J. Chloe Bulinski, PhD

To understand the changes in composition and organization of cytoskeletal elements that accompany - or possibly, permit - changes in cellular function, my laboratory investigates mechanisms by which cells alter their cytoskeleton during the cell cycle, differentiation, and in response to chemotherapeutic drugs. We are testing the hypothesis that MT-associated proteins (MAPs) modulate MT functions. We have used as a model a protein called MAP4, the most abundant MAP present in organisms ranging from the worm, Caenorhabditis elegans, to humans. We started with in vitro biochemistry, proceeded with overexpression experiments, and then tested the sequelae of depleting cellular MAP4. Our studies show that MAP4 is a major MT stabilizer in cells, it is involved in regulating MT dynamics during differentiation, it regulates tubulin and MT levels, and it impacts upon cell spreading. MAP4 is also capable of regulating MT-based transport, perhaps contributing to polarization of cells. Finally, and perhaps most exciting, MAP4 also functions in cell cycle progression; we showed that MAP4 binds to cyclin B/p34cdc2 kinase and that the kinase phosphorylates MAP4 during M-phase, making it lose its capability to stabilize MTs. We are using in vitro mutagenesis to investigate how cell cycle modification of MAP4 contributes to cell cycle timing and/or physical assembly of M-phase components.

PMAPs that alter MT stability might be expected to affect the cytotoxic actions of MT-antagonistic drugs, such as Taxol, which is used in human chemotherapy, or to participate indirectly in downstream events. A candidate MAP we identified, called ensconsin, which is present in a variety of human cells, varies in its presence, abundance, and turnover among cell types and during Taxol treatment, suggesting that it may affect Taxol's cytotoxic potential. Studying ensconsin will help us understand how Taxol acts on the MT system, and how it might be most effectively used against tumors.

To investigate ensconsin's in vivo behavior, we generated cell lines expressing chimeras of green fluorescent protein (GFP) conjugated to ensconsin (GFP-EMTB). Distribution of GFP-EMTB elucidated behaviors of ensconsin that had eluded those working only with fixed material. To follow in vivo dynamics of single MTs with GFP-EMTB by time-lapse imaging, we discovered that high levels of GFP-EMTB expression were required, but this induced artifacts such as MT bundling and mitotic abnormalities. Therefore, we conjugated ensconsin to multiple GFP molecules. Cells that expressed these conjugates showed bright MT fluorescence, while their MT system was not artifactually altered. Thus, GFP-ensconsin chimeras provide a non-perturbing MT label and also allow observation of ensconsin behavior in vivo. We are studying ensconsin behavior in Taxol-treated cells; we are also using cells expressing GFP-ensconsin chimeras to study viral and mRNA transport in a physiological context, as well as in a collaborative effort in which we developed a new technique, fluorescent speckle microscopy. This technique allows us to image single molecules in vivo; it significantly improves visibility of fluorescent MTs in living cells and reveals assembly dynamics, movement, and turnover of protein assemblies. We are using fluorescent speckle microscopy to follow the dynamics of ensconsin molecules' association/dissociation with MTs in normal and drug-treated cells.