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Microtubules (MTs) . E. D. Salmon Biology tsalmon@email.unc.edu Reading: Lodish et al., Molecular Cell Biology; Alberts et al., Molecular Biology of the Cell. Some Microtubule Functions. Provide structure, acting as an internal skeleton

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microtubules mts

Microtubules (MTs)

E. D. Salmon

Biology

tsalmon@email.unc.edu

Reading: Lodish et al., Molecular Cell Biology; Alberts et al., Molecular Biology of the Cell

some microtubule functions
Some Microtubule Functions
  • Provide structure, acting as an internal skeleton
  • Act as a polarized tracks for microtubule motor (cytoplasmic kinesins and dyneins) driven movements within cells
  • With axonemal dynein produce motility of cilia and flagella
  • With microtubule motors segregate chromosomes in mitosis
  • Give cell polarity and produce polarized organization of organelles
  • Regulate activity of actin cytoskeleton in cell motility and cell division
the tubulin dimer
The Tubulin Dimer
  • Each tubulin is about 50 kD and structurally very similar to each other and to FtsZ in bacteria
  • Microtubules are composed of a and b tubulins

b

  • Each tubulin binds GTP: the a GTP is non-exchangeable and the dimer is very stable, Kd = 10-10; the b GTP is exchangable in the dimer
  • The intracellular tubulin concentration is about 20 mM; about half in microtubules and half as dimers in the cytosol

a

slide4

Microtubules are Polarized Polymers of Tubulin

+

  • a/b heterodimers bind head-to-tail along protofilaments
  • 13 protofilaments form a hollow tube-the microtubule: 25 nm OD, 14 nm ID
  • Microtubules are polar-they have a plus and a minus end
  • *1625 dimers/mm length
  • *A mammalian tissue cell (6 pL) has 16,500 mm total microtubule length; a frog egg (1 mL) has 2,500 m

-

other microtubule geometries
Other Microtubule Geometries

Axomeme (Cilia and Flagella)

Cytoplasmic:

(10-16 pf)

13 pf is typical

Centriole or

Basal Body

polarized microtubule organization in vivo
Polarized Microtubule Organization in vivo

Centrosome

+

+

+

+

+

+

+

+

+

Mitosis

Interphase

slide9

Microtubule Assembly Occurs By End-

Dependent Association-Dissociation Reactions

a

b

  • In vitro, microtubules that self-assemble from pure tubulin
  • can grow and shorten at both ends
  • The plus end grows faster and is more dynamic than
  • the minus end
microtubule self assembly from pure tubulin in vitro
Microtubule Self-Assembly From Pure Tubulin In Vitro:
  • 2 mg/ml tubulin in 1 M PIPES, 1mMEGTA, 1mM MgCl2, 1mM GTP pH = 6.8 and 37C

(can “seed” MTs using preformed cross-linked MT fragments for example:

Cc

[tub] in polymer

slide12

b

b

At an end: dL/dt ~ [fg(kag(S-Ccg)) –fskds]

hamamatsu orca er ccd camera
Hamamatsu Orca ER CCD Camera
  • Low readout noise (~8 electrons)
  • High Quantum Efficiency
  • Broad spectral response
  • Fast readout: ~14MHz
slide17

Yokogawa Scanning Head

Nikon TE300 inverted microscope

Filter Wheel

Orca ER CCD

Focus Controller

80 mW Argon-Krypton Laser Input (fiber optic)

microtubule polarity and dynamics dynamic instability
Microtubule Polarity and DynamicsDynamic Instability

In vitro mitotic cell extract + centrosome

Living interphase animal cell; cell edge

Fluorescent Speckle Microscopy

MTs green; actin red (Salmon and Waterman)

slide20

tubulin Ring Complexes Nucleate Plus-End

Growth in Cells; Self-Assembly and Minus-End

Growth Is Rare

+

gTubulin Ring Complex

g

g

gTuRC~12-14 gtubulins,

Xgrips72,109,110,133,195

g

-

g

g

g

g

g

g

g

g

Zheng et al. 2000

Nat. Cell Biol. 2:358

centrosome is a microtubule organizing center mtoc
Centrosome is A Microtubule Organizing Center (MTOC)

MTOC’s control where microtubules are formed

Centrosomes contain peri-centrosomal nucleation complexes surrounding pair of centrioles

Centrioles within centrosomes become basal bodies, which are nucleation centers for cilia and flagella

centrosome is a microtubule organizing center mtoc22
Centrosome is a Microtubule Organizing Center (MTOC)

Centrosomes contain:

Peri-centrosomal: gTuRC nucleation complexes bound to pericentrin and centrin fibrous material plus many kinases

slide25
Microtubule Associated Proteins (MAPs) Control Microtubule Assembly and Many Stabilizing MAPs Bind to Outer Surface of Protofilaments
catastrophe factors and rescue factors many concentrate at mt plus ends
Catastrophe Factors and Rescue Factors: Many Concentrate at MT Plus Ends
  • Catastrophe: Op18, Stathmin; End-binding protein
  • Kin I (e.g.XKCM1, hMCAK); Kip3
  • Rescue: Stabilizing Microtubule Associated Proteins (MAPs)
  • -XMAP215 (hTog, S.c. Stu2 ): antagonizes KinI
  • -Brain Maps (MAP2, Tau)
  • -Growing End Binding proteins: EB1,CLIP170
  • *Note: Activity of all these factors regulated in the cell cycle
  • by phosphorylation or during cell motility
  • *Note: In budding yeast, microtubule motors can effect plus
  • end dynamics: destabilize-Dyn1, Kar3, Kip3
  • stabilize-Kip2
slide28

MT Assembly Dynamics is Regulated in the Cell Cycle

(Cyclin B/Cdk1 kinase)

(Inactive)

(Active)

Cat.

Rescue

proteins which transiently bind growing ends
Proteins Which Transiently Bind Growing Ends
  • EB1: Links to APC which can bind Beta catenin at cortex; EB1 binds Ncd kinesin motor
  • Bim1 in yeast binds Kar 9 at cortex
  • Clip 170: Links to dynein/dynactin, CLASPS
  • Dynein/dynactin-links to cortex (binds beta-catenin
  • Ncd: minus kinesin
  • MCAK: KinI depolymerase
  • Review: Vaughan KT, 2004, Surfing, regulating and capturing: are all microtubule-tip-tracking proteins created equal? Trends Cell Biol. 2004 14:491-6.
slide31
Alexa-488-EB1 Bound to the Growing Ends(10 mm/min)of Microtubulesin Early PrometaphaseSpindle in Xenopus EggExtracts

Jennifer Tirnauer

microtubule drugs
Microtubule Drugs

Bind tubulin dimer and blocks assembly: Colchicine, nocodazole, vinblastine, podophilotoxin, vincristine

Binds dimer in microtubule

lattice and stabilizes

microtubules: Taxol