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Microtubules, Actin (and intermediate filaments). Macrophage microtubulin green Intermediate filaments red DNA blue. Mitochondria, actin. Cytoplasmic microtubules Cytoplasmic m tubules brkdwn Mitotic spindle Differentiated cell types have stable m tubule structures

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Microtubules, Actin (and intermediate filaments)

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Microtubules actin and intermediate filaments

Microtubules, Actin

(and intermediate filaments)

Macrophage microtubulin green

Intermediate filaments red

DNA blue


Microtubules actin and intermediate filaments

Mitochondria, actin


Microtubules actin and intermediate filaments

Cytoplasmic microtubules

Cytoplasmic mtubules

brkdwn

Mitotic spindle

Differentiated cell types have

stable mtubule structures

Cilia, flagella form from

basal bodies


Microtubules actin and intermediate filaments

So what are Microtubules??

… hollow, rigid tubes composed of a/b heterodimers

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


Microtubules actin and intermediate filaments

Each subunit binds GTP

… but the beta

subunit has intrinsic

GTPase activity.


Microtubules actin and intermediate filaments

Microtubule networks assemble and disassemble

Assembly is initiated at MTOCs

MicroTubule Organizing Centers

(aka centrosomes)


Microtubules actin and intermediate filaments

MTOC

100’s of g subunits serve to nucleate

mtubules,

Centriole pair

(sometimes)

Similar to

Basal bodies


Microtubules actin and intermediate filaments

Anti a tubulin, anti g tubulin


Microtubules actin and intermediate filaments

Growth vs. shrinking

is related to the amt.

of free tubulin (Cc)

Growth when balanced

tipped toward >[Cc]


Microtubules actin and intermediate filaments

Microtubule assembly and disassemble is

CATASTROPHIC!!

Occurs preferentially at the plus ends

Critical Concentration, Cc, above grows/below shrinks

Does not require GTP hydrolysis for assembly

The rapid nature of disassembly is due to its constrained

internal structure (GDP tubulin)


Microtubules actin and intermediate filaments

Autumn Crocus

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?

Yew


Microtubules actin and intermediate filaments

Microtubules in the mitotic spindle of a mouse fibroblast in culture.  

Image by Steve Rogers, UNC Dept. Biology.  (Confocal Microscope)


Microtubules actin and intermediate filaments

Organelles, vesicles with

cargo move around the cell.

Why’s that?


Microtubules actin and intermediate filaments

How do we know?

Pulse-chase with radioactive amino acids injected into rat ganglion

Assay, slice up axon over time, SDS PAGE autoradiography


Microtubules actin and intermediate filaments

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.


Microtubules actin and intermediate filaments

In vitro,

Extruded

Exoplasm

ATP-dependent


Microtubules actin and intermediate filaments

Centrosomes …… away from


Microtubules actin and intermediate filaments

Video microscopy

increases resolution and

visualizes movement.

This movement requires ATP.

But AMP-PNP (analog)

Competitively inhibits


Microtubules actin and intermediate filaments

Ron Vale, UCSF

… and AMP-PNP

Michael Sheetz, Columbia

17.5 kinesin.mov

Movie from Jeff Gelles lab


Microtubules actin and intermediate filaments

Sliding microtubule assay- used to purify kinesin.


Microtubules actin and intermediate filaments

Video microscopy can be used to track the movement of a

single kinesin molecule


Microtubules actin and intermediate filaments

One example of microtubules in action…


Microtubules actin and intermediate filaments

Attached ciliary dynein


Microtubules actin and intermediate filaments

Filaments (F actin) are another important

means by which the cytoskeleton is used for trafficking.

It is important for cell shape, location and contraction


Microtubules actin and intermediate filaments

Abundant:

5% of total protein,

half as filaments

Actin monomers

in 4 colors


Microtubules actin and intermediate filaments

F actin grows at BOTH ends but the plus ends grows faster.

ATP hydrolysis is not required for assembly.


Microtubules actin and intermediate filaments

Phalloidin binds F actin, from Death Angel

Cytochalasins and lantrunculin

bind G actin


Microtubules actin and intermediate filaments

Actin filaments are rarely single …

Nets and bundles.

Profilin

thymosin

filamin

gelsolin

myosin


Microtubules actin and intermediate filaments

Filopodium (spikes) are similarly formed


Microtubules actin and intermediate filaments

Actin-related proteins

promote branching

More distally


Microtubules actin and intermediate filaments

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


Microtubules actin and intermediate filaments

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


Microtubules actin and intermediate filaments

One head (ATPase) and one tail, tail varies


Microtubules actin and intermediate filaments

Dimer

300 heads here, bind actin


Microtubules actin and intermediate filaments

Best understood example … contraction in muscle cells.

Multinucleate (formed by cell fusions

50 mm diameter and centimeters long)


Microtubules actin and intermediate filaments

… just one sarcomere.


Microtubules actin and intermediate filaments

Plus ends


Microtubules actin and intermediate filaments

Each filament has ~300 heads.

Each head binds 5X/s

32 mm in 0.1 s

Attached, No ATP, “rigor”

Released, binds ATP

Cocked, hydrolysis

Loose Pi

Power stroke, loose ADP

Attached again,


Microtubules actin and intermediate filaments

T-tubules bring PM up close to the SR …


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