Basic questions about kinetochore function

The process of microtubule attachment to the kinetochore. (a) Several microtubules grow out from, and shrink back towards, the spindle pole during prometaphase. (b) One microtubule makes a lateral contact with a kinetochore. The kinetochore attaches to it and starts moving polewards. (c) A vesicle makes a lateral contact with the same microtubule and also moves polewards
Fig. 8.10. The process of microtubule attachment to the kinetochore. (a) Several microtubules grow out from, and shrink back towards, the spindle pole during prometaphase. (b) One microtubule makes a lateral contact with a kinetochore. The kinetochore attaches to it and starts moving polewards. (c) A vesicle makes a lateral contact with the same microtubule and also moves polewards.
Two basic questions about the function of kinetochore, which are being actively pursued are :
(i) how do microtubules get attached to kinetochores? and
(ii) w.hat are the structural requirements for the generation of the force required for chromosome movement? Both these questions have been partly answered through experiments conducted in recent years (1990 and 1991).

Mechanism of kinetochore-microtubule attachment.
Following two hypotheses were earlier available for kinetochore-microtubule attachment : (i) According to first hypothesis, the microtubules are nucleated at the centrosome and later captured by the kinetochores. (ii) According to second hypothesis the microtubules are nucleated at the kinetochores, and only later interact with the spindle pole. Recently, support for the first hypothesis was provided by video contrast optical microscopy. It was possible to image individual microtubules, as they grow out from mitotic poles and first come in contact with kinetochores.
Contact is established as a microtubule grows out towards the periphery of the spindle,, brushing past the side of a kinetochore. Dynamic instability causes continuous growth and shrinkage of microtubules, allowing them to probe through the cytoplasm and thus increase the probability of finding kinetochores. The kinetochore latches into the microtubule lattice aort the chromosome is jerked polewards (Fig. 8.10). A motor protein dynein, which is now known to be a part of kinetochore, may be involved in this movement. However, the possibility that the chromosomes are pulled polewards by microtubule depolymerization, can not be ruled out.
The process of microtubule attachment to the kinetochore. (a) Several microtubules grow out from, and shrink back towards, the spindle pole during prometaphase. (b) One microtubule makes a lateral contact with a kinetochore. The kinetochore attaches to it and starts moving polewards. (c) A vesicle makes a lateral contact with the same microtubule and also moves polewards
Fig. 8.10. The process of microtubule attachment to the kinetochore. (a) Several microtubules grow out from, and shrink back towards, the spindle pole during prometaphase. (b) One microtubule makes a lateral contact with a kinetochore. The kinetochore attaches to it and starts moving polewards. (c) A vesicle makes a lateral contact with the same microtubule and also moves polewards.

Possible arrangement of dynein in the kinetochore. Two headed dynein molecules are shown tugging on the end of a microtubule, whose disassembly could govern rate of movement
Fig. 8.11. Possible arrangement of dynein in the kinetochore. Two headed dynein molecules are shown tugging on the end of a microtubule, whose disassembly could govern rate of movement.
Mechanism of anaphase chromosome movement
According to an earlier view, the kinetochore is a passive structure, which remains attached tomicrotubule ends, while these mfcrotubules are shortened. The shortening of microtubules is achieved by disassembly or depolymerization. In this hypothesis, energy released (from GTP hydrolysis) during the microtubule assembly and disassembly is sufficient to drive chromosome movement during anaphase A, and any role of motor molecules was ruled out. However,
recently kinetochore has been shown to be a dynamic structure, which may contain two force producing ATPases in the form of proteins like kinesin and cytoplasmic dynein (Fig. 8.11). The forces generated by these two motor proteins are opposite in direction, one of them directed towards the pole and the other away from it. In this hypothesis, energy for the force is released during hydrolysis of ATP involved in phosphorylation of motor proteins. The presence of cytoplasmic dynein and its association with kinetochore was recently demonstrated by immuno-fluorescence microscopy, but no such cytological evidence for the presence of kinesin could be available (although the presence of a kinesin like motor protein was inferred from other observations).
Possible arrangement of dynein in the kinetochore. Two headed dynein molecules are shown tugging on the end of a microtubule, whose disassembly could govern rate of movement
Fig. 8.11. Possible arrangement of dynein in the kinetochore. Two headed dynein molecules are shown tugging on the end of a microtubule, whose disassembly could govern rate of movement.

