Cell Division Jon Lane L1-2

Question 1

What determines time of cell cycle?

Answer 1

Varies b/w organism and developmental stage

Question 2

What are the 3 levels of control to regulate CDK/cyclin activity?

Answer 2

Inhibitory/stimulatory phosphorylations on the CDK
CDK inhibitors
Synthesis/destruction of the cyclin subunit

Question 3

How is CDK1 fully activated?

Answer 3

Cyclin B (partially)
CAK (fully)-phosphorylation on T loop changes the conf and allows the active site to be perfectly available for binding substrates.

Question 4

Wee1

Answer 4

phosphorylates CDK1
Inhibitory kinase
Inactivates CDK1/cyclin B

Question 5

What turns on cdc25?

Answer 5

PIK
Polo-like kinase
Phosphorylates cdc25 which activates this phosphatase
CDK1/cyclin B active complex also phosphorylates cdc25
POSITIVE FEEDBACK

Question 6

How do cyclin kinase inhibitors work?

Answer 6

A vareity of types exist with different functions-some prevent cyclins from binding,
others lock the complex into place and block access to the active site

eg P27 cyclin2CDKA (S phase)
Inhibitors ensure cell division only occurs when it is required

Question 7

What is cyclin ubiquitination controlled by?

Answer 7

APC complex

anaphase promoting complex

Question 8

How many subunits does APC contain?

Answer 8

15 subunits form 3 functional domains
It is an E3ligase complex that targets multiple cell cycle regulatory proteins
It has 2 regulatory subunits

Question 9

What are the two regulatory subunits of the APC complex?

Answer 9

cdc20
cdh1
These function at different cell stages

Another important E3 is the SCF complex that targets specific phosphorylated substrates for degredaion

Question 10

How does APC known when it needs to be switched on?

Answer 10

Kinetichore proteins are responsible for this

Question 11

Astral MTs

Answer 11

Don’t contact the chromosomes yet anchor on to the cortex of the cell and can use pulling forces to secure the position of the spindle and also allow the spindle to rotate in 3D space for the correct plane of division for cytokinesis

Question 12

Spindle

What type of MTs are motors located at?

Answer 12

Overlap MTs

can make contacts with ch. arms via motor proteins

Question 13

Kinetochore

Answer 13

Contain majority of proteins which are involved in signalling and structure and function of the mitotic spindle
Non coding region yet attracts proteins of certain types
MT + ends are rapidly growing and shrinking structures and become stabilised when they attach to the kinetochore-overtime more MTs attach to the kinetochore
MTs terminate at the kinetochore
Contain SIGNALLING FACTORS that couple ch. spindle alignment with mitotic exit-factors signal that kinetochores are attached by MTs - can detect tension across these 2 kinetochores

Question 14

Kinesin and kinesin related proteins

Answer 14

N terminal motor=PLUS END directed
C terminal motor=MINUS END
Central motor = MT destabiliser (PACMAN) destabilise + ends - catastrophe

Question 15

In mitosis what do motors carry?

Answer 15

protein cargoes including chromosomes, signalling factors and also MTs
Motors control the formation of the spindle, the segregation of chromosomes and the timing/position of cytokinesis
Also regulate MT dynamics by stabilising or destabilising MT plus ends.

Question 16

Kinesin 5 (BimC)

Answer 16

Homotetrameric
PLUS end directed
Slides ANTIPARALLEL MTs
Prophase and anaphase
Motor domains at both ends of the molecules so can bind to MTs in an antiparallel fashion or parallel fashion and slide them across one another

Question 17

Kinesin 6 (Mk1p1 types)

Answer 17

N terminal motor PLUS end directed
Various splice variants
Cytokinesis
Interacts with cell cortex at the cleavage furrow

Question 18

Kinesin 7 (CENP-E)

Answer 18

Kinetochore associated motor-supports bipolar attachment of MTs to kinetochores
Involved in chromatid movement towards poles during ANAPHASE

Couples the capture of MTs at the kinetochore to spindle assembly checkpoint

Question 19

Kinesin 10 (Chromokinesin)

Answer 19

Binds CHROMATID ARMS and pushes chromatids towards the spindle equator (polar ejection force)
Binds to OVERLAPPING MTs

Question 20

Kinesin 13 (MCAK type)

Answer 20

INTERNAL motor domain
Couples ATP hydrolysis to MT depolymerisation in a “Pac-man” mechanism
Required for anaphase
Accelerates MT depolymerisation about 100fold

Question 21

Mitotic spindle

Search and capture spindle assembly

Answer 21

Doesn’t necessarily need kinetochores or centrosomes to form
Chromatin mediated (as long as the cytoplasm is in a mitotic state, got active CDK1/cyclinB then overtime the MTs will self sort till you get a spindle structure)
There are factors present around mitotic chromosomes that attract MTs and promote their polymerisation

Question 22

Minus end directed motors

Answer 22

focus the spindle poles (dyneins)

Question 23

Motor activity in prophase

Nuclear env breaks down and centrosomes move to opposite ends of the poles

Answer 23

Anti-parallel MT sliding drives centrosome separation
Coordinated by homotetrameric motor BimC/Kinesin5 family (N terminal kinesins PLUS end)

