RESEARCH SUMMARY

Our lab is interested in understanding how positional information is generated within the cell. We use high resolution optical imaging and analysis techniques, the green fluorescent protein GFP and its genetically encoded variants as non-invasive fluorescent biosensors, and the fission yeast Schizosaccharomyces pombe – with its well-defined shape, size, and genetic tractability – as ideal tools for studying cellular pattern formation.

Figure 1.

Our previous studies in the fission yeast have established that the microtubule cytoskeleton defines a spatial map of positional information for the cell (Fig. 1). We aim to test various aspects of this model, particularly:

A model for the establishment of positional information. In wild-type cells, iMTOCs arrange microtubules in bilaterally symmetric bundles such that a balance of pushing forces from the microtubule plus ends positions the nucleus in the cell center. The microtubule plus ends, with regulated organization and kinetics, can mark sites of polarized cell growth, at the cell tips. The microtubule cytoskeleton thus acts as a dynamic spatial regulator, continually monitoring and defining sub-cellular domains.

1) Identification of new components of the iMTOC.

Figure 2.

The iMTOCs are novel microtubule-organizing-centers in fission yeast. We envision that components of the iMTOCs include microtubule bundling and stabilizing factors and perhaps nuclear attachment factors (Fig. 2). Previously we have characterized rsp1, the only known protein component of the iMTOC. We have identified rsp2, a mutant whose phenotype resemble those of rsp1, and will undertake molecular characterization to determine their roles in iMTOC organization and function.

rsp2 mutant cells have abnormally short microtubules, leading to nuclear positioning defects.

2) Functions of microtubule architecture in pattern formation.

Figure 3.

We have identified rnd1 and rnd2, mutants with round-cell phenotypes (Fig. 3). Characterization of rnd1 and rnd2 is underway to define the molecular pathway of pattern formation.

Results of a random insertional mutagenesis screen for morphologically round mutants. Individual fission yeast colonies were viewed and selected manually under the microscope.

 

 

3) Development and application of quantitative optical imaging and analysis methods.

We are committed to the development and application of advanced imaging and analysis techniques for our work. In conjunction with conventional fluorescence, we will also use quantitative fluorescence techniques such as fluorescence resonance energy transfer (FRET) to study protein-protein interactions, fluorescence recovery after photobleaching (FRAP) to study protein turnover kinetics, fluorescence speckle microscopy (FSM) to study protein and polymer dynamics, and transmitted-light fluorescence polarization microscopy to study protein and polymer orientation and rotational dynamics. All of these techniques will contribute to the studies of the microtubule cytoskeleton outlined above.

Our studies will impact diverse disciplines such as light microscopy, polymer science, pattern formation, cell polarity, cell motility, cytoskeleton, macromolecular self-assembly, and single molecule science.

Click here for a list of publications (searches the National Library of Medicine's PubMed database.)