Wallace F. Marshall
How Cells Count and Measure
Mission Statement: Building the Cell
Modern cell biology has made great strides in understanding cell structure and function. As with any engineering problem, however, there is a third important aspect that needs to be understood besides structure and function, and that is assembly. How are the complex three dimensional structures found within the cell specified by a one-dimensional genome? My long-term research goal is to understand how the size, number, and position of every organelle in the cell is determined by the genome, using a combination of genetic, imaging, and mathematical approaches.
So far I have concentrated on two model systems: centriole duplication as a system to study organelle inheritance, and flagellar length control as a system to study organelle size regulation. My work takes advantage of the unicellular green alga Chlamydomonas (also known as "green yeast"), which has many of the same advantages as yeast for genetic experiments, and whose genome has recently been sequenced, but which has centrioles and flagella identical to animal cells.
Control of Organelle Number: Centriole Duplication
The major research focus of my lab will be to understand the function and duplication of centrioles, using Chlamydomonas as a genetic model system. This work is motivated by the fact that centrioles are thought to be critical for proper cell division and chromosome segregation, and abnormalities of centriole number are a common feature of cancer cells.
Aim 1. Identify genes required for centriole duplication and number control.
The most general approach to understanding centriole duplication and its regulation is to identify mutants with defects in centriole number. I have already identified several mutants with apparent defects in centriole duplication, as well as a set of conditional mutants defective in centriole segregation. Identifying and characterizing additional genes required for centriole duplication will be the major focus of future screening efforts.
Aim 2. Identify the protein components of the centriole.
To understand centriole assembly, we need to know the parts list. I have developed a centriole purification procedure that yields a 3000-fold enrichment relative to total protein. 2D gel electrophoresis of these enriched centrioles reveals approximately 40 strongly staining protein spots. The next step is to identify these proteins by mass spectrometry.
Aim 3. Determine centriole function in chromosome segregation and cell division.
In an effort to understand centriole function in cell division, I have developed a genetic strategy to measure chromosome loss rates in mutants with centriole defects. I have also begun to explore the effect of centriole mutants on cytokinesis in living cells. Initial results suggest that both cytokinesis and chromosome segregation are defective in Chlamydomonas centriole mutants. In order to determine the role of centrioles in cell division in greater detail, my lab will apply four-dimensional microscopy along with genetic assays for chromosome segregation, to analyze cell division in a variety of mutants with different defects in centriole number and structure.
Control of Organelle Size: Flagellar Length
The other major goal of my lab will be to understand how organelle size it determined. Flagella are an ideal system for asking this question because their size is easily measured. I have previously shown that a simple mechanism for flagellar length control based on the inherent length-dependence of intraflagellar transport can account for most prior experimental data on length control. I am currently testing several novel predictions of this model, for example the prediction that flagellar length should vary as a function of the number of flagella present in a cell. In the near future my future work on flagellar length control will center around understanding mechanistic basis of long-flagella (lf) and short flagella (shf) mutants that have abnormal flagellar length. Ultimately I plan to test whether a similar type of mechanism can account for size control in other organelles.