One of the most important tasks facing all eukaryotes, from yeast to humans, is to precisely replicate and segregate their DNA during cell division. This gargantuan task is accomplished by packaging the replicated DNA into pairs of chromosomes and segregating the pairs to separate cells using an elaborate microtubule apparatus called the mitotic spindle. Microtubule attachments primarily occur at a single site on each chromosome called the centromere, which nucleates the kinetochore—-a large proteinaceous structure that attaches to microtubules and helps direct chromosome movements along the spindle.

The Murphy lab is interested in how a single centromere is formed on each chromosome. For many years, researchers have postulated that centromeres would function in a manner analogous to gene promoters. That is, unique DNA sequences on the chromosome would be bound by specific DNA-binding proteins, which in turn would mediate the attachment to microtubules. However, a growing body of data suggest that in many eukaryotes this is not the case. Researchers have failed to identify a “magic sequence” that is unique to centromeres, in addition some sequences can become centromere active or inactive under poorly understood conditions. These paradoxes have led researchers to propose that centromeres are partially epigenetic. This means that the centromeres are specified not by DNA sequence but by a special mark on the chromosome, such as a DNA modification or a unique chromatin structure that can be copied from the original centromere to the nascent centromeres during chromosome replication.

The Murphy lab is taking a two-pronged approach to identify the genes and proteins that are important for centromere function in Drosophila. First, they are systematically testing candidate genes using RNA interference in tissue culture cells to determine if loss of the gene product disrupts centromere function. Promising candidates are then tested to see if they disrupt centromeric chromatin or another step in kinetochore formation. Second, they are developing an assay that will directly measure the frequency at which centromeres are inactivated by analyzing the behavior of chromosomes with two centromeres (dicentric chromosomes, Fig. 1). Together, these assays will help identify the genes involved in specifying the centromere, as well as other genes connected with centromere function, such as microtubule-based motors.

Murphy’s research has also led to studying how microtubules are nucleated. Animal cells contain an organelle called the centrosome, which duplicates and nucleates the microtubules that form the mitotic spindle. Murphy found that Drosophila cells mutant for skpA, a component of the protein degradation machinery, overduplicate their centrosomes (Fig. 2). This occurs in part because skpA mutants fail to degrade cyclin E, a kinase partner that triggers centrosome duplication. However, Murphy’s findings suggest that an unknown protein is also accumulating in skpA mutant cells and acts in concert with cyclin E to trigger centrosome overduplication. Centrosome overduplication is a common feature of many cancer cells, suggesting that this unknown target of skpA may be an important oncogene.

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The typical behavior of a dicentric chromosome during anaphase is shown with the two centromeres moving to opposite poles, stretching the intervening chromatin into a dicentric bridge (arrow) that will eventually break. Chromosomes are shown in dark gray, centromeres in light gray. (Image courtesy of Byron Williams.)

Metaphase figures are shown here from wildtype (top) and skpA mutant (bottom) cells. The wildtype cell has two centrosomes (arrowheads) with the chromosomes aligned in-between, whereas the mutant cell has eight centrosomes and has failed to properly align the chromosomes.