Lecture 14. Microtubules, MAPs and Motors

Question 1

What body mechanics is the cytoskeleton responsible for?

Answer 1

Food mastication
Digestion
Blood circulation
Communication
Reproduction
Body movement

Question 2

What cellular mechanics is the cytoskeleton responsible for?

Answer 2

Organisation of organelles
Chromosome segregation
Protein and RNA transport
Cell division
Cell motility and chemotaxis
Maintaining cell integrity

Question 3

What is the structure of microfilaments?

Answer 3

Double helix of two actins and made into a polymer (7-9nm wide)
Can be 10-100,000 long

Question 4

What is the structure of microtubules?

Answer 4

Made up of two proteins, α-tubulin and β-tubulin (25nm wide)
Hole int he middle that forms a tube

Question 5

What is the structure of intermediate filaments?

Answer 5

Discovered between the microfilaments and microtubules - made up of different proteins (10nm wide)

Question 6

What energy sources are required for microfilaments, microtubules and intermediate filaments?

Answer 6

Microfilaments: actin binds and uses ATP
Microtubules: αβ-tubulin binds and uses GTP
Intermediate filaments: don’t have/need a fuel source (because not dynamic)

Question 7

What do microfilaments, microtubules and intermediate filaments do, generate or resist force?

Answer 7

Microfilaments: generate force by forming rigid gels, networks, and linear bundles
Microtubules: generate force as rigid an not easily bent
Intermediate filaments: resist force because they have great tensile strength

Question 8

What makes up microtubules?

Answer 8

Barrel/tube-like structure made up of a series of protofilaments (go along the long axis of the microtubule) made up of repetitive arrays of α,β tubulin dimer

Question 9

In which subunit of microtubules does GTP hydrolysis take place?

Answer 9

β subunit (stays GTP in α subunit)

Question 10

How does polarity exist in the protofilament and why is it important?

Answer 10

Plus end = end that ends with β subunit
Minus end = end that ends with α subunit
Gives directionality to the motor proteins to allow them to know which direction they are going in

Question 11

How many protofilaments make up a microtubule?

Answer 11

13

Question 12

What states must α-tubulin and β-tubulin be in to make a protofilament?

Answer 12

GTP-bound state (especially β-tubulin called T form)

Question 13

How does β-tubulin bind to an existing microtubule and make it grow?

Answer 13

GTP-bound β-tubulin (T form) binds preferentially to the plus end of the protofilament (and to a much weaker extent the minus end)
However, when αβ-tubulin dimers are added to protofilament, T form β-tubulin undergoes hydrolysis into GDP bound β-tubulin (D form). D form is the predominant form in the lattice. At the plus end since polymerisation is much faster than hydrolysis polymerising microtubules have a GTP cap. By contrast polymerisation is slower than hydrolysis at the minus end so the minus end is always in the D form

Question 14

How does dynamic instability work in microtubules?

Answer 14

In a catastrophe event where the GTP cap is lost, rapid depolymerisation of the microtubule takes place, leaving the plus end with D form β-tubulin until a rescue event occurs and new T form β-tubulin binds to the end and rapid growth occurs again

Question 15

How are the ends of the microtubule arranged in the cell?

Answer 15

-ve end near the nucleus with the +ve end protruding outwards to feel for the plasma membrane from the centrosome source
Sometime new microtubules will grow along existing microtubules to try and find plasma membrane

Question 16

What is the difference in the shape of the growing and shrinking microtubule end?

Answer 16

When growing, microtubule has a straight end (bamboo shoot looking)
When shrinking, microtubule has a ram horn shape caused by rapid depolymerisation where each protofilament is bending back on itself

Question 17

Why does the ram’s horn structure appear when the microtubule is shrinking?

Answer 17

D form β-tubulin has an angle of 12º between the α and β subunits (vs the 5º angle in T form (Straight heterodimer))
The D form β-tubulin causes the end to twist and coil and try to break away which is normally impossible because of the GTP cap which is straight and keeps the microtubule strained

Question 18

How can the rate of microtubule polymerisation, pausing, depolymerisation and rescue be modulated?

Answer 18

By microtubule associated proteins (MAPs) and motors

Question 19

What is EB-1 (End Binding protein-1)?

Answer 19

A plus end MT binding protein
EB-1 only has binding sites on the microtubule growing end where α,β tubulin dimer is in it’s T form, but won’t bind when dimer in D form
Acts as a marker for the growing plus end
HAs binding site that allows other proteins to utilise EB-1 and surf on the growing MT to be deposited at the plasma membrane and released as T form becomes D form

Question 20

What effect do nocodazole and colcemid have on microtubule stability?

Answer 20

Nocodazole and Colcemid are structurally unrelated small molecules that bind to the α,β-tubulin dimer to prevent its addition to microtubules. Addition of either compound to cells prevents microtubule polymerisation and causes microtubules to disassemble. Both are used frequently in biological research.

Question 21

What is the part where microtubules become nucleated called?

Answer 21

Microtubule organising centres (MTOC), way to keep the minus ends in one particular place
In animal cells this is the centrosome
The minus ends of microtubules are at the MTOC near the nucleus and the plus ends spread out towards the cell periphery

Question 22

What is the centrosome composed of?

