Microtubules Flashcards
Microtubule formation (formed from…)?
Microtubules are formed from protein subunits of tubulin.
Tubulin subunit is a heterodimer formed from 2 closely related globular proteins called α-tubulin and β-tubulin, tightly bound together by noncovalent bonds
α-tubulin “N-site”
Non-exchangeable GTP binding site
Protofilaments
Composed of alternating α-tubulin and β-tubulin
Microtubulin diameter, composition & contacts
25nm in diameter Preferably 13 (can vary from 11-16) laterally associated protofilaments. Along the longitudinal axis of the microtubule, the “top” (N-terminus) of one β-tubulin molecule forms an interface with the “bottom” (C-terminus) of the α-tubulin molecule in the adjacent dimer subunit. This interface is very similar to the interface holding the α and β monomers together in the dimer subunit, and the binding energy is strong. Lateral contacts are formed between neighbouring protofilaments. The main lateral contacts are between monomers of the same type (α-α and β-β).
The microtubule itself has a …, with α-tubulins exposed at one end and β-tubulins exposed at the other end
distinct structural polarity
… are exposed at the minus end, and … are exposed at the plus end.
α subunits, β subunits
The … is buried inside the tubulin monomers, while the … is exposed.
N-terminus, C-terminus
Length of a tubulin heterodimer
8nm
β-tubulin “E site”
Exchanhgeable (hydrolysable) GTP binding site
Experimental evidence C-terminus outside of microtubule
Using antibodies to sections of the α/β monomers, antibodies against N-termini only worked with denatured microtubules (the N-termini usually face the lumen of the microtubules).
What are the main differences between actin and microtubules?
- Subunits in tubulin form a dimer
- Tubulin binds GTP, instead of ATP
- Tubulin binds 2 nucleotides (GTP) per dimer, actin binds 1 nucleotide (ATP) per monomer.
- Form of polymer: hollow tube composed of 12 protofilaments for microtubules, 2-stranded helix for actin
- Diameter of filament: 25nm-mt, 7nm-act
- The results show that microtubule networks are easily deformed Actin filament networks are much more rigid, both rupture easily.
- In cells, dynamic instability is thought to predominate in microtubules, whereas treadmilling may predominate in actin filaments.
What are the factors needed for polymerisation, both for actin and tubulin?
Mg2+, Ca2+ and NaCl
Autoregulation of
Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs.
If there is a high concentration of the tubulin dimer → the MREI sequence on nascent β tubulin polypeptide that is being synthesised is recognised → inhibits the transcription that is in process → RNAse is released → no more formation of the tubulin dimer.
What factors contribute to tubulin heterogenity?
- Tubulins are encoded by a multigene family whose members are under strict regulation. Production of the various microtubule-specific heterodimers requires coordinate expression of the appropriate α-tubulin and β-tubulin genes → discovered through 2d gels and western blotting
- Multiple isotypes through post-translational modifications, acetylation, tyrosination, modulate stability of MT & MAP binding → isotype specific functions (e.g. β6 tubulin is required for marginal band formation in chicken erythrocytes)
- Differential expression of genes
* The tubulin sequences are highly conserved, except for the C terminus
What is dynamic instability?
Concentration of tubulin dimers between the critical concentration for the GTP and GDP form → ends that happen to be in the T form will grow, while ends in the D form will shrink → end might grow for a certain length of time in a T form, but suddenly change to the D form & shrink rapidly, even while subunit concentration is constant → rapid interconversion between growing end & shrinking state = dynamic instability
Catastrophe
Change to a rapid shrinkage
Rescue
Change to growth
MT grow out from
centrosome (MTOC)
GTP is hydrolysed after …
Microtubule polymerisation
MT do not shrink when …
polymerised in the presence of non-hydrolysable GTP
MT are only found in … (evolutionary)
eukaryotes
Tubulin hydrolysis …, therefore allowing …
weakens the bonds between tubulin molecules, depolymerisation
A … stabilises the … of a microtubule. If the concentration of tubulin heterodimers is …, dissociation of tubulin-… may expose tubulin-… at the plus end, causing that end to become unstable. … ensues.
GTP-cap, +
low, tubulin-GTP, tubulin-GDP
Rapid shrinkage
Dynamic instability is a consequence of…
the delayed hydrolysis of GTP after tubulin assembly.
Why do GDP-tubulin molecules have weaker binding?
