Further supporting this possibility, cell biological studies link NIMA to both cell tip growth and the modulation of interphase microtubule functions. suppressor colonies were isolated and spread on plates and allowed to grow either at permissive or semi-permissive temperatures (35C). The data shows that although are unable to form colonies at this temperature (A) colonies that also carry suppressor mutations are able to GDC-0834 Racemate do so (B and C).(PDF) pgen.1004248.s003.pdf (311K) GUID:?0949F642-A3E6-4B7A-90DD-4FF7884FF381 Figure S4: (A) The cell tip location of NIMA is unchanged in the absence of ESCRT complex function. NIMA-GFP is detectable at 28% of WT cell tips (n?=?117; strain KF005) and a comparable 31% of (n?=?129; strain MGH61) cell tips at 35C. (B) NIMA-GFP levels at the cell tip decrease in mitosis when NIMA displays its characteristic nuclear location. Bar, 5 m.(PDF) pgen.1004248.s004.pdf (37K) GUID:?B4151E74-0B65-47C7-970D-33B80159A43A Figure S5: Colony growth of strains expressing ectopic NIMA constructs. (A) Growth of the indicated strains carrying driven NIMA constructs under conditions when ectopic NIMA is not expressed (lactose) or is expressed (threonine) compared to WT. (B) Growth of a strain carrying cell at 35C. Delay ?=? 0.81 s. Play rate ?=? 30 fps. Length of movie ?=? 7 min.(AVI) pgen.1004248.s011.avi (2.0M) GUID:?9BE1D983-1C19-4245-8A2E-5ADF4903C3EE Table S1: Genotypes of strains used in the study.(PDF) pgen.1004248.s012.pdf (58K) GUID:?F4C7F2EA-D875-4CBC-B83B-D826774863DC Abstract The Never in GDC-0834 Racemate Mitosis A (NIMA) kinase (the founding member of the Nek family of kinases) has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus the results of a synthetic lethal screen performed in using the NIMA ortholog and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Absence of ESCRT pathway functions in combination with partial GDC-0834 Racemate NIMA ICAM1 function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP) which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel nonnuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell cycle progression with polarized cell growth. Author Summary All organisms have to integrate cell growth, and often the polarization of cell growth, with the rate of progression through the cell cycle. One of the most highly polarized modes of growth found in nature is displayed by the ubiquitous filamentous fungi. How the regulation of mitotic divisions is linked to polarized growth remains a mystery, but might involve mitotic regulators. One key mitotic regulator identified in the model filamentous fungus is the NIMA kinase, the founding member of the Nek family of protein kinases. This kinase is known to play mitotic specific roles within nuclei. Our genetic studies reported here reveal unexpected interactions between NIMA and six components of a pathway required for the turnover of cell.
