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ANAT 262 (65)

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Anatomy & Cell Biology
Course Code
ANAT 262
John Presley

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Mitotic spindle assembly and dynamics depends on two basic processes: 1) selective stabilization of MTs by chromosomes and MT motors (uses MAPs) 2) organization of MTs by motors - There are several (more than 7) different KRPs and cytoplasmic dyneins in- volves in this process - centrosome duplication and nuclear division are tightly coupled such that centrosome duplication always precedes nuclear division - the centrosome is duplicated by an unknown mechanism during interphase and splits when mitosis starts - so now the cell has two MTOCs each with a radial array of MTs called an aster - a subfamily of KRPs - bimC function in separating the duplicated centrosomes - cytoplasmic dynein is involved in spindle pole formation Early in prophase while the nuclear envelope is still intact, dynamic MTs from each aster overlap at their plus end and nuclear envelope starts to break down - KRPs of the bimC family self associate through their tails forming a bipolar motor (like myosin II) - these bipolar motors slide the oppositely oriented MTs past each other thereby pushing the spindle poles apart (sliding of antiparallel MTs) - kinesins bridge anti-parallel MTs - tails grab each other so they have two feet walking in either direction - motors move towards the + end, tails cannot move (they are locked together) In late prophase, two major events take place: 1) nuclear envelope breakdown (NEB) 2) destabilization of the MTs (10x decrease in MT half life) - both events are due to protein phosphorylation - mainly by M-phase promoting factor (m-edk cdc2/cyclinB) - kinetochore microtubules can grow and shrink faster Nuclear Envelope Breakdown: made of intermediate filaments (which get phosphory- lated and break down during cell division so that the MTs can have access to the chro- mosomes) - NEB follows phosphorylation by m-edk (M-cyclin dependent kinase) of the nuclear lamins (IFs that form a reinforced network under the nuclear envelope) - phosphorylation causes the IFs to depolymerize leaving the envelope without struc- tural support and causing it to breakdown - once NEB occurs, chromosomes becomes accessible to the dynamic MTs and each chromosome has already replicated by the start of prophase and consists of two sister chromatids joined along their length - a chromosome is a pair of two chromotids which are cinched together at the kinetochore (20-30 proteins creating a signal site at the middle) - the constriction site in the metaphase chromosomes is known as the centromere - this is the site where the kinetochores are multi-layered protein complexes con- taining structural proteins, motor proteins, MT-modulating proteins and check- point signaling proteins - kinetochore is the binding region for kinetochore MTs Destabilization of MTs: phosphorylation of MAPs and catastrophin (what severs MTs) - interphase MTs are relatively long and stable with a half-time for MT turnover of more than 10 minutes - when a cell enters mitosis the rate of catastrophe increases 10 fold - the MT population has now become short and unstable - with a half time of MT turnover less than one minute - this dramatic change in MT stability is driven by: - phosphorylation of m-ed of two classes of proteins that control MT dynamics: - MAP phosphorylation causes them to dissociated from MTs which are thus destabilizes - phosphorylation of a MT motor protein catastrophin which causes the destabilization of MT plus ends (increasing catastrophes) - activation of katanin - MT severing protein (phosphorylation of katanin will turn it on and increase severing of MTs) - increases catastrophe - MTs are much less stable The dynamic MTs generated by the action of m-edk are constantly shrinking and growing - thus exploring their surroundings - when a MT encounters a kinetochore it is stabilizes at the plus end - stabilization through a search and capture mechanism will eventually form a bipolar spindle array - each kinetochore captures 30-40 MTs - when all the sister chromatids are attached in this way to the MTs - motor proteins align the chromosomes along the metaphase plate - there is a spindle attachment checkpoint that ensures that cells do not enter anaphase until all the chromosomes are attached to both poles of the spindle - unattached kinetochores release a protein signal that puts the break on cell division until all are properly attached - the kinetochore is therefore involved in both the mechanics and control of chromosome movement Although kinetochore and polar MTs have been stabilized at their ends they are still dynamic - this movement or polar flux of tubulin through the spindle MTs is known as poleward flux and reflects the fact that spindle MTs undergo a treadmilling - which involves the net addition of tubulin subunits at the plus end balancing by net loss at the minus end (near the spindle pole) - function is not clear - this dynamic behavior is unexpected for MTs in the mitotic spindle and may reflect the association of stabilizing MT motors at the + and - ends - the motor proteins could act as sliding collars, alternatively releasing to allow for entry and exit of a subunit and binding to stabilize the end - poleward flux is not seen in astral MTs There are two distinct processes involved in the actual separation of sister chro- matids - anaphase A and anaphase B Anaphase A: the initial separation of sister chromatids (become daughter chromo- somes) towards the spindle pole is accompanied by shortening of the kinetochore MTs at the point where they are attached to the kinetochore - this depoylmerization could be due to exposure and destabilization of the plus end as dynein moves towards the minus end of the MT at the spindle pole - an alternative model indicates that MTs depolymerization can itself provide the force to move daughter chromosome towards the spindle pole - this process requires a protein with high affinity for polymerized tubulin - cell will not divide unless all chromosomes are at metaphasic plate - protein likes polymerized tubulin more than depolymerized MTs Anaphase B: overlapping of two polar MTs (in the middle is the bipolar kinesin mo- tors)...involves moving apart the two spindled by: 1) elongation of the polar MTs at the plus end and with the action of KRP (bimC) - KRP = kinesin related proteins 2) motor proteins acting in conjunction with astral MTs (dynein) pulls poles apart, anaphase A only separated the poles slightly - want to move in opposite directions, hence KRP generate force which pulls the poles apart (moves the spindles back toward the cortex) - dynein motors (with cargo of cortex) grabs onto the MT pulling the spindle to the cortex Then in telophase nuclear envelopes assemble around the chromosomes and fi- nally cell division culminates in division of the cytoplasm by cytokinesis - this is accomplished by a contractile ring composed of actin and myosin - myosin pinches the actin to divide a cell into two There is no lag phase in a cell because you are attached to gamma tubulin ring com- plexes (nucleation site) - in a test tube you do not have this they just randomly bump into each other but in a cell this does not happen (more controlled) Actin: There are six acting isoforms in mammals each encoded by a different gene - beta and gamma -- non muscle actins in all cells - tissue specific forms - alpha and gamma - smooth muscle actins - alpha cardiac muscle actin - alpha skeletal muscle actin - these different actin isoforms show size conservation (42,000 kDa) and minor AA se- quence differences which tailor their function to a specific cell type (ex: muscle actins) -- slight differences in their tails (how you get tissue specific forms) The characteristics of actin polymerization are similar to those of tubulin - ATP is hydrolyzed after the subunit is incorporated in the filament - ADP can be exchanged for ATP only after the actin subunit dissociates from the fila- ment (need ATP actin to be able to add to the growing end) - remember that ATP exchange for actin has a half time in the order of minutes, much slower than GTP exchange for tubulin (seconds) - the ATP binding site in actin is sequestered by two domains and becomes oc- cluded when the subunits is in the AF - except at the minus end - usually (in interphase) AF is turning over faster than MTs - as in the case of tubulin, actin polymerization can be studied in vitro by adding K+, Mg2+ and ATP to the buffer - this will allow AF to grow in the test tube - ATP hydrolysis after actin incorporation into AF, rendering the filament unstable by weakening the interactions between the subunits in the AF - treadmilling is more typical of AFs than MTs - AFs do not show a high degree of dynamic instability seen for MTs - more stable, gets actin from muscle The importance of dynamic properties in AF is underscored by the fact that these AF functions are inhibited by
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