Answer 22

Two centrioles surrounded by pericentriolar material (centrosome matrix) to which γ-tubulin ring complexes (γ- TURCs) are associated

Question 23

What are the γ-tubulin ring complexes (γ-TURCs) responsible for?

Answer 23

The γ-TURCs are responsible for nucleation of microtubules. Recruitment of γ-TURCs to the centrosome explains the nucleation pattern of microtubules seen previously

Question 24

What composes up the γ-tubulin ring complexes?

Answer 24

Composed of a ring of 13 subunits of γ-tubulin onto which α,β tubulin dimers bind. In addition γ-TURC contains several other accessory proteins which are also highly conserved in eukaryotes

Question 25

What role does γ-TURC have at the minus end of the microtubule?

Answer 25

Prevents depolymerisation

Question 26

What is the role of α and β tubulin?

Answer 26

α/β heterodimers form microtubules. Major constituents of microtubules. Found in all eukaryotes

Question 27

What is the role of γ tubulin?

Answer 27

Major component of γ-tubulin ring complex (γ-TURC) recruited to microtubule organising centres (MTOCs) - usually the centrosome. Found in all eukaryotes

Question 28

What is the role of δ and ε tubulin?

Answer 28

Components of centrioles and basal bodies. Found only in some eukaryotes and in protozoa such as Paramecium and Chlamydomonas (green algae)

Question 29

What is the role of ζ and η tubulin?

Answer 29

Found only in some species. Specialised function.

Question 30

What is the role of FtsZ?

Answer 30

Bacterial tubulin relative that forms polymers for cytokinesis

Question 31

What is the shared role of the Kinesin and Dynein motors?

Answer 31

Responsible for numerous microtubule-dependent transport events in eukaryotic cells. The organisation of the ER and Golgi depends on the orientation of microtubules and motor activity

Question 32

What is the main role of Kinesin motors?

Answer 32

Kinesin motors generally direct organelles (e.g. ER) and vesicles towards the plasmamembrane, for example early endosomes and secretory vesicles for exocytosis)

Question 33

What is the main role of Dynein motors?

Answer 33

Dynein directs both organelles (e.g. Golgi) and vesicles away from the plasmamembrane, for example late endosomes (for endocytosis), lysosomes and ER-Golgi intermediate compartment (ERGIC)

Question 34

What are examples of organelles and vesicles that bind to both Kinesin and Dynein?

Answer 34

E.g organelle: mitochondria
E.g vesicle pigment granules

Question 35

What are the properties of Kinesin?

Answer 35

Almost exclusively plus-end directed motors (except kinesin-14 which is minus-end directed)
14 structurally related classes
Use ATP to generate force

Question 36

What are the properties of Dynein?

Answer 36

Exclusively minus-end directed motor
Part of a very large Dynein-Dynactin complex
Structurally unrelated to kinesins
Use ATP to generate force

Question 37

What is the structure of a kinesin dimer?

Answer 37

Head region (where it binds to either ADP or ATP) that “walks” on the MT
Tail end bound to cargo (normally a vesicle)
Light chain on tail end helps bind kineisn to cargo

Question 38

What is the mechanism of kinesin movement?

Answer 38

The single dimer of kinesin can move processively along the microtubule because the action of the two heads are co-ordinated, and one of the two heads is always bound Thus, an individual kinesin molecule can transport a cargo such as a vesicle or mitochondrion a long way - for example along an axon

Question 39

What is the timeline of kinesin movement?

Answer 39

1. forward motor binds β-tubulin releasing ADP
2. Forward head binds ATP
3. Conformational change in neck linker causes rear head to swing forward
4. New forward head releases ADP, trailing head hydrolyses ATP and release phosphate (Pi)

Question 40

What is the mechanism of dynein movement?

Answer 40

Dynein moves exclusively towards the minus end of microtubules and is a much faster motor than kinesin. It cannot bind cargo itself. This is mediated by an 11 subunit complex called the Dynactin complex which contains Dynactin and other proteins including Arp1 (an actin related protein) which forms a mini filament that is responsible for cargo binding. Dynactin also has a microtubule binding domain to ensure processivity of the Dynein-Dynactin complex, when the Dynein motor heads lose contact with the microtubule.

Question 41

What does the heavy chain of dynein contain?

Answer 41

The Dynein heavy chain (motor) contains an N-terminus tail which binds the Dynactin complex (to bind cargo) and a C-terminus composed of 6 AAA ATPase domains which are arranged in a wheel
The C-terminal ATPase domain closes the wheel by forming contacts with the first AAA domain (red) which is the major ATPase that generates the conformational change which alters the position of the tail relative to the ATPase wheel

Question 42

How does bidirectional organelles or vesicle transport work?

Answer 42

Melanosomes in fish pigment are altered depending on whether the fish is scared or not
Contain large pigment granules that can change their location in response to hormonal or neuronal stimulation. The second messenger in these signalling pathways is cAMP. The pigment granules aggregate or disperse depending on the concentration of cAMP in the cell (through the action of cAMP-dependent protein kinase). Kinesin drives the melanosomes to the plasma membrane when cAMP increases and Dynein moves them back when cAMP decreases