Cytoskeleton: Microtubules and Intermediate Filaments

Difference between alpha and beta subunits

beta subunit can hydrolyze to GDP, both can bind to GTP

Which side do microtubules grow from

plus end

what forms the building blocks of microtubules

tubulin

what are protofilaments

linear polymers formed by the polymerization of tubulin heterodimers

microtubule polarity

polar

microtubule dynamics influenced by

binding and hydrolysis of GTP

Tubulin subunits at end of microtubule depends on

Rates of GTP hydrolysis and tubulin addition

T form conditions

rate of subunit addition is high, likely new subunit will be added before nucleotide in previous subunit is hydrolyzed, forms GTP cap

D form conditions

Rate of subunit addition is low, hydrolysis may occur before next subunit is added

What happens when concentration of free tubulin is intermediate, above concentration necessary for T form assembly, but below that for D form?

Dynamic instability

Since the concentration is below the threshold for D form assembly, the microtubules formed are relatively short lived and unstable

MT continuously alternate between polymerization and shrinkage

Dynamic instability

Where microtubules undergo rapid and stochastic transitions between phases of polymerization (growth) and depolymerization (shrinkage).

GTP Hydrolysis:

enzymatic cleavage of GTP to GDP that occurs on beta tubulin subunits after they are incorporated into the microtubule lattice.

Why do microtubules grow and shrink (proposed purpose)?

o   May reduce time required to find a target (formation of mitotic spindle, microtubules search for chromosomes). Microtubules could explore the interior of the cell.

Explain how a microtubule is polar

due to the structural arrangement of α- and β-tubulin subunits, with a distinct plus end where polymerization and depolymerization occur and a minus end where polymerization is less favored.

What keeps tubulin together in the microtubule

non-covalent interactions

How does Taxol kill dividing cells?

Makes them resistant to depolymerization

Taxol binds to beta tubulin subunits in microtubules and promotes their assembly into stable microtubule structures.

prevents proper chromosome segregation, causes apoptosis

Define nucleation

involves the assembly of tubulin subunits into a small, stable structure that serves as the starting point for microtubule growth.

what part of the microtubule is anchored to the MTOC?

minus end

Gamma tubulin small complex

2 accessory proteins bind to gamma tubulin

What is the purpose of the gamma ring complex?

o   Formation of spiral ring of gamma tubulin molecules, serves as template (creates a microtubule with 13 protofilaments)

o   Spiral associates with additional accessory proteins to form gamma tubulin ring complex: nucleate from minus end

What is the MTOC called in animal cells? 

Centrosome: well defined MTOC in many animal cells, located near nucleus, from which MT are nucleated at their minus ends

What are centrioles and where are they found?

Centrioles: Embedded within centrosome, cylindrical structure consists of MTs arranged in barrel shape

Structure of intermediate filaments

o   Non-polar

o   Alpha helical region of monomer -> coiled-coil dimer -> staggered tetramer of two coiled-coil dimers

o   Tetramers pack laterally to form filament

o   Large number of polypeptides lined up together, with strong lateral hydrophobic interactions, gives a ropelike character

o   Easily bent, difficult to break

o   Provide mechanical strength

Differences between IF and MT

o  IF made of fibrous protein, regulated through phosphorylation, provide support

Where are IF found

vertebrates, nematodes, mollusks

What are IF regulated by

phosphorylation/dephosphorylation events

Nuclear lamins:

meshwork lining the inner membrane of the nuclear envelope

Progeria

o   point mutation in the lamin A gene

§  Fragile nuclear membrane, low tolerance to mechanical stress

§  Accelerated aging

Keratins

o   Mechanical strength to epithelial tissue by anchoring the intermediate filaments at sites of cell-cell contact (desmosomes) or cell-matrix contact (hemidesmosomes)

Examples of IF

Lamins and Keratins

Mutations in keratin genes:

§  Defective keratins in basal cell layer of epidermis: causes epidermolysis bullosa simplex

§  Skin blisters in response to very slight mechanical stress, which ruptures basal cells