Molecular Mechanisms Of The Regulation Of Non-centrosomal Microtubule Nucleation

Microtubules are one of the main cytoskeletons,and play critical roles in many fundamental cellular processes,including material transport,cell migration,and cell division.Microtubules are cylindrical polymers composed of 13 protofilament that made of αβ-tubulin heterodimers.The inner and outer diameters of microtubules are 15nm and 24nm,respectively.Microtubule nucleation is the first step of microtubule array formation.The core component that mediates microtubule nucleation is the γtubulin complex(γ-TuC),including γ-tubulin small complex(γ-TuSC),which exists in lower eukaryotic organisms,and γ-tubulin ring complex(γ-TuRC),which exists in higher eukaryotic organisms.γ-TuC is composed of different GCPs(γ-tubulin complex proteins)and γ-tubulin.γ-TuC displays as a cone-like structure serving as a template for recruiting αβ-tubulin heterodimers to promote microtubule nucleation and assembly.Different regulatory proteins,such as CDK5RAP2 in human,Mtol in fission yeast,and centrosomin in fly,activate microtubule nucleation by associating with γ-TuRC.Specific subcellular sites where microtubule nucleation takes place are termed MTOC(Microtubule Organizing Center).In general,two types of MTOCs are present:centrosomal MTOC(cMTOC)and non-centrosomal MTOC(ncMTOC).The centrosome is cMTOC while multiple subcellular sites,including the nuclear envelope,the microtubule lattice,the Golgi apparatus,chromatin,kinetochores and the cell cortex could function as ncMTOCs.The mechanisms underlying centrosomal microtubule nucleation have been well established.However,the mechanisms underlying noncentrosomal microtubule nucleation has remained elusive.Therefore,we focused on non-centrosomal MTOC subcellular sites and dissected the molecular mechanisms regulating non-centrosomal microtubule nucleation.In the first part of this study,we studied the mechanisms of microtubule regrowth from the nuclear envelope.By live-cell microscopy,we found that,the nuclear evelope is main site initiating microtubule regrowth.We further found that Alp7,but not Alp 14,is required for the proper localization of Mto1 to the nuclear envelope and that Alp7 interacts with Mto1.Alp7,but not Alp 14,is required for microtubule regrowth from the nuclear envelope.Furthermore,we found that,Alp7 is required for the localization of Alp14 to the nuclear envelope,but not vice versus.Tethering Mtol to the nuclear envelope restores microtubule number.Finally,we demonstrated that the localization of Alp7 and Alp 14 to the nuclear envelope depends on Mto1.In the second part of this study,we demonstrated an inhibitory mechanism underlying microtubule-dependent microtubule assembly.First,we identified Klp2 as an interacting protein of Rspl,and both proteins colocalize on interphase microtubules.Next,we showed that Rspl depends on Klp2 to localize to the microtubule lattice,but not vice versus.Furthermore,we found that loss of Klp2 significantly increases the length of Ase1 foci and Ase1 foci number per microtubule or per cell.In addition,the absence of Klp2 increases non-centrosomal microtubule nucleation frequency.Although loss of Klp2 does not affect Mto1,the colocalization of Rspl and Mtol on the microtubule lattice is impaired.Finally,we showed that loss of Klp2 enhances colocalization of Mtol and Asel,and non-centrosomal microtubule nucleation.Taken together,this present study elucidates the molecular mechanisms of microtubule regrowth from the nuclear envelope and the microtubule-dependent microtubule assembly.Therefore,this study provides new insights into understanding of the molecular mechanisms of non-centrosomal microtubule nucleation.