Cell Cycle Control of Centrosome Reproduction and Function

Edward H. Hinchcliffe      Assistant Professor Ph.D.: University of Minnesota, School of Medicine Post-Doctoral: Worcester Foundation for Biomedical Research, and University of Massachusetts Medical School

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Work in my laboratory focuses on how eukaryokic cells undergo the process of cell division. In particular we are interested in the cell cycle regulation of centrosome reproduction, which as a necessary step in the assembly of a bipolar mitotic spindle, is crucial for a successful mitosis. The centrosome is the organelle responsible for the nucleation of the interphase microtubule network. Prior to the onset of mitosis, this single centrosome reproduces or duplicates exactly once to form a pair of sister centrosomes. The pair of daughter centrosomes disjoins and separates to the opposite sides of the cell, where each forms a pole of the mitotic spindle. Failure in either centrosome reproduction or separation can lead to the formation of a monopolar spindle. Such cells will not divide and become polyploid. Conversely, if the centrosome undergoes more than one reproductive event per cell cycle (a process termed centrosome amplification, or endoduplication) then the cell has the potential to assemble a multipolar spindle. Such abnormal spindles have been implicated in the accumulation of genomic instability associated with the onset and progression of neoplasia.

We have developed an in vitro system to study centrosome reproduction based on a cytoplasmic extracts made from frog eggs. Previously, we used this system to demonstrate that the reproduction of the centrosome is controlled by the activity of the Cdk2-cyclin E complex. Cdk2-E is an enzyme that is also involved in driving the replication of DNA during S phase, the time during the cell cycle when centrosome reproduction takes place. Whether Cdk2-E acts directly in driving centrosome duplication (by, say phophorylating key centrosome components) or indirectly through other pathways is not clear. Currently, we are using this extract system to determine the role played by other cell cycle regulatory pathways in controlling centrosome reproduction.

My lab is also exploring the role played by the centrosome in controlling the progression of the cell cycle, in assembling a bipolar spindle, and in regulating cytokinesis. For these studies we use cultured mammalian somatic cells, time-lapse videomicroscopy, and microtechnique to remove centrosomes from living cells.

Figure 1. Centrosome reproduction in Xenopus egg cytoplasmic extracts.

Frames from a time-lapse video sequence showing the increase in aster number over time. This increase is due to the one-to-two duplication of the centrosomes. Polarization optics. 10 m per scale division.

Figure 2.  Examples of mitotic figures containing multiple centrosomes seen in p53-/- mouse embryonic fibroblasts.

a. Bipolar spindle with multiple centrosomes at each pole. Microtubules are in green, -tubulin is in red.

b. Multipolar mitotic spindle, each pole has a focus of -tubulin (red).

c. A cell containing both a bipolar spindle (lower left) and two supernumerary asters (upper right). Note that the bipolar spindle has one normal spindle pole with associated astral microtubules and a focus of -tubulin, whereas the other spindle pole is abnormal: it is anastral (i.e. does not contain astral microtubules) and is not stained by the -tubulin antibody.