Regulatory Mechanism Of The Num1 Protein In Spindle Localization In Saccharomyces Cerevisiae

The budding yeast Saccharomyces cerevisiae reproduce in the form of asymmetric division.Spindle orientation is key to ensure that the fate determinants are distributed accurately to the daughter cells during cell division.It is known that the Kar9 and Dynein pathways regulate the orientation of mitotic spindles cooperatively.Num1 is a membrane protein that plays an important role in the Dynein pathway.It provides an anchoring site at the cell membrane for Dynein,a dynamic protein that is offloaded from the plus end of cytoplasmic microtubule to the cell membrane,thus facilitating Dynein to produce pulling forces for positioning the spindle.Studies have reported the molecular structure and function of Num1.However,its regulatory mechanism is still unclear.This study explored the function of its interaction protein Sik1 and the regulatory mechanism of Num1 protein in spindle positioning via analyzing the phosphorylation of Num1.Num1 is a multi-domain macromolecular protein with a molecular weight of 313 kD.In order to detect the phosphorylation of Num1 protein by SDS-PAGE,Phos-tag was used in electrophoresis and 0.5%agarose was added to the gel,which successfully separated the phosphorylated and non-phosphorylated Num1 in electrophoresis.The main results are as follows:1)The full-length Num1 protein is a phosphorylated protein.Lacking its N-terminus,C-terminus,CC domain and the fragment between CC domain and PH domain affected its phosphorylation to variable extents.2)The phosphorylation of Num1 was examined in cells arrested at G1,S,G2-M,late anaphase and telophase by?-factor,hydroxyrea,nocodazole and the temperature-sensitive mutants cdc5-2,cdc15-2 and cdc14-1,respectively.Among these,cdc15-2 and cdc14-1 arrested cells to similar stages,namely telophase.The results showed that the phosphorylation of Num1 showed dynamic changes during the cell cycle.It was hyper-phosphorylated during G1 to S phase,and became non-phosphorylated during G2 to M phase,and then re-phosphorylated by late anaphase and telophase.3)In order to test whether the protein kinase Cdc5 plays a role in Num1phosphorylation,the cdc5-2 temperature-sensitive mutant was used to detect the effect of inactivating Cdc5 on Num1 phosphorylation.Compared to wild type cells,the level of phosphorylation of Num1-GFP in cdc5-2 cells was lower during 50?150 min after release at37?from a G1 arrest at 25?,indicating that Cdc5 might be the protein kinase responsible for the phosphorylation of Num1 at this stage.Sik1(Suppressors of I?B)is an interacting protein of Num1 screened by our pull down experiment and mass spectral analysis.Although it is a conserved nucleoprotein,it has been reported that overexpression of Sik1 can lead to defects in spindle orientation.The present study examined the role of Sik1 in spindle orientation from the following aspects:Firstly,strains expressing GAL1p-3HA-SIK1 along or expressing both GAL1p-3HA-SIK1 and GAL1p-NUM1-YFP were constructed,and the interaction between Num1 and Sik1 in vivo was confirmed by co-immunoprecipitation experiments.Secondly,the effect of Sik1 overexpression on spindle orientation and nuclear migration was examined.The overexpression of Sik1 resulted in a deviation of spindle alignment along the mother-bud axis prior to M phase.The average angle?between the spindle and the mother-bud axis was 48.0°(48.0±29.7°)in GAL1p-3HA-SIK1 cells,32.4°(32.4±24.5°)in wild-type cells and 41.1°(41.1±24.5°)in num1?cells.The difference was significant between GAL1p-3HA-SIK1 and wild type cells(P<0.001),but not significant between GAL1p-3HA-SIK1 and num1?cells or between num1?and wild type cells(P>0.001).However,examination of nuclei stained by DAPI revealed no binucleated cells in GAL1p-3HA-SIK1 strains,suggesting that,although the over-expression of Sik1resulted in certain degree of defects in spindle positioning,it was not sufficient to cause the formation of binucleated cells.Then,the effects of overexpression of SIK1 on the growth of Kar9 and Dynein pathway mutants were detected.The growth of num1?,GAL1p-3HA-SIK1,num1?GAL1p-3HA-SIK1,kar9?,kar9?GAL1p-3HA-SIK1 and wild type cells were compared at30?,16?and 37?,respectively.It was found that the viability of these strains varied under the stress of low temperature at 16?.Overexpression of SIK1 weakened the viability of kar9?cells,but partially restored the viability of num1?cells,indicating that Sik1 might be involved in the regulation of Dynein/Num1 pathway.Finally,the effect of SIK1 overexpression on the offloading of Dynein from the plus-end of cytoplasmic microtubules to the cell membrane was examined.By comparing the fluorescence intensity of DYN1/DHC-3GFP foci at the plus-end of cytoplasmic microtubules in WT,GAL1p-3HA-SIK1 and num1?cells,it was found that the overexpression of SIK1 led to the accumulation of Dynein at the plus-end of microtubules,similar to what was observed for deleting NUM1.The average fluorescence intensity of DYN1/DHC-3GFP foci at the plus-end of cytoplasmic microtubules in GAL1P-3HA-Si K1cells was 1.9×10~4(A.U.),lower than that in num1?cells(2.6×10~4 A.U.),but significantly high than that in wild-type cells(1.1×10~4 A.U.)(P<0.001),indicating that overexpression of Sik1 interfered with the offloading of Dynein from the plus-end of cytoplasmic microtubules to the cell membrane.In summary,this study confirmed the phosphorylation of full-length Num1 protein in cell cycle,and preliminarily proved that Cdc5 may be a protein kinase involved in the phosphorylation.Meanwhile,several lines of experimental evidence showed that Sik1 was involved in the Dynein pathway to regulate spindle orientation,including:Sik1 protein interacted with Num1 both in vitro and in vivo,and overexpression of Sik1 led to defect in spindle orientation and affected the offloading of Dynein from the plus-end of cytoplasmic microtubules.In addition,cell growth experiments under the stress of low temperature confirmed the genetic interaction between SIK1 and NUM1.Together,these results lay a good foundation for further understanding the molecular mechanism of the Dynein pathway controlling spindle orientation.