Microtubules

1
Introduction
Microtubules Microfilaments Intermediate
filaments

Cytoskeleton

Kinesin Dynein Myosin
Motor proteins
2
Introduction
The sub-cellular biological world is full of
phenomena that challenge physical intuition
single-molecule machines, self-assembling
architectures and spontaneous information
processing. These phenomena derive from the
physical character of biological macromolecules,
which have passed through the evolutionary
design process and acquired the character of a
technology. Deborah Fygenson

3
Scenario 1 Mitosis Microtubules
capture kinetochores
Scenario 1

4
Scenario 1

5
Scenario 1
Kinetochore (Kc) is a docking site for
microtubules located at the centromere, one per
sister chromatid Centromere is heterochromatic
region of DNA Simplest model would be that mt
ends attach to Kc Favoured theory side
attachment via a motor protein, leaving the end
free to grow or shrink

6
Scenario 1

7
Scenario 1
spindle checkpoint If just one kinetochore is
unattached to spindle, progress to anaphase is
halted. Cell may just arrest at M, or may
apoptose Protection mechanism against aneuploidy?

8
Scenario 1
Compare size of a single typical protein
molecule e.g 5nm with distance from spindle pole
to chromosomes e.g. 50mm Distance to be explored
in random search Is 10,000 (104) x
greater Fly-fishing distance-cast analogy

9
Scenario 1
Colchicine, poison from autumn crocus (Colchicum
autumnale) blocks mitosis In presence of
colchicine, chromosomes condense and nuclear
membrane dissolves, but no spindle
forms (Colchicine known as a spindle poison) 3H
-labelled colchicine identified a
colchicine-binding protein, tubulin, which was
found to be building block of microtubules

10
Scenario 1

11
Scenario 1
Sub-unit for assembly of microtubules is a
heterodimer two homologous proteins, a- and
b-tubulin bound together non-covalently Each
monomer binds a guanine nucleotide a-tubulin
GTP, unimportant. b-tubulin binds another GTP
and can hydrolyse it to GDP, i.e. has GTPase
activity GDP can switch back to GTP by exchange
with free GTP in solution

12
Scenario 1
Bacterial protein FtsZ Fts Filamentous
temperature sensitive FtsZ is a bacterial
protein required for septation (completion of
cell division) Some sequence similarity and
identical fold to eukaryotic tubulins. May be
ancestral to a,b and g tubulins and small
G-proteins such as p21-ras

13
Scenario 1

14
Scenario 1
Arrangement of tubulin in microtubules Heterodime
rs in a shallow helix, form the wall of a
cylinder. Overall diameter 25 nm. Wall is 5 nm
thick. Cylindrical structure for
rigidity. Heterodimers form straight lines along
the cylinder axis. protofilaments Protofilame
nts have alternating a and b and all heterodimers
point same way 13 protofilaments form the
cylinder

15
Scenario 1

16
Scenario 1

17
Scenario 1
Isolation of assembly-competent tubulin from
brain. Homogenise brain in buffer containing
GTP Incubate at 370 to assemble microtubules On
centrifugation, microtubules are in pellet,
with Much other insoluble material Resuspend
pellet, cool to 0o, microtubules melt,
releasing tubulin as a soluble protein On
centrifugation, remaining insoluble material is
removed. Purified tubulin now in
supernatant. Recycle - go back to assembly step
(370 and GTP)

18
Scenario 1

• Self-assembling protein structures
• The instructions for precise way in which
  protomers fit together are encoded in the primary
  sequence
• The sequence generates docking sites
• e.g. hydrophobic patches, opposite charges,
• H bond donors/acceptors on the surface of the
  protomer
• Lego analogy!
• Provide the right solution environment (esp small
  ion concentrations) and structure assembles
• No template or scaffold required (or small child)

19
Scenario 1

20
Scenario 1
Dynamic instability Microtubules in equilibrium
with GTP tubulin Found to be in 2 different
states Most are growing slowly, most growth at
one end. (Known as plus end) A subset are
shrinking rapidly. Overall amount of tubules
constant GTP is required for assembly but not
its hydrolysis Since GDP-NP forms stable tubules

More follows..
21
Scenario 1
Dynamic instability - continued Random switching
between states Growth to shrinking
catastrophe Shrinking to growth rescue These
phenomena also observed in cells.

22
Scenario 1
Text follows

23
Scenario 1

• GTP cap hypothesis of D.I.
• Heterodimers with GTP on b-tubulin bind to
  plus end
• (with GDP, different conformation, dont bind)
• After addition GTP is hydrolysed to GDP
• Is a-tubulin a GAP for b-tubulin
  (GTPase-Activating Protein)?
• GDP tubulin is stable within lattice, but not at
  end
• Late arrival of GTP tubulin (random
  fluctuations) allows end to become GDP and start
  catastrophe

24
Scenario 1

• Why might DI be needed?
• To defeat the law of mass action!
• For a simple protomer/polymer equilibrium
• Kontub koffmt i.e.
• tubulin/mt koff/kon
• On rate Kon is limited by diffusion
• So you can either have lots of assembled mt OR
  fast dissociation, but not both.

25
Scenario 1

26
Scenario 1

• MTOC
• Microtubule Organizing Centre
• Centrosome origin of cytoplasmic and
  interphase microtubules
• In fungi, mitosis takes place within intact
  nuclear envelop, MTOC is inserted in nuclear
  membrane, and known as Spindle Pole Body (SPB)
• Microtubules are nucleated from a starting
• ring of g-tubulin molecules, in complex with
• specific proteins