Microtubules, Actin (and intermediate filaments) Macrophage microtubulin green Intermediate filaments red DNA blue.

Microtubules, Actin(and intermediate filaments)

Macrophage microtubulin greenIntermediate filaments redDNA blue

Mitochondria, actin

Cytoplasmic microtubules

Cytoplasmic tubulesbrkdwn

Mitotic spindle

Differentiated cell types have stable tubule structures

Cilia, flagella form from basal bodies

So what are Microtubules??

… hollow, rigid tubes composed of heterodimers

POLAR.. w/o polarity, it can not function.

Each subunit binds GTP

… but the betasubunit has intrinsicGTPase activity.

Microtubule networks assemble and disassemble

Assembly is initiated at MTOCs

MicroTubule Organizing Centers(aka centrosomes)

MTOC100’s of subunits serve to nucleatetubules,

Centriole pair(sometimes)

Similar to Basal bodies

Anti Anti tubulin tubulin, , anti anti tubulin tubulin

Growth vs. shrinkingis related to the amt.of free tubulin (Cc)

Growth when balanced tipped toward >[Cc]

Microtubule assembly and disassemble is

CATASTROPHIC!!

Occurs preferentially at the plus endsCritical Concentration, Cc, above grows/below shrinks

Does not require GTP hydrolysis for assemblyThe rapid nature of disassembly is due to its constrained

internal structure (GDP tubulin)

Toolbox:

Colchicine (from crocus) binds free tubulin

Taxol (from yew) stabilizes microtubules

Both cancer therapy drugs, both block mitosis.

How? If opposite modes of action?

YewYew

Autumn CrocusAutumn Crocus

Microtubules in the mitotic spindle of a mouse fibroblast in culture.  Microtubules in the mitotic spindle of a mouse fibroblast in culture.   Image by Steve Rogers, UNC Dept. Biology.  (Confocal Microscope)Image by Steve Rogers, UNC Dept. Biology.  (Confocal Microscope)

Organelles, vesicles with cargo move around the cell.

Why’s that?

Pulse-chase with radioactive amino acids injected into rat ganglionPulse-chase with radioactive amino acids injected into rat ganglionAssay, slice up axon over time, SDS PAGE autoradiographyAssay, slice up axon over time, SDS PAGE autoradiography

How do we know?How do we know?

Microtubules are needed for trafficking vesicles (10 cm/day) but this is dramatically faster than diffusion in the axon.

…in a differentiated cell- dynamic instability is suppressed… by capping proteins.

In vitro,ExtrudedExoplasm

ATP-dependent

Centrosomes …… away from

Video microscopy Video microscopy increases resolution and increases resolution and visualizes movement. visualizes movement.

This movement requires ATP.This movement requires ATP.

But AMP-PNP (analog)But AMP-PNP (analog)Competitively inhibitsCompetitively inhibits

Ron Vale, UCSF

Michael Sheetz, Columbia

… and AMP-PNP

Movie from Jeff Gelles lab

17.5 kinesin.mov

Sliding microtubule assay- used to purify kinesin. Sliding microtubule assay- used to purify kinesin.

Video microscopy can be used to track the movement of a Video microscopy can be used to track the movement of a single kinesin moleculesingle kinesin molecule

One example of microtubules in action…One example of microtubules in action…

Attached ciliary dynein

Filaments (F actin) are another important means by which the cytoskeleton is used for trafficking.

It is important for cell shape, location and contraction

Actin monomersin 4 colors

Abundant:5% of total protein, half as filaments

F actin grows at BOTH ends but the plus ends grows faster. ATP hydrolysis is not required for assembly.

Phalloidin binds F actin, from Death Angel

Cytochalasins and lantrunculinbind G actin

Actin filaments are rarely single …

Nets and bundles.

Profilinthymosin

filamin

myosin

gelsolin

Filopodium (spikes) are similarly formed

Actin-related proteins promote branching More distally

Racs (Cdc42 is a member) are small GTPases related to the Ras members discussed before.These can be activated by recptors and in turn activate actin assembly

Myosin is a motor that runs along (or tugs at) actin.

Actin-based movement of vesicles

Myosin-driven cell shape changes

Muscle contraction

Myosin I and myosin I

One head (ATPase) and one tail, tail varies

Dimer

300 heads here, bind actin

Best understood example … contraction in muscle cells.

Multinucleate (formed by cell fusions50 m diameter and centimeters long)

… just one sarcomere.

Plus ends

Each filament has ~300 heads.Each head binds 5X/s32 m in 0.1 s Attached, No ATP, “rigor”

Released, binds ATP

Cocked, hydrolysisLoose Pi

Power stroke, loose ADP

Attached again,

T-tubules bring PM up close to the SR …T-tubules bring PM up close to the SR …