Different functions played by centrosomal protein CEP135 and centriolar satellite proteins SSX2IP and WDR8

Abstract

The centrosome of animal cells is a crucial organelle that regulates cell division, cell migration, ciliogenesis, intracellular transportation and polar determination with microtubule organizing center as its primary function. Misfunction of centrosome associated proteins can lead to ciliopathies, microcephalies and cancers. In recent years, novel proteins which locate within or surrounding the centrosomes have been discovered every year. The detailed roles of these proteins and how they contribute to centrosome functions are, however, often still unclear. Our lab previously discovered two centriolar satellite proteins WDR8 and SSX2IP, that can interact with each other as well as with the centrosomal protein CEP135. The dependency and inter-regulations of these three proteins and how much they can affect cell division and ciliogenesis are not fully understood. <br / > I firstly exploited the large amount of proteins accumulated in eggs to survey the interctome of wdr8 using immunoprecipitation and observed a tight interaction of cep135, wdr8 and ssx2ip. I also show different subpopulations of cep135 by immunoprecipitation in Xenopus with different cep135 antibodies. I further demonstrate that ssx2ipí expression level is slowly increased during oocyte growth and maturation and that cep135 only expresses after egg maturation. Both ssx2ip and cep135ís expression in oocytes and eggs are under the regulation of CPEB. Consistent with previous results on ssx2ip, cep135 depletion also affected bipolar spindle formation in Xenopus egg extract. <br / > Secondly, I checked the functions of SSX2IP, CEP135 and WDR8 in human somatic cells. I generated CRISPR knockouts using human RPE-1 cells. While wdr8 and ssx2ip knockout cells show growth abnormalities, cep135 knockout proliferated normally although none of the knockouts of did show any defects in cell division and ciliogenesis. The protein expression and centrosomal localizations of SSX2IP and WDR8 are dependent on each other and on CEP135. However, Cep135ís centrosomal localization is not affected in wdr8 and ssx2ip knockout cells. Importantly, cep135 knockout cells show premature centrosome splitting. <br / > Thirdly, I further analyzed premature centrosome splitting caused by the loss of CEP135. Premature centrosome splitting in cep135 knockout cells is associated with diminished pericentriolar material as exemplified by reduced levels of gamma tubulin and CDK5RAP2. The imbalanced distribution of CDK5RAP2 between mother and daughter centrioles showed that splitting happens without the completion of centriole to centrosome conversion. The different aster patterns formed in a MT regrowth assay in cep135 knockout cells showed a diminishment and scattering of pericentriolar material. <br / > Lastly, I provide evidence for regulation of satellite proteins and centrosomal proteins by phase separation.