Mechanics of Mitosis

Kinesin 5

(+) end directed motor that cross links overalp MTs and controls the separation of the spindles via MT sliding. (Pushes MTs apart).

Kinesin 14

(-) end directed motor that cross links overlap MTs and controls spindle separation/length by MT sliding (Pulls opposing MTs together).

What are the three classes of MTs in the spindle?

1) Astral MTs 2) Kinetochore MTs 3) Overlap MTs

What are the three cell cycle checkpoints

1) Before S (start) - is the environment favorable? 2) Before M (G2/M) - is the DNA replicated, is the environment favorable? 3) Metaphase-to-Anaphase - Are all chromosomes attached and properly alligned

Discuss the attachment process of the chromosome to the microtubule

1) Chromosome bumpts into a MT 2) Laterally associates with the MT 3) MT grows outward (from the positive end) and the chromosome subsequently slides towards the spindle pole 4) Chromosome converts to an end-op unipolar attachment to the kinetochorewhere now the MT makes a stable connection 5) + end motor proteins and MT growth now push the MT to the center of the cell 6) Other free kinetochore captures a MT from the opposite spindle to convert from a unipolar to a bipolar attachment

Discuss what occurs during metaphse

1) Chromosomes align at the metaphase plate (spindle equator) 2) Bipolar spindle assembly is complete

Discuss the events of prophase

1) Chromosomes condense 2) Interphase MTs break down and interpolar-spindle MTs assemble 3) Centrosomes start to separate through the action of polo light kinases and activating proteins that induce the anti-parallel MTs coming from each of the centrosomes to slide against one another and push each other apart *NUCLEUS STILL INTACT

Discuss what occurs during cytokinesis

1) Contractile ring assembly (pinching off through myosin mediated activity on actin) 2) Delivery of new membrane to the cytokinetic furrow (adds the necessary membrane so that the cells can detach).

Discuss what occurs during telophase

1) Daughter chromosomes arrive at the poles 2) Nuclear envelope starts to reassemble 3) Contractile ring starts to assemble (its position is completely dependent on the interpolar MTs and where they overlap, they dictate localization of the rho-GTPase that will initiate contraction)

What two major factors effect assembly of the mitotic spindle?

1) MT dynamic instability (its ability to rearrange) 2) Opposing motor proteins and MTs

What two cytoskeletal elements play a role in cell division and what is their role?

1) Microtubules - mitotic spindle 2) Actin (and myosin) - contractile ring

Discuss the events of pro-metaphase

1) Nuclear envelope breaks down 2) Chromosomes attach to the spindle MTs and begin to move towards the center (via motor proteins and MT growth)

What three specific kinases (involved in the mitotic spindle) are activated by M-Cdk?

1) Polo like kinases (required for assembly of the bi-polar spindle via phosphorylation of proteins involved in separation of spindle poles) 2) Aurora A kinase (controls proteins invovlved in spindle assembly and stability) 3) Aurora B kinase (controls attachment of sister chromatids to the MT in the spindle --> transition to anaphase)

Discuss what occurs during anaphase

1) Sister chromatids separate suddenly (breakdown of cohesin protein by separase) and move synchronously towards the poles 2) Spindle poles move apart (mediated by motor proteins and MT dynamics)

Anaphase occurs when ______

APC is activated via binding with Cdc20 so that it can ubiquinate securin and release separase. Separase then degrades cohesins holding sister chromatids together and subsequently MTs can pull chromotids to the opposite poles.

What makes up the contractile ring?

Actin and myosin

What is a cellular requirement for segregation of the DNA?

Assembly of a bipolar mitotic spindle

Describe the process of cytokinesis in a very general way.

Assembly of actin/myosin contractile ring that contricts the furrow greatly. There is then delivery of new membrane to the furrow from the exocytosis of post-golgi and recycling vessicles (syntaxin 2) so that the daughter cells have sufficient membrane to fuse and close off from one another resulting in scission.

Kinetochore MTs?

Attach to the kinetochore at the centromere of chromosomes

In order to process to anaphase, what must occur?

Both spindle poles must be bound to a chromosome. The cell must sense the tension at the centromere.

Sensores of the cell cycle checkpoints which activate Cdk proteins.

Cyclins

Myosin phosphatase

Dephosphorylates myosin regulatory light chains thereby inhibitin its activity.

What is the use of FRAP in the context of studying the mitotic spindle?

Determined that MT instability is markedly increased during mitosis. Bleached a portion of the spindle and measured the time of recorvery (determines how long it takes the MT to reassemble). Found that time was 15 sec. vs. 5 min in interphase.

Discuss the formation of the bipolar spindle.

Driven by both MT growth and opposing motor proteins. Overlap MTs grow towards the center. Once they start to overlap, motor proteins bind and crosslink opposite MTs. These motors (kinesin 5) then start to walk towards the POSITIVE ends thereby pushing the spindles away from one another. This continues as the MTs continue to growth pushing the spindles further and further apart! During metaphase (as the chromosomes are lining up) the cell wants to maintain a constant distance between the poles. So the pushing balanced out by a MINUS directed motor (kinesin 14) which brings the spindles closer togther. Therefore a balance between the two ensures the spindles at just the right distance from one another.

What allows for the reformation of the nuclear envelope.

During this anaphase process, M-Cdk is inactivated causing dephosphorylation of the nuclear lamins. The nuclear membrane vessicles begin to assoicate with the chromosomes and as they do so, they begin to fuse and the nuclear lamina reassembles

Is the metaphase spindle dynamic or static?

