The molecular mechanism of cytokinesis

One critical step in cell multiplication is the subdivision of a single cell into two cells. This process, cytokinesis, is coupled with the nuclear division cycle so that the two daughter cells are each endowed with a complete set of chromosomes and other essential organelles. Although the critical driving force for cytokinesis is an actomyosin based contractile ring that is associated with the plasma membrane, there is strong evidence that the central spindle also plays a critical role in cytokinesis. Embryos that do not form the central spindle are able to form ingressing cleavage furrows, but they are not able to complete cytokinesis. The CeMKLP1/ZEN-4 kinesin like protein and the CYK-4 RhoGAP are essential for the assembly of the central spindle. We have found that these proteins associate in vivo and in vitro and have define the domains that mediate this interaction. To further our molecular understanding of cytokinesis, it is necessary to understand the mechanism of central spindle assembly. The aim of this project is to reconstitute central spindle assembly in vitro and to use this in vitro system to understand this process at a mechanistic level. In addition, since there is evidence that, in vivo, the formation of the central spindle is regulated by the Aurora B kinase complex, we propose to use the in vitro system to dissect the mechanism by which Aurora B regulates central spindle assembly. Additionally, we propose to map the sites of phosphorylation and use both C.elegans and mammalian cultured cells to determine whether phosphorylation of CYK-4 and/or ZEN-4 at specific sites is required for its in vivo function.

The cell is the basic unit of life, since it is has the capacity for self propagation. Cell multiplication requires DNA duplication to make two identical copies of the instructions to make the molecular machines critical for cell function. The two copies of the DNA, the genome, are then partitioned into two separate cells, this partitioning is called mitosis. The machinery responsible for segregating the chromosomes is known as the mitotic spindle. The major component of the mitotic spindle are microtubules, small filaments with a distinct polarity that allows the cell to transport objects over relatively long distances. The microtubules act as tracks for motor proteins that travel along the tracks in one direction. Motor proteins can bind to "cargo" to deliver it to different places in the cell. For example, in mitosis, motor proteins work together with microtubules to move the chromosomes to opposite sides of the cell. After mitosis, the cell itself has to divide, a process known as cytokinesis. Division of the cell is accomplished by a different type of filament, actin, and a different kind of motor, myosin, which is similar to the motor that allows muscles to do work. In order for the cell to divide in the right place, actin filaments and myosin need to be concentrated in the center of the cell, between the dividing chromosomes. The microtubules of the mitotic spindle instruct the actin and myosin where to assemble. In addition, the microtubules are important for the two cells be finally separate. This project focused on a specific microtubule motor protein that is important for the organization of a structure called the central spindle. The central spindle forms after the chromosomes have moved apart from one another and it consists largely of microtubules that form antiparallel bundles that overlap at one end. These bundles form a scaffold that concentrates proteins that are important for cytokinesis. One important result is that we discovered a molecular mechanism that controls the timing of formation of this scaffold. Specifically, the motor protein that is critical for forming these bundles is modified, phosphorylated, by the enzyme that regulates mitotic phase of the cell cycle. When this motor is modified, it does not bind well to microtubules and therefore these bundles can not form during mitosis. However as soon as the cell leaves mitosis, the motor is activated and this begins the process of cytokinesis or cell division. Why do cells make this motor inactive during metaphase? We found that if the motor remains active during mitosis, then the chromosomes are not faithfully segregated to the two daughter cells.