Spindle Pole Bodies and Centrosomes
Errors in chromosome segregation lead to aneuploidy, which results in birth defects, cancer or cell death. Accurate chromosome segregation is performed by a large molecular machine called the mitotic spindle. The mitotic spindle contains many smaller machines including the centrosome, the microtubules themselves, the kinetochores where the microtubules attach to the chromosome and a multitude of microtubule motors.
The centrosome, first recognized over one hundred years ago as a key organizer of cellular structure, remains one of the great mysteries of modern cell biology. From the molecular composition, to the structural organization, to the intriguing process of duplication, our understanding is rudimentary. Cells start the cell cycle with a single centrosome, which duplicates only once each cycle. The two centrosomes organize the microtubules that make up the mitotic spindle and thereby play a crucial role in ensuring that each daughter cell receives exactly one copy of each chromosome. In cancer cells, this process goes awry, resulting in cells with multiple centrosomes as well as abnormal mitotic spindles.
The Kinetochore and Spindle Dynamics
The functional equivalent of the centrosome in the unicellular eukaryote Saccharomyces cerevisiae is a cylindrical multilayered structure called the spindle pole body (SPB). As the name suggests, the two SPBs form the poles of the mitotic spindle. They also organize the cytoplasmic microtubules that reach to the cortex of the cell and position the nucleus. Although SPBs do not resemble centrosomes on the ultrastructural level, many known SPB components have homologues found in the animal cell centrosome. The centrosome and the SPB perform similar functions and contain similar components. Thus, a detailed understanding of the regulation, structure and assembly of the yeast SPB will yield important insights into animal cell centrosomes. We are exploiting our ability to manipulate yeast genes at will to define the properties of this central and essential organizer. Finally, we are applying what we learn in yeast to an analysis of the centrosomal and chromosomal abnormalities that occur in human carcinomas.
The ultimate goal of mitotic spindle assembly is to arrange each chromosome with its sister chromatids attached to opposite poles via microtubule fibers. This ensures that when anaphase occurs, the two sister chromatids are pulled apart and partitioned one into each daughter cell. The assembly process is done by trial and error. Incorrect attachments are detected and detached to allow another attempt at correct attachment. Classic experiments have shown that tension is a key aspect of the error detection mechanism. The tension is sufficient to stretch the chromatin but not enough to shred the DNA or pull apart the spindle. How is tension established and maintained at the right level? How do the kinetochores and the motors work together to balance the forces in the spindle?
The kinetochore is a machine with a difficult task. It attaches the chromsomes to the dynamic microtubule fibers and must do so against ~20 pN of force, much more than is required to drag a chromosome through the cellular milieu. All chromosome segregation depends on this connection. The checkpoints are tuned to check for this connection and the tension it generates. We are studying the kinetochore as a molecular machine. Much of our work focuses on yeast, where we have a nearly complete parts list for the kinetochore and many tools to exploit. We use a wide range of approaches including genetic analyses, quantitative microscopy, biochemical assays and computational modeling.
FRET - Fluorescent Resonance Energy Transfer
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