Binding of BimC to cytoplasmic dynein/dynactin is important at the centrosome (keeps MTs bound and firmly attached at spindle poles)

Question 24

Motor activity in prometaphase

Sister chromatids are only attached to one spindle pole at this stage

Answer 24

CENP-E/Kinesin7 (a kinetochore associated motor) supports bipolar attachment of MTs to kinetochores -involved in chromatid movement towards the poles during anaphase (MINUS END)

Also important for BubR1 in the spindle assembly checkpoint

Chromosome arms are also attached to MTs via “chromokinesins” (kinesin10)/Kid. This pushes the chromosome arms away from the poles-polar ejection force

Tendancy for kinetochore MT to shorten
Chs pulled in two directions

Question 25

Motor activity in metaphase

MTs from both spindle poles are bound to the kinetochores on both sides

Answer 25

Poleward forces/polar ejection forces + chromatid cohesion balanced-no net movement

Correct ch. alignment receives the spindle assembly check point

activating APC/cdc20 ANAPHASE to degrade CyclinB/CDK1…. securin….and kid removing the polar ejection force facilitating anaphase onset

Question 26

How are cohesions between new and old chromosomes broken?

Sister chromatids become attached to one another during S-phase by a COHESION complex
SECURIN keeps SEPERASE inactive

Answer 26

Indirect function of M-CDK is to trigger dissolution of the molecular “glue” that holds sister chromotids together in the metaphase-to-anaphase transition

At spindle assembly check point APC/cdc20 is activated and ubiquitinates securin which is degraded by the proteasome to release active SEPARASE which then destroys the cohesions

APC/cdc20 also degrades chromokinesin by ubiquitination which removes the polar-ejection force in spindle

Question 27

What does Mad2 do?

Answer 27

In prophase Mad2 protein occupies each and every kinetochore
During pro metaphase kinetochores attached to the spindle lose Mad2
Unattached kinetochores retain Mad2 and continue to generate “wait anaphase” signals

Mad2 has a safety belt which hooks over the protein and locks a ligand in place when either Mad1 or cdc20 are present

Cytoplasmic dynein carry away this protein

Question 28

What is the Mad2 “safety belt” hypothesis?

potent and diffusibile cascade of Mad2 priming and cdc20 binding

keeps APC/cdc20 inactive

Answer 28

When Mad2 is bound to its ligand Mad1 on the kinetochore…..this primes soluble Mad2 by loosening its safety belt

Primed Mad2 can then bind to cdc20 at the kinetochore and this complex dissociates from the kinetochore and forms a template for the priming of soluble, cytoplasmic Mad2

Question 29

What does BubR1 detect?

Answer 29

tension of MTs at kinetochores via CENP-E (kinesin7) and down regulation of cdc20 activation

If both MTs are attached CENP-E arm region becomes extended and this is transduced via BubR1 and the block on cdc20 activation is removed

BubR1 binds CENP-E motor protein which is bound to the kinetochore

BubR1 itself inhibits cdc20
BubR1-Bub3-Bub1 also inhibits cdc20

Question 30

Motor activity Anaphase A

Chromatids move to the spindle poles as cohesions are degraded

Answer 30

Pacman motors such as MCAK (kinesin 13) reduce the length of kinetochore microtubules by nibbling away the plus ends (chewing)

Cytoplasmic dynein at the kinetochore may translocate towards the spindle poles due to its MINUS end directed activity (pulling)

Continued MT flux

Question 31

Motor activity Anaphase B

Movement of chromatids continue and the spindle itself becomes longer and the centrosomes are pulled to the cortex of the cell

Answer 31

Cortrically-anchored cytoplasmic dynein pulls astral MTs thereby pulling the centrosomes apart
Kinesin 5 class motors cross-link MTs in the midzone sliding them apart pushing apart the spindle poles
CENP-E also functions here...

Question 32

Cytokinesis

(in telophase lots of overlapping MTs are in the middle and form a CENTRAL spindle important for the recruitment of signalling complexes)

Chromatids have reached spindle poles, start to decidedness and nuclear envelope reforms

Answer 32

Contractile ring pulls membrane inwards at the site of the cleavage furrow at the CENTRAL spindle
As cytokinesis proceeds an electron dense structure forms-the midbody where all the signalling complexes are present
Cytokinesis terminates in a process called cellular abscission
Often only one cell inherits the midbody and the other one doesn’t-creates developmental memory for which cell is the mother and which is the daughter

Question 33

Motor activity during cytokinesis

Answer 33

Motors of kinesin6-MKLP
Organise MIDZONE MTs and regulate interactions with the cortex

Have MT binding affinity but main role appears to be in localising signalling molecules to the cleavage furrow
(MKLP1 forms the centralspindlin complex which regulates RhoA GTPase-controls actin/myosin)
RhoA: ECT2(Rho GEF)/RacGAP50C (Rho Gap)

Plk1 protein returns and collects the central spindle and phosphorylates targets right in the central domain of the spindle.