Lecture 21: Microtubules

What is the structure of microtubules?

• alpha and beta-tubulin dimer

• alpha is always in GTP form

• beta is mostly in GDP form

• alpha is the minus end

• beta is the plus end

• contains a seam

When is GTP hydrolyzed on a MT?

after monomer adds to a polymer

When can GDP be exchanged for GTP?

after monomers are released from a polymer (GDP is locked in as long as a monomer holds it in)

True or False: The MTs in flagella and cilia are identical in structure.

true

What are centrosomes?

MT organizing cnters

True or False: Nuclei are not in the middle of the cell but centrosomes are.

true

Where are the minus ends of MTs anchored?

the centrosome

What do spindle poles do?

reach out and grab sister chromatids to faithfully separate them

What are basal bodies?

MT organizing centers

Where do plus ends extend in basal bodies?

towards the tip

True or False: Mitotic spindles are a dynamic process because they are continually breaking and building.

true

What and when were MTs defined by?

EM in 1963

Are MT dimers stable?

yes, they rarely dissociate

True or False: Alpha and beta-tubulin have about 40% of identical amino acid residues.

true

True or False: Tubulins are highly conserved and are more than 75% identical between plants and animals.

true

How many protofilaments are in 1 MT?

13 protofilaments

What is the seam in a MT?

the region where alpha GTP and beta GDP meet

How are dimers assembled in MTs?

in a linear/head-to-head fashion

Is there polarity in tubulin polymerization?

yes, subunits add preferentially to the plus ends

Does the alpha-tubulin or the beta-tubulin have a higher critical concentration?

alpha-tubulin does

What did the study about the dynamics of pure tubulin (Mitchison and Kirschner) reveal?

the total amount of monomers in the polymer is constant, but the number and length of filaments is not constant

What happens to MTs as time progresses in the study?

there are fewer MTs but a constant amount of actin

What is catastrophe?

the change from growing to shrinking

What is rescue?

the transition from shrinking to growing

What is dynamic instability?

a process of multiple catastrophes and rescues

What does catastrophe result from?

the accidental loss of the GTP cap

What does rescue result from?

regaining the GTP cap

True or False: The site of GTP hydrolysis is close to polymerization.

true

What shape are filaments in when assembling?

linear

What does GTP beta tubulin form?

linear filaments

What does GDP beta subunits form?

splayed filaments

What shape are filaments in when disassembling?

in a “splayed” formation

What causes structural strain to MTs?

GTP hydrolysis

What are the roles of GTP hydrolysis?

• makes the reaction steady state

• puts strain on protofilaments

What is structural strain constrained by?

the GTP cap at the plus end

What do MAPs stand for?

MT-associated proteins

What do MAPs play a role in?

• assembly

• disassembly

• stability

• spacing and arrangement of MTs

• search and capture

What does EB-1 do?

• binds to the seam of the GTP cap

• stabilizes the GTP cap

• promotes elongation

• slows rate of GTP hydrolysis

What are XMAP215 and CLASP, and what do they do?

TOG domains that bind to the GTP cap to promote assembly

What is the function of Kinesin-13 and Stathmin?

promote disassembly by binding to the curved form of tubulin (GDP tubulin)

What does Katanin do?

promotes disassembly by severing GDP MTs

Where does MAP2 localize, and what does it do?

localizes to dendrites and cross-links MTs to MTs and IFs

Where does tau localized, and what does it do?

localizes to axons and assembles, stabilizes, and cross-links MTs

True or False: MAPs can dimerize with other MAPs,

true

True or False: MAPs can regulate MT spacing, and MT-spacing depends upon MAP that is expressed and the length of the arms.

true

True or False: MAPs often connect to IFs.

true

What are the drugs that disrupt MT dynamics?

colchicine and colcemid

What is important to know about colchicine?

• binds alpha-beta dimers

• prevents polymerization

• blocks cells in metaphase when drugs are in low concentrations

• used to count chromosomes in metaphase and treat gout

What drug disrupts MT dynamics?

Taxol

What does taxol do?

binds and stabilizes MTs

What does Taxol treat?

cancers such as breast cancer

What type of cells does Taxol target?

rapidly dividing cells

How are many MAPs regulated by?

phosphorylation, specifically cyclin-dependent kinases

True or False: Cell cycle regulation of the kinases responsible for phosphorylating MAPs allow for rapid disassembly and rebuilding of the MT structures associated with the different phases of the cell cycle.

true