Identifying Proteins in the Microtubule Lumen Microtubules are ubiquitous, dynamic filaments of the cytoskeleton that participate in practically any cellular activity that involves movement. Associated with these structures in cells are a diverse group of proteins that regulate microtubule function or use microtubules as tracks to transport materials throughout the cell. The tubes are composed entirely of a single protein:a,b- tubulin. All of the transporter proteins and virtually all of the regulatory proteins bind to the outside or to the ends of the tubulin polymers. To date there is only one example of a regulatory process that takes place in the microtubule lumen: acetylation of lysine-40 of a-tubulin. Cryoelectron tomography studies have demonstrated that many cellular microtubules are not empty - they are in fact filled with particles. Just one posttranslational modification with a relatively small enzyme cannot account for such a large amount of material. There must be additional proteins with the microtubule lumen to account for the particles. The identity, purpose and function of these particles are not known. Understanding the nature of luminal proteins is important in fundamental biology of microtubules. It may also provide insight into the molecular pathology of microtubule-associated disease and lead to important new targets for chemotherapy. The first step toward understanding the purpose and function of the particular matter in microtubules is to identify the material. Thus, the goal of this project is to identify proteins associated with the lumen particles of cellular microtubules. Our approach is to harness the unique microtubule binding properties of the drug paclitaxel, which binds with high affinity to a single, luminal site on assembled tubulin, to place photoaffinity labels in the interior of microtubules in living cells. Paclitaxel derivatives will be synthesized in which a photolabeling functional group will be linked to paclitaxel through a long polyether tether. The probes are designed such that the photoaffinity label will be projected deep within the microtubule lumen. Photolysis of the microtubule-bound probe in live cells will crosslink paclitaxel to nearby proteins, which will be identified in the complex mixtures of cellular proteins by detecting the paclitaxel tag. Photolabeled proteins will be analyzed by mass spectrometry. We hypothesize that some of the proteins identified will be entirely new discoveries.

Public Health Relevance

Susan Bane, principal investigator Title: Identifying Proteins of the Microtubule Lumen Microtubules are a very important target for cancer chemotherapy. In addition, defects in microtubule-protein interactions are involved in diseases such as Alzheimer's. Identifying proteins inside of tube will yield a new place in the cell that can be the target for chemotherapeutic drugs against cancer and other diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Gindhart, Joseph G
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State University of NY, Binghamton
Schools of Arts and Sciences
United States
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Dilek, Ozlem; Lei, Zhen; Mukherjee, Kamalika et al. (2015) Rapid formation of a stable boron-nitrogen heterocycle in dilute, neutral aqueous solution for bioorthogonal coupling reactions. Chem Commun (Camb) 51:16992-5
Mukherjee, Kamalika; Chio, Tak Ian; Sackett, Dan L et al. (2015) Detection of oxidative stress-induced carbonylation in live mammalian cells. Free Radic Biol Med 84:11-21
Mukherjee, Kamalika; Bane, Susan L (2013) Site-specific fluorescent labeling of tubulin. Methods Cell Biol 115:1-12