Microtubules are dynamic structures that serve to organize the cytoplasm in interphase cells and to provide the structural framework and possibly the motive force for the spindle in mitotic cells. The broad objectives of this proposal are to understand microtubule dynamics in vivo and vitro and to determine whether selected mitotic events are dependent upon the dynamic properties. A variety of experimental approaches will be used including fluorescent and hapten tagging of proteins, microinjection, hapten- mediated immunocytochemistry, fluorescence photobleaching, and digital imaging microscopy. These methods allow determination of the sites of tubulin subunit addition into an loss from pre- existing microtubules. They also permit a determination of the kinetics of turnover of the microtubule network. Posttranslationally modified subunits will be identified by indirect immunofluorescence with peptide specific antibodies. The principal specific aims will be to complete our analysis of anaphase dynamics; to determine the mechanism of turnover in metaphase; to identify sites of subunit addition and loss during congression in prometaphase; to investigate further microtubule dynamics in the leading lamellum of fibroblasts and their relationship to cell motility; to obtain direct evidence for the mechanism of microtubule turnover near the cell center; to test the role of two posttranslational modification, detyrosination and acetylation, on microtubule turnover; to evaluate the stability of microtubules in a highly differentiated cell type, the PC-12 neuronal cell line; and to directly test the structural hypothesis of slow axonal transport. The results should help elucidate the precise nature and significance of microtubule lability. The results should also be of relevance for mechanisms regulating motility and division in normal cells. An understanding of normal cell motility and division may assist in the analysis of cancer cells where the mechanisms restricting division are aberrant.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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University of Wisconsin Madison
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United States
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Lee, Ho-Sup; Komarova, Yulia A; Nadezhdina, Elena S et al. (2010) Phosphorylation controls autoinhibition of cytoplasmic linker protein-170. Mol Biol Cell 21:2661-73
Komarova, Yulia; De Groot, Christian O; Grigoriev, Ilya et al. (2009) Mammalian end binding proteins control persistent microtubule growth. J Cell Biol 184:691-706
Komarova, Yulia; Lansbergen, Gideon; Galjart, Niels et al. (2005) EB1 and EB3 control CLIP dissociation from the ends of growing microtubules. Mol Biol Cell 16:5334-45
Kandere-Grzybowska, Kristiana; Campbell, Christopher; Komarova, Yulia et al. (2005) Molecular dynamics imaging in micropatterned living cells. Nat Methods 2:739-41
Lansbergen, Gideon; Komarova, Yulia; Modesti, Mauro et al. (2004) Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J Cell Biol 166:1003-14
Kojima, Shin-ichiro; Vignjevic, Danijela; Borisy, Gary G (2004) Improved silencing vector co-expressing GFP and small hairpin RNA. Biotechniques 36:74-9
Bertorello, Alejandro M; Komarova, Yulia; Smith, Kristen et al. (2003) Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells. Mol Biol Cell 14:1149-57
Komarova, Yulia A; Vorobjev, Ivan A; Borisy, Gary G (2002) Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary. J Cell Sci 115:3527-39
Komarova, Yulia A; Akhmanova, Anna S; Kojima, Shin-Ichiro et al. (2002) Cytoplasmic linker proteins promote microtubule rescue in vivo. J Cell Biol 159:589-99
Maly, Ivan V; Borisy, Gary G (2002) Self-organization of treadmilling microtubules into a polar array. Trends Cell Biol 12:462-5

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