Study Of The Nucleoporin NPP-3 Function In The Spatial Organization Of Chromosome In C. Elegans Embryo

Nuclear pore complexes(NPCs),composed of multiple copies of 30 different nucleoporins in vertebrates,persist in featuring an eightfold rotational symmetry structure.Most nucleoporins are highly conserved in C.elegans,plant and vertebrates.NPCs can b e divided into six subcomplexes according to their spatial distribution.Cytoplasmic filaments and the nuclear basket form ring structure at the sides of nuclear envelope(NE).NPCs maintain the integrity of NE and facilitate the transportation of molecules including mRNA and molecules by nuclear transport receptor(NTR)between cytosol and nucleus,which is mainly controlled by the peripheral nucleoporins.Up to now,the controlling mechanism of central nucleoporins remain poorly understood.Besides the classical function,NPCs have been reported to participate in modulating chromosome architecture,preserving genome integrity and also regulating DNA replication,gene expression and cell divis ion.In this study,we investigate the roles of NPCs in addition to its physical barrier function.Nucleoporins alteration has been found to be related to various diseases accompanied with their specific function in different tissues.For example,atrial fibrillation(AF),an inherited clinical arrhythmia leading to sudden cardiac death,is caused by NUP155 mutation in atrium.Moreover,NUP133 can regulate the neural lin eages during the embryonic development and the mutation of NUP133 can cause defect in di fferentiation from neural progenitors to terminally differentiated neurons.The molecular mechanisms of nucleoporins alteration in these abnormalities are still unclear.Recently,studies performed in Caenorhabditis elegans providing an advancement method to overcome obstacles in nuclear pore complexes research.Our recent study demonstrated that one of the nucleoporin members,NPP-3,regulated chromosome localization in prophase.We observed that after the depletion of NPP-3,almost all chromosomes gather ed in the nuclear periphery and formed a ring structure in prophase and also the late stage of embryos and oocyte s development.Previous studies reported that nucleoporins depletion could alter cell cycle,spindle rotation and cytoplasmic and nuclear trans port.For example,depletion of NPP-7 and NPP-10 can affect the DNA condensation and the chromosome segregation.Moreover,it has been found that NPP-1,NPP-13 and NPP-4 can regulate the rotation of spindle.However,these phenotypes are different to the N PP-3 depletion phenotype observed in our study.NPP-3 depletion did not affect the DNA condensation and the chromosome segregation but caused the longer time of cell cycle and this defect caused lethality in C.elegans.According to the prospective experi ments,we found NPP-3 depletion affected the localization of some proteins in the nuclear envelope.For example,NPP-3 depletion changes the localization of NPP-2 and NPP-7,which means NPP-3 depletion can disrupt the integrity of nuclear envelope.In addition,NPP-7 depletion also causes all chromosomes gathering in the nuclear periphery and the phenotype is existent in the early and post-mitotic embryos.Our data demonstrated that NPP-7 is downstream of the NPP-3 to regulate the spatial organization of ch romosome.It has been reported that when the nematode embryo is put into hypoxia for 24 hours,all the chromosomes in the nucleus will also be aligned around the nuclear membrane,and the phenotype can be restored after re-oxygenation.At the same time,th e article found that CDK-1 will co-localize with the chromosome at the edge of the nuclear membrane in anoxia;CDK-1 is normally located in the cytoplasm and nucleus at the interphase,and more than in the nucleus.In the early stage,the signal of CDK-1 will be weakened in the nucleus,and NPP-16 depletion can rescue half of the phenotype of chromosomes alignment to the nuclear membrane,but the paper does not study CDK-1 in depth and the mechanism of NPP-16 in the regulation of chromosome distribution,so in my thesis I will also explore whether NPP-3 will also affect the distribution of CDK-1,and whether NPP-16 depletion will rescue the NPP-3 depletion phenotype.Since no relevant paper has reported the mechanism of chromosome aggregation around the nuc lear membrane,we can only explore it by ourselves.First,we start with the NPP-3 depletion phenotype that has been discovered.NPP-3 depletion changes the permeability of the nuclear membrane.Referred to papers,we found that the permeability change caused by NPP-3 depletion is basically the same as the permeability change caused by NPP-4 and NPP-13 depletion,so we depleted NPP-4 and NPP-13.NPP-4 depletion did not affect the distribution of chromosomes,which proves that the change in permeability is n ot the cause of chromosome distribution around the nuclear membrane.NPP-13 depletion can also cause all chromosomes