motility mcbo lecture

8/8/2019 Motility MCBO Lecture

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MCBO-I core lecture

Intracellular Transport / CytoskeletonCell motility

David Teis, PhD

Biocenter

Membrane Traffic and Signaling Group

[email protected]

Cytoskeleton determines cell shape / movement

Vic Small Lab

Mouse fibroblastMoving into wound (3h)

Chicken fibroblastMoving alone (3h)

m.melanomacell

20 min

trout

epidermalKeratocyte

4min

15 Qm/min

Which biological processes require cell migration ?

UsainBolt

How do cells move ?

on extra-cellular matrix (made by cells)

Integrin Matrix Interaction

Integrins tie the outside of the cell to the intracellular cytoskeleton

Integrins are part of a fundamental architectural toolkit characteristic for all

mutlicellular species

Integrins: Outside-in / inside out signaling

E and 8 F-chains combine into 24 specific Integrindimers

different specificities for ligands

Short intracellular domain (13-70aa) large extracellular domains (800aa)

Hemidesmoses in Epithelia contain E6F4 (binds to laminin in the basal lamina)

Separation of intracellular tails


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Mutations in EIIbIntegrin and failure

to activate Integrins results in

Glanzmanns disease

Talin: a key regulator for integrin activation

FERM domain: F0-Ezrin, Radexin, Moesin

Tail: recruits Vinculin, which in turn binds to

actin

C-term: binds directly to actin

After integrin activation..

Legate K R et al. Genes De v. 2009;23:397-418

Focal

complexes

Focal

adheasions


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Integrins -> Focal contacts / adheasion

Integrin based dynamic multi-protein complexes (app. 160 proteins)link the actin network with the ECM

Transducea variety of signals that regulate proliferation, differentation survival

Israel Patlae t al 2010 NCB


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Repeat:72nm

14 subunits/strand

28 subunits total

ACTIN, an ATPase

Assembly properties of actin: steady state treadmilling

Barbed end Pointed end

F-ActinG-Actin

Profilinmaintains treadmilling

Thymosin:

sequesters Actin as monomer

Profilin (when activated)

Binds to actinpromotes binding to +end,

change in Conformation.

Profilin competes with Thymosin

Cytochalasin

LatruculinA


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Capping proteins:

Stabilize the end

Arp2/3 complex:

Branches and nucleates

Cofilin:Binds to ADP actin and

Destablizes actin->

Induces twisting

Easier dissociation

WASP:

Formins:

Control actin assembly

Is actin really branched ?

Urban et al 2010 NCB


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MVID

What makes the 'dilute' mouse have a dilute coat color


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Nature 1997

What makes the 'dilute' mouse dilute (in coat color)?

ActinActin--bindingbinding

motormotor -- 'head''head'

Lever ArmLever Arm

GlobularGlobular

tailtail

domaindomain

YeastsYeasts

MyosinMyosin--VVheavy chains:heavy chains:

Myo2pMyo2p (essential)(essential)

& Myo4p& Myo4p (not essential)(not essential)

CoiledCoiled--coilcoil


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How does Myosin walk ?

Monty Python Ministry of silly walks

36nm

How is Myosin moving ?

Always towards (+)


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Summary

Actin assembly

Factors: Profilin, Capping proteins

Actin nucleation and branching

Arp2/3 complex

Myosin movement on actin

Toward (+) end

Pi release triggers power-stroke

ATP binding detaches Myosin from actin,

ATP hydrolysis (ADP+Pi) recocks the lever into pre-

stroke state


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Interphase Metaphase

Ciliated cell

Nerve Cell

Proliferating cell

Interphase

Mitosis

Microtubule-based motility Tubulin, a GTPasedimer


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GTP cap model for dynamic instability

From Desai& Mitchison. 1997. Annu. Rev. Cell Dev. Biol. 13:83-117.

GTP MTs are stable

GDP MTs are unstable

Why is there a GTP cap?GTP hydrolysis is stimulated by incoming subunit

Taxolcolchicine


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+TIPs regulate MT dynamics

XMAP215-A microtubule Polymerase

XMAP215 is a MT-associated protein from Xenopus

similar proteins exist in virtually all eukaryotes

Addition ofXMAP215 to in vitro MT assembly assays

1) increases MT polymerization rate at plus-ends

2) has little effect on catastrophe frequency

=> increases MT length = MT stabilizing protein

XMAP215 tracks with dynamic MT ends

Brouhard et al. 2008. Cell 132:79-88

Kymogram


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Model for tubulin polymerization by XMAP215

Brouhard et al. 2008. Cell 132:79-88

MTOC

Microtubule Organizing Center

Centrosome

Theodor BoveriNamed the centrosome in 1888

3D reconstruction of the MTOC

2 Centrioles

Centriole Matrix

KTubulin


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Nucleation of MTs by KTuRC

+ KTuRC

- KTuRC

Model for nucleation of MT growth by K-TuRC

+

-


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Biophysical studies show that kinesin takes 8nm steps -

stepping from one F-subunit to the next -

It is highly processive. How does it move?

Models forhow kinesin moves:

Symmetrichand-over-hand

Aysmmetric hand-over-hand

Inch-worm

Yildiz et al. Science 303, 676-678 (2004) Yildiz et al. Science 303, 676-678 (2004)

E215


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Yildiz et al. Science 303,676-678 (2004)

In this trace, they used an E215C homodimer with

substoichiometric labeling of the cysteine with Cy3 fluorophore

so that only one cysteine was labeled.

Yildiz et al. Science 303, 676-678 (2004)

Since the step size of the whole kinesin (two heads) is 8nm,

but the step size of ONE head is 16nm, the hand-over-hand

model must be correct.

EE EEFF FF

+-

Leading head


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Walking Kinesin

From ron vales lab (UCSF)


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Kinesin binds

Myosin V

KIF13A Binds

AP-1 adaptin

KIF17 binds

LIN10, a PDZ-

containingprotein for

basolateral

sorting; similar

interaction for

delivery of

NMDA receptor

to dendrites

KIF1C binds

14-3-3 proteins

Rab6 binds

Rabkinesin-

6For Golgi to

ER traffick

MT-binding

AAA Repeats


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Summary III Microtubule assembly

EFTubulindimer

Factors: Tip trackers /

Microtubule nucleation

MTOC, K-Tubulin

Kinesin moves towards + end of Microtubules

ATP binding (leading head) strengthensF-Tub interaction

Results in conformational change and step

Lagging head (ADP+Pi) -> leading head

Dynein moves towards minus of Microtubules

Maybe shuffling

Uses a large cargo adaptor complex Dynactin

motility mcbo lecture

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