Dynamic. In order to maintain the chromosomes on the metaphase plate there must be a balance of forces between motor protein movement towards the + end (kinesin 5) and MT depolymerization at the - end which pulls the chromosome back.

When do centrosomes move/separate?

Early mitosis (prophase/pro-metaphase)

FRAP

Fluorescent recovery after photobleaching. Label MTs with GFP-tubulin. Apply a laser to photobleach . Measure the time it takes to recover fluoresence.

Why is the centrosome duplicated?

For the formation of the bipolar mitotic spindle and to establish the plane of cell division.

Dyenin at the cortex

Holds astral MTs to maintain spindle position in the cell. Attaches to the PM and walks towards the (-) end thereby creating tension.

What are overlap MTs?

Inter polar MTs that are not involved with the MTs but instead extend from each pole towards the center and interacts with MTs radiating from the opposite poles.

Aurora B kinase

Kinase that is apart of the kinetochore. Acts to sense tension on the chromatids as MTs attach and tug them in opposite directions. Regulates transition to anaphase (want to ensure there IS appropriate attachment).

Anaphase occurs in two phases. What happens in the first phase.

Kinetochore MTs shorten (depolymerization at the - end) causing movement towards the pole.

What causes the breakdown of the nuclear envelope?

M-Cdk phosphorylates nuclear pore proteins and lamins A/B/C thereby releasing lamins A and C into the cytoplasm (structural framework of the NE) and causing vessiculation of the NE with lamin B bound.

In mitosis, MT dynamic instability is a balance between what two factors?

MAPs (which stabilize) and Catastrophin factors. *Note: During mitosis there are more catastrophies per min. than interphase resulting in shorter and fewer MTs. Overall there is more dynamic instability because of the vast rearrangments taking place.

Centrosome.

Major organelle in the assembly of the mitotic spindle. Contains a pair of centrioles oriented perpendicular to one another (mother and daughter).

Most important cell cycle checkpoint.

Metaphase to anaphase transition. Controlled by the spindle. Ensures all of the chromosomes are properly attached and aligned.

How can you differentiate between a mother and daughter centriole?

Mother (which originates from the original centriole) has whispy appendages which make it better at nucelating MTs (centrioles acquire these as they age). Daughter does not have these appendages and thus does not do much nucleating UNTIL the onset of mitosis.

How is the golgi partitioned during mitosis?

No one understands how equal amounts of the golgi are partitioned to daughter cells. But during prophase when the interphase MTs breakdown this causes fragmentation of the golgi so that it can more easily be segregated.

Which enzymes are responsible for sensing the bipolarity of the mitotic spindle and acting to move the spindles to their respective poles?

Polo like kinase

Major functions of M-Cdk

Promotes nuclear envelope breakdown, reorganization of actin and the golgi (necessary so that the golgi and other organelles are distributed equally between the daughter cells), triggers chromosome condensation (via condensin), and induces assembly of the mitotic spindle.

During what stages of mitosis are M-Cdk active?

Prophase, pro-metaphase, and metaphse

Kinetochore

Proteins which bind to the centromeric region of the chromosome. These function to capture the MTs. Also act as a sensor, detecting a good strong interaction between the MTs and the chromosomes (important for the metaphase-anaphase transition).

Astral MTs

Radiate towards the cell cortex. Make contact with dyenin (?) at the end which helps to hold them in place and position the mitotic spindle so that it is centered in the cell.

What regulates formation of the contractile ring?

Remnant interpolar MTs between the future daughter cells. Their position localizes rho-GTPases that trigger the assembly of actin filaments at the appropriate site of the furrow and activate myosin contraction.

Discuss how rho-GTPase regulates cytokinesis.

Rho-GTP activates formins to form actin stress fibers and activates Rock which phosphorylates and activates myosin. Together this leads to assembly and contraction of the contractile ring.

Rock

Rho-activated kinase. Phosphorylates the regulatory myosin light chain thereby activating myosin. Rock itself is activated by rho-GTPase.

When is the centrosome duplicated?

S phase

What regulates centrosome duplication?

Similar factors (i.e. G1/S-Cdk) that initiated DNA replication

Anaphase occurs in two phases. What happens in the second phase.

Sliding forces between interpolar MTs from opposite poles to push the poles apart (these MTs elongate during this time to continue this process) while a simulatenously pulling force acts directly on the poles to move them apart

When does the nuclear envelope reform?

Telophase

How does centrosome duplication occur.

The centrioles separate from one another and a new centriole buds off of/forms from this "parent" such that the new centrosome contains a pair of centrioles, one old and one new.

How does the spindle attachment checkpoint work?

There are a large variety of kinetochore proteins that sense both MT attachment at the centromeres and tension on the sister chromatids. Once these are sensed, then the checkpoint is silenced (certain proteins removed) and APC is activated leading to the degradation of securin and M-Cdk.

Checkpoints of the cell cycle are binary switches. What does this mean?

They are either on/off. Once on, it will go to until it reaches the next checkpointn.

In what cases are astral MTs no necessary?

When there is asymmetric division (i.e. oocyte)

Kinesins 4 and 10

move sister chromatids toward MT + ends on spindle MTs

Syntaxin 2

t-Snare that localizes in the midbody during cytokinesis which is needed for the fusion of vessicles delivering new membrane and essential for the generation of two separate daughter cells.