gathering in the nuclear membrane and the phenotype is present in early and late embryos.NPP-3 and NPP-13 belong to the NUP93 complex and can simultaneously regulate the function of the nuclear pore complex,such as the output of certain specific proteins or the size of the output molecules.Therefore,we will start with the structure of NPP-3 and explore whether the depletion of NUP93 compl ex can regulate the spatial distribution of chromosomes in the early stage of cells.Nuclear basket proteins have been shown to interact with chromosomes.For example,the meiotic automatic region of Nup2/NPP-16 can directly bind to specific sequence of ch romosome and regulate the homologous pairing.In this study,we studied whether NPP-3 depletion could change the localization of nuclear basket proteins and regulate chromosome distribution.In addition,chromosome accumulating in the nuclear periphery regulate the level of gene transcription.Therefore,we also investigated the relationship between NPP-3 and nuclear lamina.In order to gain a clearer understanding of the function of NPP-3 in chromosome periphery,we used C.elegans as the research model to investigate the NPP-3/NUP205 role in the chromosome movement and provided the scientific basis for further analyzing the nuclear pore protein function.In addition,there is limited studies concerning the relationship between nucleoporins and chromosomes,in which our study could provide a novel perspective to uncover the functions of nucleoporins.For the spatial distribution of nucleoporins and chromosome,a candidate must be existed to link the nucleoporins and chromosome.Referring to studies working o n the chromosome and nuclear envelope,we speculate that NPP-3 may affect the telomere anchoring and the spindle assembly checkpoint to regulate the chromosome localization in the nuclei.During the meiosis I prophase,telomere at the both sides of chromosome can bind to the KASH/SUN1 protein in the nuclear envelope for homologous pairing,which has been proved in C.elegans and human cells.We speculate whether depletion of NPP-3 can affect the function of SUN-1 and POT-1 to activate this pathway in mitosis.Centromere in the C.elegans is different from the vertebrates.In C.elegans,centromeres align at the both sides of chromosome and anchored by the kinetochore to completer spindle microtubules assembly.If this process is not accurate,the spindle assembly checkpoint will active and make the unattached kinetochore chromosomes move to nuclear periphery for repairing.Therefore,we wonder whether depletion of NPP-3 can overactive the spindle assembly checkpoint to regulate chromosome pe riphery.So we will deplete NPP-3 and spindle checkpoint proteins to observe the chromosome dynamics.Nup153 is reported to regulate the activity of spindle assembly checkpoint.Overall,we conclude that NPP-3 and NPP-7 function in the same pathway to regu late the organization of chromosomes.So we will further explore the relationship between NPP-3,NPP-7 and spindle assembly checkpoint proteins.Our spindle assembly checkpoint results showed that co-depletion of NPP-3 and MAD-1,MDF-2,and BUB-3 can rescue most of the nucleus with chromosomal abnormalities;depletion of NPP-3 and other non-related spindle assembly checkpoint kinesin does not affect the chromosomal distribution,which proves that NPP-3 regulates the spatial distribution of chromosomes by affecting the spindle assembly checkpoint.Our next step is to explore how NPP-3 regulates spindle assembly checkpoints.Our results suggest that depletion of NPP-3 and NPP-7 prolongs the localization of CDK-1 in the nucleus,but since CDK-1 is a cyclin-dependent kinase,its depletion will severely affect cell division.The first cell division embryo could not be found.Most of the embryos showed a phenotype with only one large nucleus in the embryo,so we also need to design other experiments to verify wheth er the duration of CDK-1 is the cause of chromosomes accumulating around the nuclear membrane.In addition,CDK-1 is a protein kinase that phosphorylates some proteins,and the activation of the spindle assembly checkpoint is controlled by the level of phosphorylation of spindle assembly checkpoint proteins.While CDK-1 and PLK-1 mainly regulate nuclear membrane rupture,but there is no report on the interaction between CDK-1 and MDF-1,we will also explore the interaction between CDK-1 and MDF-1,and then improve Our guess model.For the further studies,we can use different RNA inference in transgenic strains and the spinning disk confocal microscope to investigate NPP-3 function in chromosome movement.We will design transgenic strains of GFP::NPP-3 and NPP-3::GFP by Crisper Cas9 to investigate the dynamics of NPP-3 in the cell cycle.Then,we can offer a clearer understanding of chromosome periphery and the meaning of this behavior.This study provides theoretical support for medical practice and basis fo r further application.