The broad objectives of this proposal are to understand the mechanism of microtubule dynamics in vivo and the relationship between this dynamic activity and important cell biological functions including organization of the cytoplasm, the generation of cell form, cell division and cell locomotion. The conceptual framework, underlying the proposal is that the organization of microtubules and their turnover in cells result from an interplay of cellular components. Two important classes of component are: (1) the centrosome as a nucleating and anchoring structure and (2) cytoplasmic factors that effect the dynamic activity of the minus and plus ends, respectively. Of particular focus in this proposal will be a novel, recently formulated hypothesis for microtubule turnover which we call the minus-end pathway.
The specific aims targeted for this grant period are designed to test elements of the minus-end pathway hypothesis and to determine the mechanism by which it operates.
The aims are: (1) to determine whether minus end stabilization is dependent on the centrosome in fibroblasts but independent of the centrosome in epithelial cells; (2) to evaluate whether the difference in MT behavior between fibroblasts and epithelial cells is regulated by cell-cell contacts; (3) to determine the status of the MT minus end after its release from the centrosome; (4) to determine the relative contributions of the minus end pathway and plus-end dynamics to the process of MT turnover; (5) to investigate properties of MT treadmilling in vivo and determine the involvement of plus-end factors; and (6) to develop an in vitro MT treadmilling system as an assay for the discovery of minus (and plus) end factors. Our research strategy will employ a combination of kinetic, structural, biochemical, molecular biological and cellular approaches. Novel methods and approaches include the use of centrosome-free cytoplasts as an assay system for microtubule treadmilling; a cell-free system for microtubule release; correlative digital fluorescence imaging and replica electron microscopy; and single microtubule labeling for minus and plus end factors. The results will contribute to an understanding of basic mechanisms of cytoplasmic organization which underlie the maintenance, motility and division of normal as well as malignantly transformed cells.
| 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 |
| Chen, Xinyu; Kojima, Shin-ichiro; Borisy, Gary G et al. (2003) p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions. J Cell Biol 163:547-57 |
| 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 |
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