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Microtubules, from tubulin to transport

Dynamic instability
Centrosomes
Motors

Today:
Cell division (mitosis)
-Chromosome condensation
-Mitotic spindle assembly (attachment to bipolar spindle)
-Chromosome segregation to daughter cells
-Cytokinesis

ECB, Chap. 18
Overview of mitosis and cell cycle
Synchronized mitoses in the syncytial Drosophila embryo

https://vimeo.com/202222028
Histone-GFP puts emphasis on chromosomes:
condensation, separation to daughters, decondensation
 Mitosis highlight 1:Chromosome condensation
Figure 4-72 Molecular Biology of the Cell (© Garland Science 2008)

Interphase DNA is diffuse in the nucleus, in

extended chromatin

How do we get this nicely packaged

for mitosis?
Mitotic chromosome organization is organized
by structurally related molecules: Condensins and Cohesins

Cohesins link sister

chromatids together

The Condensin complex drives folding

of chromatin

Cohesin rings keep sister chromatids

tethered until anaphase, when separation
is initiated by the destruction of Cohesin.

Essential Cell Biology, Figure 18-18 See also http://www.wehi.edu.au/wehi-tv/molecular-visualisations-dna

Mitotic events:

chromosomes condense

centrosomes duplicate
bipolar spindle forms
Nuclear envelope breaks
down

Essential Cell Biology, Figure 18-21

 Mitosis: emphasis on MTs

Fixed human cell

MTs, DNA, KTs

Live sand dollar embryo

fluorescent tubulin
 Mitosis highlight 2: construction of the mitotic spindle

MTs are 10x more dynamic in mitosis than interphase

From Lodish, Molecular Cell Biology 6e

Formation of a bipolar mitotic spindle:

MT dynamic instability

stabilization of interpolar MTs

Essential Cell Biology, Figure 18-22

Poll: Which type of
microtubule motor, if
localized in the overlap
zone of interpolar
microtubules, would
push the spindle poles
apart:
A. Kinesin (+ end
directed)
B. Dynein (- end
directed)
Three classes of MTs
in the mitotic spindle:

Essential Cell Biology, Figure 18-24

Mitosis: emphasis on kinetochores (KTs)

Cultured rodent cell,

red fluorescent tubulin,
chromosome attachment sites (kinetochores)
and spindle poles labelled in green
Chromosomes attach to MTs at the kinetochore:

Essential Cell Biology, Figure 18-23

 Metaphase MT dynamics:
Even when spindle appears stable:
1) MTs grow & shrink; chromosomes remain attached and oscillate
2) Poleward MT flux (treadmilling, b/c loss from – end at centrosome)
These maintain tension on kinetochores

How do we know?
‘photoconvert’ GFPvariant-Tubulin in central spindle:
UV illumination converts to red fluorescence
Mitosis highlight 3: anaphase separation of chromosomes
Once spindle forms, problem is to move the chromosomes to different poles
https://www.youtube.com/watch?v=sBcbXzamai4

Fly embryo, Histone-GFP

Initiating anaphase: how does the cell know that it is ready to divide?
how does it separate sister chromatids?

If sister kinetochores not attached to opposite poles: delay anaphase

If sister kinetochores are attached to opposite poles: allow anaphase

APC ubiquitylates Securin, which

triggers Securin degradation and
releases Separase

Separase protease
cleaves Cohesin

sister chromatids separate

Essential Cell Biology, Figure 18-28

 MT dynamics during anaphase
1) Shortening of kinetochore MTs 2) Elongation of interpolar MTs
pulls chromosomes apart separates poles

2, 3) Molecular motors
enable spindle movement
Anaphase chromosome movement mechanisms:
1A
1A) Kinetochore MTs depolymerize,
but kinetochore stays attached to
shrinking MT + end

1B 1B) Depolymerization at – end pole

chromosome reeled in by flux
(treadmilling)

3 2) Motors push poles apart

21
3) Dynein anchors + end of astral
MTs to plasma membrane

From Lodish, Molecular Cell Biology 6e

 actin and myosin return
Mitosis highlight 4: cytokinesis in the contractile ring

Mitosis is complete!
2 daughter cells with
identical genomes

Actin-GFP

Actin & Myosin

contraction
From Lodish, Molecular Cell Biology 6e
 Mitosis interpretation
Filmed by Will Luddington (former MCB Fellow)

https://www.youtube.com/watch?v=eFuCE22agyM