|
Marcel JANSON, Isabelle LOIODICE, and Phong TRAN (University of Pennsylvania USA) The microtubule cytoskeleton is essential for cellular processes such as mitosis, organelle transport, and cell polarity. The key organizer of microtubules is the microtubule organizing center (MTOC). All MTOCs are composed of multi-protein complexes and share three general properties: a) They nucleate microtubules. b) They arrange the microtubules into functional patterns. And c) They attach the microtubules to their proper organelle targets. In recent years the molecules that are localized to the centrosome, the primary MTOC in animal cells, have been catalogued and much progress has been made in understanding the mechanism by which the gamma-tubulin ring complex (g-TuRC) located at the centrosome nucleate microtubules. However, most cytoplasmic g-TuRCs are not located at the centrosome, and their cellular functions are unknown. Furthermore, while the canonical centrosome arranges radial arrays of microtubules which are attached to the nucleus, many highly differentiated cell types neurons, myotubes, and polarized epithelial cells have linear arrays of microtubules which are not attached to the nucleus. The mechanisms which generate linear arrays of microtubules are unknown. The fission yeast Schizosaccharomyces pombe is an ideal organism to study linear arrays of microtubules and MTOCs because they are genetically tractable, optically convenient for high spatial-temporal resolution imaging and analysis, and have MTOCs which organize four linear arrays of microtubules. There are three classes of MTOCs in the fission yeast: the centrosome-equivalent spindle pole body (SPB), the equatorial MTOC (eMTOC) which forms during cytokinesis, and multiple interphase MTOCs (iMTOCs). Works from ours and several other labs have identified mto1p/mto2p, ase1p, and klp2p as playing key roles in the organization of the interphase microtubule arrays. Using a combination of in vivo live cell imaging and in vitro motility assays with purified proteins, we are investigating the molecular mechanism of how antiparallel linear arrays of microtubules are formed. We discovered that: 1) mto2p recruits -TuRCs to pre-existing microtubules and activates de novo nucleation of a new microtubule on the pre-existing microtubule, 2) ase1p preferentially bundles the new and old microtubules into an antiparallel array, and 3) this new antiparallel microtubule array is then pulled to the site of the iMTOC by the minus end kinesin klp2p. Our results suggest a model where microtubules, MAPs, and motors interact in a coordinated manner to organize linear and dynamic microtubule structures. |
|