GTP tubulin has a straight conformation → GTP hydrolysis → GDP tubulin is curved → curverd protofilaments interact weaker leading to catastrophe (due to mechanical stress on MT lattice in absence of GTP cap) → energy of GTP is used to depolymerise molecules.
While α- and β-tubulins are the regular building blocks of microtubules, another type of tubulin, …, is involved in the … of microtubule growth.
γ-tubulin, nucleation
Microtubules are generally nucleated from a specific intracellular location:
microtubule-organizing centre (MTOC)
Discovery γ-tubulin
Genetic screen for proteins that interact with MT in A. nidulans → mutations in gene MIPS that acts as extragenic suppressor of a heat sensitive β-tubulin mutation benA33
- MIPA gene product has 30% homology with α- and β-tubulins
- Found in humans, maize, frog…
- In A. nidulans MIPA attaches MT to SPB (spindle pole body
Microtubules are nucleated at their … end, with the … end growing outward from each … to create various types of …
minus, plus, MTOC, microtubule arrays
What is a γ-TuRC?
A γ-tubulin ring complex (γ-TuRC) has been is an impressively efficient nucleator of MT growth. This ring of γ-tubulin molecules is thought to serve as a template that nucleates a microtubule with 13 protofilaments.
Consists of γ-tubulin and accessory proteins.
The MTOC in animals is the …, in yeast/fungi … and in plants is found …
centrosome, spindle pole body, throughout MT array (a poorly defined electron-dense material)
Embedded in the … is the …: a pair of cylindrical structures, which become the … of cilia & flagella in motile cells and organise the …
Centrosome, centrioles, basal bodies
centrosome matrix
What is the pericentriolar material?
Pericentriolar material (PCM) is an amorphous mass of protein which makes up the part of the animal centrosome that surrounds the two centrioles.
Spindle Pole Body
Functionally equivalent to the centrosome, unlike the centrosome the SPB does not contain centrioles. The SPB organises the microtubule cytoskeleton which plays many roles in the cell. It is important for organising the spindle and thus in cell division.
What are MAPs and what are their main properties?
- Proteins that bind along the sides of microtubules
- Group of proteins that co-purify with MT
- Promote assembly and stabilise MT
- Different sizes, can be subdivided into:
1. High molecular weight (200-300kD)
2. Tau (55-62kD) - Different thermostability:
1. Boiling doesn’t affect activity
2. Sensitive to boiling
… binds to the microtubule lattice at both its N- and C-termini, with a … projecting loop. Gives …nm projections on MT
Tau, short, 35nm
… binds along the microtubule lattice at one end and extends a … with a second microtubule-binding domain at the other end. Gives …nm projections on MT
MAP2, long projecting arm, 90
What are the specific properties of Tau?
- 1 per 12 tubulin dimers
- 55-62kD
- give closely packed MT
- found in axons (long, parallel MT)
What are the specific properties of MAP2?
- …
- 200-300kD
- give widely spaced MT
- found in dendrites (short, parallel & antiparallel)
What are some properties of both Tau and MAP2?
- Promote assembly & stabilise MT
- Thermostable
- Bundles & stabilises MT
- Strongly homologous structure
What is the main difference between MAP2 and Tau?
The N-terminal extension in tau is 35nm, in MAP2 it is 90nm.
What is the MAP2/tau structure?
N-terminal projection (long in MAP2, short in tau) – KSP (phosphorylation site) – positively charged amino acids (3 repeats), interspersed with PGGG sharp turns – H-phobic helix C-terminal
How is MAP2/tau regulated?
They’re are negatively regulated by phosphorylation.
What is the MAP2/tau main function?
They promote assembly and stabilise MT (promote nucleation)
In addition to … at the … ends, there are several groups of …binding proteins that help localize the growing microtubule end to specific target proteins in the cell cortex.
γ-TuRC, minus, plus-end-
End Binding Proteins properties
- Binds to growing + ends of MT by calpolin homology domains
- Doesn’t bind to shrinking + ends
- Has a higher affinity for GTP-tubulin → promotes MT polymerisation
- Interacts with a network of other + end binding proteins, through EB-homology and EEY/F domains.
Structure EB 1,2,3
Calpolin homology – coiled coil – EB homology – EEY/F
Discovery of motor proteins
First identified in the giant axon of the squid, where it carries membrane-enclosed organelles away from the neuronal cell body toward the axon terminal by walking toward the plus end of microtubules.
Soluble proteins were bound to MT and released into solution by addition of ATP → two classes separated by size & density:
dyneins & kinesins.
