CytoskeletPrednáška 6

Mikrotubuly a mitóza

Polarity of tubulin polymerization

Nuclei

Tubulin > CC

Tubulin < CC

Preferential addition of tubulin ar (+) ends

Preferential loss of tubulin ar (+) ends

Kinesin – another molecular motor

Globular head

Heavy chain Coiled-coil α helix

ATP hydrolysis;Binding to MT

Binding to transported vesicle

10 nm Lightchain

Kinesin versus myosin cycle

Dynein [(-)-end directed motor] and dynactin complexHeavy chains

Intermediate chains

Light chains

Mitosis in an animal cell

A course of mitosis in a typical animal cell

Animation mitosis

A typical time course for mitosis and cytokinesis (M phase) in a mammalian cell

time (minutes)

CYTOKINESIS

0 20 40 60 80

The cytoskeleton in M phase

Three sets of MTs in the mitotic apparatus

(polar)

Three sets of MTs in the mitotic apparatus

A model for formation of bipolar mitotic spindle by the selective stabilization of microtubules

Astral MT

Polar MT

centrosome

Mitotic apparatus in Saccharomyces cerevisiae

Chromosome

Polar MT

Kinetochore MT

Nucleus

Spindle pole body (SPB)

The spindle pole body in yeast

A chromosome in metaphase

Kinetochore microtubules

Centromeric attachment of MTsChromatids

Kinetochore

Outer plate

Inner plate

Centromeric chromatin

Fibrous coronaMicrotubule

The kinetochoreDAPI kinetochore

anaphase chromatidkinetochore

MTs embedded inkinetochore

direction of chromatidmovement

1 µm

Kinetochores in cultured cells

Scleroderma patients produce anti-kinetochore antibodies

10 µm

Three problems of prometaphase:

1. Spindle pole formation2. Chromosome capture

3. Chromosome line-up at the equator

Centriole replication

A newly replicated pair of centrioles

Fluorescence-recovery after photobleaching (FRAP)

McNally et al. (2000). Science 287: 1262-1265.

FRAP can be used for understanding dynamics of protein complexes

Microtubules in an M-phase cell are much more dynamic than the microtubules at interphase

Dynamic instability and capture of chromosomes

end capture

kinetochoreGrowing MTs

Shrinking MTs

SPB

Dynamic instability and capture of chromosomes

Side capture

Chromosome slidesto the (+) end

The dynamic behavior of microtubules in the metaphase spindle

The dynamic behavior of microtubules in the metaphase spindle

Blue-DNA; Red-rhodamine-tubulin; Yellow-caged fluorescein-tubulin

The dynamic behavior of microtubules in the metaphase spindle

CENP-E

Dynein

(+) (-)

Demonstration of the astral exclusion (pushing) force

Demonstration of the astral exclusion (pushing) force

Two possibilities for how chromosomes line up at the metaphase plate

PUSH Astral exclusion force decreases with distance from pole

Pulling force proportional to length of kinetochore MTsPULL

ANIMATION:MT behavior during prometaphase

The two processes that separate sister chromatids at anaphase

Separation of chromatids at anaphase A

Movement of chromosomes during anaphase A

Movement of chromosomes during anaphase A is mediated by shortening of MTs at (+) ends

Involvement of kinetochores in chromosome movement during anaphase A

The two processes that separate sister chromatids at anaphase

How microtubule motor proteins act in anaphase B

ONWARD PUSH ON SPINDLE POLES

ONWARD PULL ON SPINDLE POLES

cell cortex

Sliding of overlap microtubules at anaphase

Anaphase checkpoint – important tool ensuring a “healthy” cell cycle

Mitosis in animal cells video

How do two blind people equally split 46 pairs of socks?

