The Study On The Mechnisms Of KIBRA Regulating Exosome Secretion

Background:Exosomes are nanovesicles of 30-150 nm in diameter that secreted by most cell types.Exosomes could be involved in intercellular communication,allowing exchange of proteins and lipids between the exosome-producing cells and target cells.Exosomes develop from in-budding of early endosomes,which,in turn,forms multivesicular bodies(MVBs)that contain intraluminal vesicles(ILVs).Some MVBs then fuse with the plasma membrane(PM)to release ILVs to extracellular environment as exosomes.Alternatively,some MVBs are delivered to lysosomes where their cargo,such as proteins,is degraded and parts of degraded products are recycled.The molecular mechanisms that directly govern exosome secretion and trafficking have been extensively studied.Recent studies have identified several essential regulators of exosome biogenesis and secretion in diverse cell types.Endosomal sorting complexes required for transport(ESCRTs)proteins(e.g.,HRS and Tsg101),lipids(e.g.,ceramide),and tetraspanins(e.g.,CD81 and CD9)have been demonstrated to regulate exosome secretion by regulating MVB biogenesis.Some Rab GTPases(e.g.,Rab11,Rab27,and Rab35)have also been shown to regulate exosome release,probably by affecting transport or docking of MVBs to the target PM.Furthermore,soluble N-ethylmaleimide-sensitive factor-attachment protein receptor(SNARE)complexes are instrumental in allowing fusion of the lipid bilayers after docking of two different intracellular compartments.KIBRA is predominately expressed in the kidney and in the memory-related brain regions,where it functions as a scaffold protein in various cell processes,such as cell polarity,cell migration,and membrane trafficking.KIBRA knockdown accelerates the exocytosis of the apical protein to the cell surface through inhibition of aPKC kinase activity.In neurons,KIBRA plays a critical role in regulating?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor(AMPAR)trafficking underlying synaptic plasticity and learning.It has been reported that KIBRA regulates the long-range transport of early endosomes through an interaction with dynein light chain 1,which is a component of cytoplasmic dynein.In addition,the KIBRA-aPKC pathway seems to be important in directional migration.However,the potential roles for KIBRA in regulating exosome or extracellular vesicle(EV)secretion have yet to be elucidated.Aims:1.To analyze the effects of KIBRA on exosome secretion.2.To investigate the molecular mechanisms by which KIBRA regulates exosome secretion.Methods:1.Construct KIBRA-downregulated or-overexpressed cell lines.In mammals,KIBRA is predominately expressed in the kidney and the brain.Thus,we performed in vitro experiments using a mouse hippocampal neuronal cell line(HT22)and a mouse podocyte cell line(MPC5).KIBRA was downregulated or overexpressed in these two cell lines,and the results were confirmed by western blot and quantitative real-time-polymerase chain reaction(qRT-PCR)analysis.2.Analyze the effects of KIBRA on exosome secretion in KIBRA-downregulated or-overexpressed cell lines.To analyze the effects of KIBRA on the exosome secretion,KIBRA-knockdown or-overexpressed cells were cultured in DMEM or RPMI-1640 medium containing 10%exosome-depleted fetal bovine serum(FBS)for 48 h,and the EVs were isolated from the conditioned media by sequential differential centrifugation.Briefly,the medium was centrifuged at 300 × g for 10 min to remove cells.The supernatant was centrifuged at 2,000 × g for 10 min,and then at 10,000 × g for 30 min.The resulting supernatant was filtered through a 0.22 ?m filter,and the exosomes were pelleted by ultracentrifugation at 100,000 × g(Beckman Type 90Ti)for 70 min.The exosome pellet was washed in cold PBS and collected by ultracentrifugation again at 100,000 ×g(Beckman Type 90 Ti)for 70 min.Finally,the exosome pellet was resuspended in PBS or lysis buffer before further analysis.73.Isolated and purified exosomes from the extracellular space of the brain and the kidney in mice.To define the role of KIBRA in exosome secretion in a more physiological context,we isolated and purified exosomes from the extracellular space of the brain and the kidney in KIBRA-knockout(KO)mice and their wild-type(WT)counterparts by sucrose density gradient centrifugation.The contents of the sucrose step gradient fractions(a-g)were immunoblotted for proteins known to be either enriched(CD63 and CD9)or absent(Calnexin)in exosomes.Immunoblotting of the sucrose step gradient fractions(a-g)demonstrated that CD63 and CD9 were present mainly in four of the fractions(b-e)but not in the other fractions(a,f,and g).To explore whether KIBRA also regulates exosome secretion in vivo,fractions b-e isolated from the extracellular space of the brain or kidney in KIBRA-KO and-WT mice were blotted for the exosomal markers.4.Investigate the molecular mechanisms that KIBRA regulates exosome secretion.To investigate the molecular mechanisms by which KIBRA regulates exosome secretion,we performed mass spectral(MS)analysis and an isobaric tag for relative and absolute quantitation(iTRAQ)assay to screen the differentially-expressed proteins in the brains of KIBRA-KO mice compared with their WT littermates.The mechanisms of exosome biogenesis and secretion have been extensively studied;thus,we analyzed those proteins reported to be involved in exosome formation or secretion.As shown,Rab27a was the most significantly changed protein with the maximum fold change among all proteins that were statistically significant(P<0.05).To further verify the protein profiles,we compared the protein and mRNA levels of Rab27a in KIBRA-KO and-WT mice by immunofluorescence analysis,western blot and real-time PCR analysis.5.Explore the Rab27a degradation mechanisms.To explore the Rab27a degradation mechanisms,KIBRA-KD and