein is involved in DNA segregation

MreB

In bacteria the Tubulin-like _____ protein is involved in regulating division

FtsZ

In bacteria _____ protein is involved in regulating cell shape (curving of cell)

Crescentin

Super resolution microscopy. What is it used for + how does it work?

For really small things, obtain HIGHER RESOLUTION than microscope can offer using GAUSIAN Distribution (esp. bacteria where things are really small)

What is the largest of the cytoskeleton components?

Microtubules

How do microtubules look?

Ends are not flat bc protofilaments line up in spirals.

What are the 2 types of microtubules?

1. Cytoplasmic

2. Axonemal

What are cytoplasmic microtubules? What do they mainly do? (3 things)

1. Moving chromosomes around during cell division (mitosis + myosis spindles)

2. Maintaining/ altering shape

3. Placement + movement of vesicles

What are Axonemal microtubules? What do they mainly do?

Mainly in Motility organelles structures

• cilia + flagella

What is the axoneme?

Central shaft of a cilium or Flagellum

• highly ordered bundle of microtubules

What are protofilaments?

The longitudinal arrays of polymers that make up Microtubules (hollow cylinder shape)

What is the basic subunit of a protofilament?

Heterodimer

• Alpha tubulin

• Beta tubulin

What Interaction/bond holds the alpha-beta hetero dimers together? Do they ever come apart?

Non-covalent bonds

• never come apart

• one of the strongest non-covalent bonds in biology

Alpha and beta subunits are very _____ (similar/different) in their 3D structure and very ______(similar/different) in their A.A. sequence

Very SIMILAR 3D structure but only 40% a.a. identity

• DIVERGENT IN SEQUENCE but FOLD INTO SAME STRUCTURES

What can Alpha + Beta subunits bind to?

GTP they are NUCLEOTIDE BINDING PROTEINS

Where does GTP bind to in Alpha + Beta subunits?

N-terminus

What does GTP binding do?

Conformation change

Both alpha + beta are GTP binding proteins are both of them GTPases? Why or why not?

No both of them are not GTPases. ONLY BETA is GTPase

• GTP bound to alpha = trapped between B + a and can never be hydrolyzed

• GTP bound to beta is exposed and can be hydrolyzed

What does the C-terminus of a-B subunits associate with?

MAPs (microtubule associated proteins)

Microtubules have polarity: + and - because of the dimer orientation resulting in polarity in the protofilaments. Which subunit of the dimer is associated with the + end and which is associated with the - end?

PLUS end = Beta tubulin (GTP exposed)

MINUS end = Alpha tubulin

Which end ±/-has a higher affinity for new dimers? 

Plus end = higher affinity therefore called the plus end

Minus end = where subunits are preferentially misused

What are the GTP states at each end on the polymer? Why?

Plus end = GTP bound because they are being added

Minus end = GDP bound = fall off

Which of the 2 types of microtubules can form doublets or triplets?

Axonemal

• cytoplasmic are only singlets (A)

In doublets and triplets, how many protofilaments do each of the additional microtubules have?

NOT 13

• A (singlet) = 13

• B/C = 10 or 11

How are Microtubules formed. What 2 things are needed for the polymerization of tubulin dimers?

1. GTP

2. Mg2+

= polymerization

What role does GTP play in microtubule assembly?

GTP = dictates how tightly bound dimers are

1. GTP state = more sticky

2. GDP state = less sticky = fall apart

How does changing the [heterodimers] affect the rate of microtubule assembly?

Higher concentration = more collisions = faster assembly

Is the polymerization of tubulin subunits reversible?

Yes

What is the polymerization process of dimers into protofilaments called?

Nucleation

What is Elongation + how does it differ from nucleation?

Elongation = the addition of subunits onto a partially formed microtubules

Nucleation = initial formation of protofilaments

What does the in vitro assembly of microtubules depend on?

concentration of tubulin dimers

What are the 3 phases of In vitro Microtubule assembly? What is the speed at each phase + why?

1. Lag: Nucleation process is slow

2. Elongation = very fast

3. Plateau = slow down: Amount of available tubulin is low. Rate as assembly = disassembly

What is Critical concentration?

Tubulin concentration at which Microtubule assembly is exactly balanced by disassembly

What occurs above + below critical concentration?

Above = tubule gets longer

Bellow = tubule gets shorter

How does microtubule disassembly occur? Why does it happen when subunit concentrations are low?

GTP bound Subunits bind + add = form a GTP CAP

• Hydrolysis comes from the back and travels towards the cap like a wave

• If assembly is too slow, hydrolysis will catch up + reach the end = the GDP subunits will fall off

Why is the GDP bound state of the microtubule subunits fall off while GTP stays stuck?

