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Today:

Microtubule structurePrinciples of microtubule dynamics

Microtubule-based motorsMicrotubules in cell organization and organelle movement

ECB Chap. 17

Last Time:

Actin structures in cellsDynamics of purified actin in vitro

Actin polymerization in vivo

Actin driving cell movement

Myosin motors, Actin-regulating signals

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What do MTs do?

Mitosis

Organelletransport

Beating of ciliaand flagella

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interphase mitosis

Microtubules adopt different organizationsin different cell cycle stages

microtubules

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Microtubules are dynamic

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Properties of microtubules: the tubulin heterodimer

From Lodish, Molecular Cell Biology 6e

Subunit= tubulin= stable heterodimer of !-tubulin and "-tubulin

"-tubulin can exchange/hydrolyze GTP, !cannot

non-exchangeable !

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Properties of microtubules:Microtubule (MT) structure a hollow polymer of tubulin subunits

Figure 17-9 Essential Cell Biology ( Garland Science 2010)

13 linear protofilamentsMTs are polar-subunits uniformly oriented(alternating !/": "toward + end)

(+) end- fast growing(-) end- slower growing

Lateral interactions betweenprotofilaments form tube ofmicrotubule

Polymerization kinetics:Ccnucleation,elongation,steady state

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MT assembly is coupled to GTP hydrolysis:

GTP tubulin adds to (+) ends, GTP then hydrolyzed to GDP

if [tubulin] >> CcTon >> Toff " GTP cap

if [tubulin] < CcTon# "GTP cap lost

hydrolysis catches up

GDP latticeGTP cap stabilizes filaments

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curved protofilaments

GTP hydrolysis destabilizes microtubules:

From Lodish, Molecular Cell Biology 6e

straight protofilamentsGTP cap(+) end stable

"Assembly

catastrophe rescue

GTP cap lost(+) end unstable

"Disassembly

(GDP subunits released)

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Microtubule dynamic instability at steady state:

For a single microtubule:

From Lodish, Molecular Cell Biology 6e

if [tubulin] = Ccsome MTs grow, some shrink

single MT can alternate between growing and shrinking states

~

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The structural basis of dynamic instability

http://cryoem.berkeley.edu/moviesnogales_400x300_narrated.mov

MCB ProfessorEva Nogales

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Microtubules are nucleated from centrosomes:

centrosome-

nucleated MTs

add MT depolym drug wash out drug

MTs grow overtime

To purify tubulin:Purified tubulindoes not form MTsin vitro

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Microtubule organization in mammalian cells:

K

entMcDonald

From Lodish, Molecular Cell Biology 6e

MTs nucleate at centrosome

(-) ends capped by #-Tubulin ring complex:nucleator of MTs

(+) ends toward cell periphery

Where

nucleationoccurs

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http://www.youtube.com/watch?v=PCI_GUHJJaY&NR=1

GFP-EB1 +-end tracking protein: shows growing MTs

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Panel 16-3 Molecular Biology of the Cell ( Garland Science 2008)

Microtubule function in cells: controlled by binding proteins

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Figure 17-16a Essential Cell Biology ( Garland Science 2010)

Microtubule motor proteins:mechanochemical enzymes that harness ATP hydrolysis to drive

movement along microtubules

Many KinesinsMost (+) end directed

e.g. kinesin 1

Some (-) end directede.g. kinesin-14

Some depolymerize MTse.g. kinesin-13

Dyneinaxonemal-cilia, flagella

cytoplasmic"moves cargo #(-) enddirected

cargo

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http://www.youtube.com/watch?v=lLxlBB9ZBj4

Kinesin mechanism:Use energy of ATP hydrolysis to walk along MT

Kinesin 1 motility assay

Kinesin attached to slide; purified labeled MTs + ATP

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Microtubule motors in cell organization: Kinesins andDyneins function in organelle positioning

From Lodish, Molecular Cell Biology 6e

Dyneinmovescargo towardcentrosome

Kinesinmoves cargo

to periphery

(recall,generally -end-directed)

(recall,generally +end-directed)

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Microtubule motors in cell organization:kinesins and dyneins function in organelle dynamics

http://www.youtube.com/watch?v=9iXoXzgmEXw http://www.youtube.com/watch?v=_w_nN2_-SWM

Endoplasmic reticulum(green, MTs are red)

Exocytic vesicles for secretion(green, MTs are red)

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Melanosome positioning: a fun example of the role of MTmotors

From Lodish, Molecular Cell Biology 6e

Kinesin Dynein

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Melanosome positioning:a fun example of the role of MT motors

High cAMPLow cAMP

From Lodish, Molecular Cell Biology 6e

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Actin and Microtubule drugsused to evaluate their roles

For Actin- phalloidin- binds to F-actin, stabilizes latrunculin, cytochalasin- depolymerize

For MTs- taxol- binds MTs + stabilizes them nocodazole, colchicine- depolymerize