cytoskeleton - MT and intermediate filaments

Purpose of cytoskeleton (with examples discussed in class)

§  Cell shape:

• Neurons branch out

• amoebas change shape when they move towards a food source

§  Strength:

• mechanical strength

• intermediate filaments support nuclear envelope

§  Cell movement

• Organelles move around within cell while attached to cytoskeletal structures.

• Motor proteinscan move vesicles along cytoskeletal filaments

§  Solid state biochemistry:

• Metabolic pathways anchored to cytoskeletal platform in liquid medium (incr. rxn rate of enzymes)

key players of cytoskeleton

• Microtubules: largest (25 nm diameter), composed of tubulin protein

• Intermediate filaments: mid-sized (10 nm diameter), constructed from a number of different subunit proteins

• Actin filaments: smallest (6 nm diameter), made up of actin protein

  • Typically form helical structure – flexible but becomes more rigid w/ other actin filaments

  •   Provides structural support for plasma membrane; necessary for whole-cell mvmt; drive pinching of 1 cell into 2

• Accessory proteins: regulate and link the filaments to other cell components, as well as to each other

functions of microtubules

determine the positions of organelles, direct intracellular transport, and form the mitotic spindle that segregates chromosomes during cell division

Describe the heterodimer tubulin

• MT are polymers of the protein tubulin

• tubulin subunit is alpha tubulin and beta tubulin globular proteins

  • each alpha or beta monomer has 1 binding site for 1 molecule of GTP

  • GTP bound to alpha tubulin cannot be hydrolyzed or exchanged

  • beta tubulin can be bound to GTP or GDP

protofilaments

composed of alpha-beta tubulin heterodimers stacked head to tail then folded into a ring

• 13 parallel protofilaments make up a MT (hollow cylinder)

• ring then sheet then tube

How is a microtubule polar?

o   Microtubule has a plus side and a minus side

o   Subunits point in same direction (head to tail orientation)

• alpha at minus end - favors elongation

• beta at plus end - biased against elongation

Define T vs D form, what conditions affect these forms

o   T form: beta tubulin is bound to GTP

o   D form: beta tubulin is bound to GDP

• rate of subunit addition high (filament growing rapidly) - new subunit added before nucleotide in prev subunit is hydrolyzed → tip remains in T form, forms GTP cap

• rate of subunit addition is low - hydrolysis may occur before next subunit is added → tip in D form

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

any end that happens to be in T form will grow

and any end that happens to be in D form will shrink

Define dynamic instability. What are catastrophe and rescue?

• Dynamic instability: sudden loss or gain of GTP cap and subsequent conversion from growth to shrinkage or vice versa (at uniform free tubulin conc.)

  • catastrophe: sudden loss of GTP cap → change from growth to shrinkage

  • rescue: gain of GTP cap → change from shrinkage to growth

Why do microtubules grow and shrink (proposed purpose)?

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

How does Taxol kill dividing cells?

• taxol: dried bark of Pacific yew tree

• binds to and stabilizes MTs, causing a net incr in tubulin polymerization

• MT dynamics crucial for correct functoin of mitotic spindle - Taxol kills dividing cells

• can be used to kill certain type of tumor cells

Define nucleation and MTOC (Microtubule organizing center). What is the purpose of the gamma ring complex? What part of the microtubule grows outward?

• Microtubule nucleation: de novo (new) formation of microtubules; takes place in intacellular location known as MTOC

• MTOC high in gamma tubulin. Gamma tubulin + 2 accessory proteins = gamma tubulin small complex. Formation of gamma tubulin ring complex (gamma-TuRC) serves as template to create a MT w/ 13 protofilaments

• nucleation occurs from minus end (plus end “exposed”)

What is the MTOC called in animal cells? What are centrioles and where are they found?

o   Centrosome – MTOC in animal cells, located near the nucleus, from which MT are nucleated at their minus ends

o   Centrioles – embedded within the centrosome. Cylindrical structure consists of MTs arranged in barrel shape

What part of the microtubule is anchored to the MTOC?

minus end of MTs are to MT-organizing centers

Structure of intermediate filaments (focus on differences between microtubule structure and intermediate filaments), how is the structure regulated, general function

• 2 alpha-helical monomers forma coiled-coil dimer (str. hydrophobic interactions)

• 2 dimers form a staggered tetramer

• 8 tetramers pack laterally, form growing filament

• easily bent, difficult to break

• often regulated by phosphorylation / dephosphorylation events

• provide mechanical str.

nuclear lamins - where are they found, diseases associated w/ mutations

meshwork lining the inner membrane of the nuclear envelope

progeria: point mutation inthe Lamin A gene:

• fragile nuclear membrane, low tolerance to mechanical stress

• accelerated aging

keratins - where are they found

• mechanical str. to epithelial tissue by anchoring the intermediate filaments at sites of cell-cell contact (desmosomes) or cell-matrix contact (hemidesmosomes)

• tough coverings for animals: skin, hair, nails, claws, scales

keratins - diseases associated with mutations

Mutations in keratin genes: defective keratins in basal cell layer of epidermis - causes epidermolysis bullosa simplex

• skin blisters in resonse to very slight mechanical stress, which ruptures basal cells