Cell 109: 9-12 (2002)

Cell ensures bipolar attachment of each chromosome

Monopolar attachment Unipolar attachment Bipolar attachment

Kinetochores occupiedTension not established

Kinetochores unoccupiedTension not established

Kinetochores occupied Tension established

Incorrect MT capture Correct MT capture

Cohesins and anaphase

Cohesin dimer Sister chromatid cohesion by cohesin

Gruber et al. (2006). Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge. Cell 127: 523-537.SMC: Structural Maintenance of Chromosomes

Cohesins and anaphase

∅30-40 nm

Anaphase-promoting complex (APC)

cohesin

separasePds1 (securin; inhibitor of separase)

Ub

Ub ubiquitin

APC

Mad2

Mad2

Nature 434: 575-576 (2005); Cell 120: 739-746 (2005)Sun, Y.,Kucej Martin et al. (2009). Separase Is Recruited to Mitotic Chromosomes to Dissolve Sister Chromatid Cohesion in a DNA-Dependent Manner. Cell 137, 123–132.

Meiosis: even more challenging type of cell divisionMitosis

Meiosis

Metaphase

Anaphase

Metaphase I

Metaphase II

Anaphase I

Anaphase II

Journal of Cell Science 117, 4017-4023 (2004)

Juraj Gregáň :

http://www.mfpl.ac.at/index.php?cid=441

Gregan, J., et al (2007). Current Biology, 17(14): 1190-1201.

Petronczki, M., Matos, J., Mori, S., Gregan, J. et al. (2006). Cell, 126(6): 1049-64.

Riedel, C.G., Katis, V.L., Katou, Y., Mori, S., Itoh, T., Helmhart, W., Galova, M., Petronczki, M., Gregan, J. et al. (2006). Nature, 441(7089): 53-61.

Gregan, J. et al. (2005). Current Biology, 15(18): 1663-69.

...

Meiosis: even more challenging type of cell division

Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation.Cell 132, 925-928.

Novel role(s) of cohesinS. cerevisiae: SMC1 & SMC3 are required to prevent spreading of heterochromatin from silenced HMR locusH. sapiens: Mutations in Scc and SMC cause Cornelia de Lange Syndrome (autosomal dominant; 1-10,000-1:30,000) (some cases (SMC1A exhibit X-linked inheritance)

NO DEFECTS IN SISTER CHROMATID COHESION

Gullerová Monika & Proudfoot, N.J. (2008). Cohesin Complex Promotes Transcriptional Termination between Convergent Genes in S. pombe. Cell 132, 983–995.

Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation. Cell 132, 925-928.

Novel role(s) of cohesin

Nonrandom distribution of cohesin throughout the chromosomes(acummulation of cohesin between convergent genes in G2 phase)

Read-through transcription in G1 leads to RNAi-dependent formation of heterochromatin and recruitment of cohesin

So similar…

… yet so different

Sci. Amer. May 2009 issue

Santino(Furuwik ZOO)

Osvald. M. (2009). Spontaneous planning for future stone throwing by a male chimpanzee Curr. Biol. 19: R190

How to identify loci in human genome, which are under positive selection?

Examples of human loci under apparent positive selectionwt

aspm

har1

HAR1, brain, possible affects size of cerebral cortex

FOXP2, transcription factor, speech production

AMY1, digestion of starch, exploration of novel food (?)ASPM, control of brain size (mutated in microcephalic patients)

LCT, digestion of lactose provided by domesticated animals

HAR2, development of wrist and thumb

Spindle rotation during development

Neuroeipithelial (NE) cells: primary neural progenitors

Lineage relationships between neuroepithelial cells (NE), radial-glial cells (RG), basal

progenitors (BP) and neurons (N).

Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17: 648-657

NE RG

BP

N

Types of division:

Symmetric proliferative: NE à NE + NENEUROGENIC:Asymmetric self-renewing: NE à NE + neuronAsymmetric bi-differentiative: NE à radial glial cell + neuron

Ventricular germinal zone: A layer of intensive division of neuroepithelial cells

Purves et al. (2001). Neuroscience, 2nd Edition, Sinauer Associates, Inc.

neural tube

Ventricular germinal zone

Intermediate zoneMarginal zone

Symmetric versus asymmetric division of neuroepithelial and radial-glial cells with vertical cleavage plane orientation

Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17: 648-657Fish a kol. (2007). Proc. Natl. Acad. Sci. USA 103: 10438-10443

apical p

lasm

a mem

bran

e

Aspm

symmetric asymmetric