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
• 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
•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
•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
b
a
Microtubules are Polarized Polymers of Tubulin
• a/b heterodimers bind head-totail 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
Axomeme (Cilia and Flagella)
Cytoplasmic:
(10-16 pf)
13 pf is typical
Centriole or
Basal Body
Microtubule Organization in
Interphase and Mitosis
Centrosome
25 mm
5 mm
Polarized Microtubule Organization
in vivo
Centrosome
+
+
+
+
+
+ +
+
+
Interphase
Mitosis
Microtubules are Abundant in
Neurons:Brain is the best source
of tubulin
-
+
Microtubule Assembly Occurs By EndDependent 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:
• 2 mg/ml tubulin in 1 M PIPES, 1mMEGTA,
1mM MgCl2, 1mM GTP pH = 6.8 and 37C
Cc
(can “seed” MTs using
preformed cross-linked MT
fragments for example:
Microtubule Ends Exhibit
Dynamic Instability, Not Simple
Equilibrium Assembly
+ End
Microtubule Dynamic Instability
GROWTH PHASE
CATASTROPHE
GDP
RESCUE
SHORTENING PHASE
At an end:
b
b
dL/dt ~ [fg(kag(S-Ccg)) –fskds]
GTP-TUBULIN
GDP-TUBULIN
GTP
Microtubule Ends Change
Conformation Between Growth
and Shortening Phases
Recording Microtubule
Dynamics in Living Cells
Or, express
Tubulin-GFP
Digital-Imaging Fluorescence
Microsocpy
CCD
Hamamatsu Orca ER CCD
Camera
•
•
•
•
Low readout noise (~8 electrons)
High Quantum Efficiency
Broad spectral response
Fast readout: ~14MHz
Yokogawa Scanning Head
Nikon TE300 inverted microscope
Filter Wheel
Orca ER
CCD
Focus
Controller
80 mW Argon-Krypton
Laser Input (fiber optic)
Schematic of the CSU-10
Microtubule Polarity and Dynamics
Dynamic Instability
In vitro mitotic cell extract + centrosome
Living interphase animal cell; cell edge
Fluorescent Speckle Microscopy
MTs green; actin red (Salmon and Waterman)
tubulin Ring Complexes Nucleate Plus-End
Growth in Cells; Self-Assembly and Minus-End
Growth Is Rare
+
-
 

 


 

Tubulin Ring Complex
TuRC~12-14 tubulins,
Xgrips72,109,110,133,195
Zheng et al. 2000
Nat. Cell Biol. 2:358
Centrosome is A Microtubule
Organizing Center (MTOC)
MTOC’s control where
microtubules are formed
Centrosomes contain pericentrosomal 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 (MTOC)
Centrosomes contain:
Peri-centrosomal: TuRC
nucleation complexes
bound to pericentrin and
centrin fibrous material
plus many kinases
Centrioles within a pair of centrosomes before mitosis
Basal Bodies are Nucleation
Centers for Cilia and Flagella
Microtubule Associated Proteins (MAPs)
Control Microtubule Assembly and Many
Stabilizing MAPs Bind to Outer Surface of
Protofilaments
Neuronal MAPs Initially Most
Studied
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
MT Assembly Dynamics is Regulated in the Cell Cycle
(Cyclin B/Cdk1 kinase)
(Inactive)
(Active)
Cat.
Rescue
A Catastrophe Factor:
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 betacatenin
• 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.
Alexa-488-EB1
Bound to the
Growing Ends
(10 mm/min)
of
Microtubules
in Early
Prometaphase
Spindle in
Xenopus Egg
Extracts
Jennifer Tirnauer
Microtubule Drugs
Bind tubulin dimer and
blocks assembly:
Colchicine, nocodazole,
vinblastine, podophilotoxin,
vincristine
Binds dimer in microtubule
lattice and stabilizes
microtubules: Taxol