GTP bound subunits are in ALIGNMENT = stay stuck

GDP bound = OUT OF ALIGNMENT. Without cap = curl outwards + start to peel off

What is the function of the GTP CAP?

GTP CAP holds the out of alignment GDP together. If hydrolysis reaches end = no CAP to hold together = fall + peel apart

• Stabilizes the Microtubule

The addition of tubulin dimers occurs more quickly at which end of the microtubule? How can this be visualized?

Quick = Plus end

• visualize with Basal body

Are the Critical concetrations’s the same for the plus + minus end of microtubules?

NO

• Minus end is less sticky = need higher critical concentrations to meet the rate of polymerization at the plus end

• at same concentration minus end polymerizes slower

What is Treadmilling?

Addition of subunits at plus = removal at minus

• MT length doesn’t change

What [tubulin'] does there have to be in relation to each end for treadmilling to occur?

[tubulin subunit] = higher than critical concentration for Plus end but lower than minus end?

Is it possible to have a net addition of tubulin subunits at the minus end?

YES

• just need a ton of subunits in order to exceed the critical concentration

What is Microtubule catastrophe?

switch from growth to loss

What is Microtubule Rescue?

Switch back from loss to growth

If the Microtubule disassembles how does that affect the [dimers]?

[dimers] increases

• rescues the MT

Dynamic Cyclization between Catastrophe and rescue as disassembly increases dimers available driving growth

Drugs can affect the assembly of Microtubules. What are the 2 drugs talked about?

1. Colchicine

2. Nocodazole

What does Colchicine do?

Binds to tubulin heterodimers + inhibits their assembly + promotes disassembly

• decreases the concentration of assembly competent heterodimers = amount of collisions that result in assembly = low

often used for gout

What does Nocodazole do?

Inhibits MT assembly

Which effects are more easily reversed? Colchicine or Nocodazole?

Nocodazole

What are Antimitotic drugs + what do they do? What are they useful for? Give 2 examples.

Interfere with spindle formation = inhibit cell division

• Useful in cancer treatment

eg. Colchicine + Nocodazole

Where do microtubules originate from?

MTOC

• microtubule organizing centers

What is a MTOC that many cells have near the nucleus

Centrosome

What are centrioles?

Inside Centrosomes. 2 of them are part of Centrosome in animal cells

What is the structure of centrioles?

9 pairs of triplet microtubules oriented at right angles to each other

What are the functions of centrioles? (2)

1. Organizing microtubules

2. Basal body formation for Cilia + flagella

What do cells without centrioles often have?

Poorly organized mitotic spindles

What is Gamma Tubulin? What does it do?

Involved in NUCLEATION of a+B tubulin

• where MTs grow from

What is gamma tubulin part of/ where is it located?

Part of Ring shaped protein complexes in the CENTROSOME

Which end of the MT is anchored to the gamma tubulin?

minus end

• plus end is growing outward

What are these Ring shaped protein complexes called + what do they do?

Gamma-tubulin ring complexes

• nucleate the assembly of new MT

In what direction does the MT assemble in relation to the centrosome?

away

What are the functions of MAPs + what are they?

Microtubule Associated proteins

• bind at regular intervals along MT

• Allows for interaction with other cellular structures + filaments

What are 2 examples of MAPs?

1. Tau

2. MAP2

What does Tau proteins do?

Cause MTs to form tight bundles in axons

• Bundling proteins (prevents them from getting tangled)

What does MAP2 do?

Bundles MT in dendrites ( a little looser bundling)

What are +TIP proteins? What do they do?

PLUS end tubulin interacting proteins

• they RECRUIT + collect tubulin dimers tot he plus end = increase collision rate

• Stabilize the dimers at plus end + prevent peeling back of heterodimers

DECREASE likelihood of MT catastrophic subunit loss/ disassembly

What are some examples of MT destabilizing proteins?

1. Catastrophins (MCAK)

2. Katanin’s

Why are MT destabilizing proteins important?

needed for mitotic spindle functions

What do Catastrophins do?

Promote peeling of subunits at plus end

What do Katanin’s do?

Sever/cut through MTs

• generate 2 pieces out of one

How does MT disassembly help in mitotic spindle formations

Peeling of MT = forces a ring around the MT in the direction of depolymerization

• Centromere of DNA = attached to ring = getting pulled along as MT depolymerizes

What is Speckle microscopy? How does it work? Why/what is it used for ?

Look at large assemblies made up of tiny pieces + see their specific functions

• eg. since tubulin so small, if all were tagged with fluorescence. Everything would just look green you cant distinguish them and look at their functions

Only tag a small number of proteins in the polymer + look at their function + movement in the larger assembly