Ctrl-KD cells were treated with the protein synthesis inhibitor(cycloheximide,CHX),the proteasome inhibitor(Lac)or the lysosome inhibitor(Baf).Western blot analysis showed that Rab27a was enormously degraded when treated with CHX for 12 h,and this degradation was restored by the proteasome inhibitor Lac but not by the lysosome inhibitor Baf.Furthermore,IP experiments were performed to analyse the level of ubiquitinated Rab27a.To further confirm the direct interaction between KIBRA and Rab27a,we performed cross IP and immunofluorescent experiments.6.Investigate whether overexpression of Rab27a rescues exosome secretion.To further explore whether KIBRA regulates exosome secretion via Rab27a,KIBRA-KD and Ctrl-KD cells were transfected with DsRed-Rab27a plasmids,and G41 8 was used to select the neomycin-resistant transformants.The cells were cultured in DMEM medium containing 10%exosome-depleted FBS for 48 h,and the exosomes were isolated for further detection.Results:1.KIBRA regulates secretion of exosomes in vitroKnockdown of KIBRA in HT22 cells led to a significant decrease of Alix,Tsg101,CD63,and CD9 in the ultracentrifuged pellets Consistent with these results,the nanoparticle tracking analysis(NTA)of the ultracentrifuged pellets revealed a decrease in the number of particles secreted by KIBRA-KD cells compared with control cells.In contrast,after overexpressing KIBRA in HT22 cells via infection with KIBRA-expressing lentivirus,exosome secretion was remarkably enhanced,as indicated by both measuring the total amount of exosomal proteins and immunoblotting analysis of exosome markers.To further explore whether KIBRA regulated exosome secretion is cell specific,we used CRISPR-Cas9 gene editing system to down-regulate KIBRA in MPC5 cells(referred to as MPC5-KD cells).The results showed that the total amount of exosomal proteins as well as levels of exosomal markers(Alix,CD63,Tsg101,and CD9)decreased significantly in MPC5-KD cells compared with control cells,which was consistent with the results observed in HT22 cells.2.Knockdown of KIBRA increases the size and number of MVBs.To gain further insight into the mechanisms for impaired exosome secretion in KIBRA-KD cells,we performed an electron microscopic analysis to investigate the number and morphology of ILVs and MVBs.Interestingly,although exosome secretion significantly decreased when KIBRA was depleted,the number of MVBs per cell and the number of ILVs per MVB dramatically increased compared with control cells,indicating that absence of KIBRA may result in the abnormal accumulation of ILVs in MVB(Fig.2a-d).Meanwhile,the morphology of ILVs did not show any significant change,suggesting that KIBRA may only affect the exosome secretion rather than its generation(Fig.2a).3.KIBRA regulates exosome secretion in vivo.To explore whether KIBRA also regulates exosome secretion in vivo,fractions b-e isolated from the extracellular space of the brain or kidney in KIBRA-KO and-WT mice were blotted for the exosomal markers CD63 and CD9.Consistent with the in vitro results,western blot analysis showed a decrease of extracellular exosomes(CD63 and CD9)both in the brain and kidney of KIBRA-KO mice compared with their WT counterparts.Accordingly,NTA of the exosome pellets isolated from the same volume of serum showed that the number of exosomes isolated from KIBRA-KO mice was only about 52%of that from the WT mice.These results suggested that KIBRA regulates exosome secretion in vivo.4.Depleting KIBRA promotes degradation of Rab27a.To further verify the protein profiles,we compared the levels of Rab27a in the cortex and hippocampus of KIBRA-KO and-WT mice by immunofluorescence analysis.The results showed that depleting KIBRA resulted in a dramatic decrease of Rab27a expression in the cortex and hippocampus area(Fig.4b).In addition,western blot analysis indicated that Rab27a decreased significantly in the cortex,hippocampus,kidney,muscle,and liver of KIBRA-KO mice compared with those in their WT counterparts.The real-time PCR results showed that the Rab27a mRNA level remained unchanged in the kidney,muscle,and liver of KIBRA-KO mice,or even increased in the cortex and hippocampus,which was probably due to a compensatory mechanism for excessive degradation of Rab27a protein.Thus,it is reasonable to believe that the decrease in the Rab27a protein level in KIBRA-KO mice is due to increased degradation rather than decreased synthesis of Rab27a.5.KIBRA prevents Rab27a from being ubiquitinated.Western blot analysis showed that Rab27a was enormously degraded when treated with CHX for 12 h,and this degradation was restored by the proteasome inhibitor Lac but not by the lysosome inhibitor Baf.Furthermore,IP experiments showed that Rab27a was more easily ubiquitinated when KIBRA was depleted,and the Lac treatment dramatically increased the levels of ubiquitinated Rab27a.These results indicate that Rab27a was degraded mainly through the ubiquitin-proteasome pathway.The cross IP and immunofluorescent results collectively suggest that Rab27a and KIBRA interact directly with each other.Taken together,these data indicate that Rab27a is degraded mainly through the ubiquitin-proteasome pathway rather than the lysosome pathway.Rab27a becomes stabilized through an interaction with KIBRA,which,in turn,regulates exosome secretion.6.Overexpression of Rab27a rescues impaired exosome secretion.The exosomal protein level increased significantly when Rab27a was overexpressed in KIBRA-KD cells.Immunoblotting of exosomal markers(Alix,Hsc70,and Tag101)showed similar results.These data indicate that overexpressing Rab27a rescues impaired exosome secretion in KIBRA-KD cells.Conclusions:1.KIBRA controls exosome secretion both in vitro and in vivo.2.KIBRA plays an essential role stabilizing Rab27a against being degraded which,in turn,regulates the exosome secretion.