Dramatic evidence for rapid chromosome movement along microtubules in vivo has also been presented, which suggests a role of motor molecules in chromosome movement during anaphase A. One of the obvious candidates for such a motor molecule is cytoplasmic dynein, which produces force in the appropriate direction to make the chromosomes move towards the spindle poles. However, the rates of chromosome movement during metaphase and anaphase (1-3 μmin-1 or .01-.05 μm-sec-1) are much slower than those expected from dynein.

In vitro association and relative movement of microtubules and kinetochores Recently the forces involved in chromosome movements have been studied using in vitro techniques involving association of isolated chromosomes and microtubules.
The association and chromosome movement were recorded using video microscopy. Since, it is difficult to examine the movement of relatively large chromosomes along very thin- microtubules, in this recent in vitro study the movement of microtubules against the chromosomes (adsorbed on a slide) was examined. In order to detect the movement of microtubules and its direction, the microtubules were assembled from a mixture of fluorescently labelled and unlabelled tubulin subunits, so that the minus (-) or pole ends and the plus (+) ends of microtubules could be distinguished by the intensity of fluorescence.

Association in presence of ATP.
It was shown that microtubules become associated with kinetochores and in presence of ATP (to add phosphate group) travel towards the minus end (of in poleward direction). The rate of movement was 0.46 μm sec-1 (28 μmmin-1), similar to that in early prometaphase, but much greater than the known rate of chromosome movement during anaphase. This was explained on the basis of the assumption that chromosomes were perhaps isolated from cells arrested at prometaphase. The movement can be attributed to cytoplasmic dynein.

Association after pretreatment with ATP-v-S.
When the chromosomes and microtubules were pretreated with ATP-ν-S (with thiophosphate group) before adding ATP, the movement was much slower (.048 μm/sec-1) and in opposite direction i.e. towards the centre.
ATP-ν-S will add a thiophosphate group preferentially to one motor protein, which can not be removed by a phosphatase so that the protein is modified irreversibly, thus turning off the minus-end directed motors and turning on the plus-end directed motors^ Using an antibody (anti-thiophosphate), this thiophosphate group could be located in the kinetochore, suggesting that phosphorylation of a component protein of kinetochore actually leads to reversal of the direction of chromosome movement. It is suggested that both a kinase (it could be cdc-2 kinase) and a phosphatase may be associated with the kinetochore (kinase adds phosphate group and phosphatase removes it). Further, there may be two motor proteins involved in the development of forces in opposite directions.

Role of phosphorylation in motor activity.
Using in virto studies, it could be shown that there are two typesof motor proteins at the kinetochores, one developing minus-end directed force and the other developing a plus-end directed force. ATP analogues (e.g. ATP-ν-S) were used which are known to be used differently by known motor proteins, dynein and kinesin, both ATPases. The minus-end motor gives a response similar to that given by dynein, which has now been shown to be located at kinetochore. But plus-end motor protein seems to be different from kinesin (GTP is used by kinesin, but not by plus-end motor protein; also kinesin makes microtubules move much faster), which unlike dynein could not be located in kinetochore. Thus it may be a new protein, likely to be identified in the near future. It has also been shown that differential phosphorylation and dephosphorylation of two ATPases (motor protons) in kinetochores are responsible for chromosome movements during cell division.
The phosphorylation is facilitated by a kinase enzyme and dephosphorylation is facilitated by a phosphatase enzyme, and a delicate balance between kinase and phosphatase is maintained. Although it is not known how phosphorylation controls direction of motor movement, but a parallelism has been drawn with similar control system in melanophores, where pigment movement is effected by two motors, whose relative activities are controlled by phosphorylation. Besides phosphorylation and dephosphorylation, microtubules grow and shrink (due to polymerization and depolymerization) at the kinetochore. This suggests that motor activity and microtubule dynamics are co-ordinated in a delicate manner regulating chromosome movement, away from and towards the spindle pole during cell division.

Reversible phosphorylation within the cell is such an important phenomenon in cellular regulatory mechanisms, that the 1992 Nobel Prize for medicine was awarded to two U.S. bio-chemists (Edmond Fischer arid Edwin Krebs) for their researches in this area.

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