Our long term goal is to understand intracellular transport processes such as: the movement of chromosomes during mitosis and the joining of male and female pronuclei at fertilization. These processes are microtubule-based and require proper assembly and reorganization of the microtubule cytoskeleton. When these basic intracellular transport processes do not function properly, serious health problems result including cancer and aneuploidy. The focus of this study is to understand how microtubule assembly in cells is regulated by microtubule associated proteins (MAPs). The array of microtubules found in cells consists of two subpopulations: one subpopulation is dynamic and exchanges subunits rapidly with the subunit pool, while the other subpopulation is much more stable. Assembly of the dynamic cellular microtubules and of purified tubulin is characterized by a unique behavior termed dynamic instability where microtubules transit between one of two phases: elongation and rapid shortening. The transitions between these phases are abrupt and stochastic. Compared to pure tubulin, factors must exist in the cell that: increase elongation velocity, increase transition frequencies, block minus end assembly and prevent non-nucleated assembly. In addition, the activities of transition frequency regulators are likely regulated during the cell cycle. Although these types of MAPs must be present, few MAPs have been identified and analyzed at the level of individual microtubules required to measure the parameters of dynamic instability (rates and transition frequencies). In addition, the functions and mechanisms responsible for generating stable microtubules are not known. Our goals are to use functional assays with the ability to visualize individual microtubules to: (1) characterize the effects of previously identified MAPs on microtubule dynamic instability; (2) isolate and characterize MAPs regulating dynamic instability both in sea urchin egg extracts (microtubule dynamic instability in this cell free system is similar to that in the cell) and in human monocytes; (3) develop a cell free system to study the formation of stable microtubules. For goal (1) we will use video enhanced differential interference microscopy (DIC) to record, in real time, the dynamic instability of purified tubulin in the presence and absence of the following MAPs: XMAP from Xenopus, nucleotide diphosphate kinase, and the mammalian brain MAPs, MAP2 and tau. For goal (2), we will use video and immunofluorescent microscopic assays to fractionate sea urchin egg extracts and isolate the factors present that regulate dynamic instability. These studies will focus on biochemical fractionations including microtubule affinity chromatography and substraction experiments where we will assay for loss of activity. We will also use these assays to functionally screen a human monocyte cDNA library. For goal (3), we will again use microscopic functional assays to examine stable microtubules in a cell free system. We will first develop a cell free system where stable microtubules form in vitro. Next we will use this system to characterize the assembly and disassembly of these stable microtubules and to isolate the factors responsible for generating stability.
|Howell, B; Deacon, H; Cassimeris, L (1999) Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo. J Cell Sci 112 ( Pt 21):3713-22|
|Spittle, C S; Cassimeris, L (1996) Mechanisms blocking microtubule minus end assembly: evidence for a tubulin dimer-binding protein. Cell Motil Cytoskeleton 34:324-35|