Research On The Mechanism Of PRKCA Regulating The Formation Of Type Ⅰ Nuclear Plasmic Network

China is a big producer and consumer of pork.Stress can affect the performance of pigs,leading to disorders of cellular metabolism,suppression of immune responses,and severe even death of pigs.Stress at the cellular level is mainly manifested in DNA damage and programmed cell death due to increased unfolded proteins.The nucleus is the center of genetic control that regulates the life processes of cells.The nuclear membrane(NE)is a unique membrane structure that physically distinguishes the nucleus from the cytoplasm,which helps maintain the integrity of the nuclear structure.As an extension of the nuclear membrane,the nucleoplasmic reticulum plays a very important role in regulating the entry and exit of substances into and out of the nucleus.The nucleoplasmic reticulum is closely related to intranuclear lipid droplets,which may participate in the regulation of cellular lipid metabolism,regulate the synthesis of neutral lipids and phospholipids in cells,and improve the adaptability of cells.Intranuclear lipid droplets(n LDs),as organelles in the nucleus that store and balance energy metabolism homeostasis,have a unique structure,generally neutral lipids such as mainly triglycerides(TG)and cholesterol esters(CE)composed of a hydrophobic core,which is wrapped by a phospholipid monolayer,which has a specific set of protein modifications on the phospholipid monolayer.By changing the number and morphology of type Ⅰ nucleoplasmic reticulum to regulate the formation of n LD,cells can alleviate nuclear lipid stress,thereby reducing the damage caused by stress to cells.However,the cause and regulatory mechanism of the formation of the type Ⅰ nucleoplasmic network itself are unknown.The mechanism of type Ⅰ nucleoplasmic reticulum formation remains to be further elucidated.The formation of type Ⅰ nuclear plasmic reticulum and protein transfer between type Ⅰ nuclear reticulum and intranuclear lipid droplets were revealed by methods such as choline deficiency,immunoprecipitation,immunoblotting detection,signaling pathway analysis,transcriptome analysis,nuclear lipid analysis,laser confocal detection,etc.The main findings are as follows:1.Choline deficiency regulates the formation of type Ⅰ nucleoplasmic reticulum by changing nuclear morphology.(1)Choline deficiency leads to an increase in the number of type Ⅰ nucleoplasmic reticulum and intranuclear lipid droplets in cells.Using BODIPY493/503 to label lipid droplets,NUP98 and Lamin B1 co-labeled type Ⅰ nucleoplasmic reticulum,fluorescence microscopy showed that the number of type Ⅰ nucleoplasmic reticulum and intranuclear lipid droplets in the choline deficiency treatment group increased significantly.Transmission electron microscopy showed significant deformation of the nuclear membrane in the choline-deficient treatment group.(2)Lipid analysis of the nucleus showed that choline deficiency would induce changes in the phospholipid composition of the nuclear membrane,and the content of phosphatidyl ethanolamines(PE)and phosphatidic acid(PA)increased.(3)Choline deficiency leads to changes in lamin LaminA/C-S392 phosphorylation levels.LaminA/C binds to PRKCA to regulate the phosphorylation level of LaminA/C.Co-immunoprecipitation assays showed that LaminA/C binds to PRKCA and increases the degree of binding after choline deficiency treatment.(4)Rab8a participates in the extension of the type Ⅰ nuclear plasmic reticulum,and live cell observation shows that during the formation of the type Ⅰ nuclear plasmic reticulum,Rab8 a will gather at the end of the type Ⅰ nuclear reticulum and initiate the extension of the type Ⅰ nuclear reticulum.2.Transcriptome analysis shows that PRKCA regulates type Ⅰ nuclear reticulum formation through the RhoA pathway after choline deficiency.(1)Transcriptome analysis found that choline lacked gene enrichment in cell cycle,DNA,and RNA repair pathways.Detecting the cell cycle reveals that choline deficiency causes cell cycle arrest in the S phase.(2)The addition of MAZ51(cell proliferation inhibitor)and CCG-1423(Rho pathway inhibitor)respectively inhibited cell proliferation,and it was found that simple inhibition of cell proliferation did not lead to the formation of type Ⅰ nuclear reticulation,while inhibition of Rho pathway increased the number of type Ⅰ nuclear plasmic reticulum.(3)Detection of related proteins found that choline deficiency treatment led to downregulation of cell RHOA and CDK1;The addition of CCG-1423 detected the protein,and the results showed that RHOA and CDK1 expression were down-regulated.Co-immunoprecipitation assays showed that PRKCA binds to RHOA and increases in degree of binding after choline deficiency treatment.3.Type I nucleoplasmic reticulum promotes the formation of intranuclear lipid droplets.Fluorescence bleaching assays found that Rab8 a has fluidity on type Ⅰ nuclear plasmic reticulations.After the formation of type Ⅰ nuclear plasmic reticulum,the lipid droplet formation related protein SEIPIN will be displaced to the type Ⅰ nuclear reticulum to promote the formation of intranuclear lipid droplets.