Hemidesmosomes Regulate SHSP-mediated Protection Against Heat Stress In The C.elegans Epidermis

Small heat shock proteins(sHSP)belong to the alpha-crystallin family and act as the first line of defense against environmental heat stress in organisms.sHSP has a structurally conserved ACD domain whose main function is chaperone activity,which refolds the substrate protein by binding to misfolded proteins.Previous studies have found that some sHSPs are localized in the cell adhesion structure,but the association between the sHSP and the cell adhesion structure is currently limited to the effect of sHSP on the dynamic assembly of attachment structure.Hemidesmosomes is an important attachment complex that links epidermal cells to the extracellular matrix.The composition and structure of hemidesmosomes in the epidermis of C.elegans is similar to mammals,and are therefore ideal models for studying the hemidesmosome function in regulating the function of small heat shock proteins.In this thesis,we used C.elegans hemidesmosomes(CeHD)as a research model to further explore the mechanism of CeHDs in protection against heat stress.By constructing hsp-43 transgenic strains,we found the colocalization of HSP-43 with CeHDs.Then we further studied the spatial and developmental expression pattern of HSP-43,and found that HSP-43 was mainly located in the basal CeHDs,and the CeHD pattern of HSP-43 was strictly regulated by development.To clarify the function of HSP-43 in heat shock condition,we found that HSP-43 deficiency significantly decreased survival rates of worms after heat shock treatment.When overexpressing HSP-43 in hsp-43(ok647)mutant with the epidermal specific promoter of dpy-7,the ability of hsp-43(ok647)to resist heat stress was well-rescued,suggesting that HSP-43 exerted an important role in heat shock protection in epidermal cells.After determining the role of HSP-43 in heat shock protection,we examined the localization of HSP-43 under heat shock conditions,and found that HSP-43 could detach from the hemidesmosome into the cytosol following heat shock treatment.Interestingly,loss of HSP-43 did not affect the stability of the CeHDs during heat shock,indicating that HSP-43 is likely to function downstream of hemidesmosomes rather than upstream.We next looked for the specific CeHD components interacting with HSP-43.After the non-critical binding proteins and major structural proteins of CeHDs were disrupted by mutant and RNAi methods,we found that the pattern of HSP-43 were severely affected by the basal membrane receptor LET-805,indicating that HSP-43 might bind with LET-805.To investigate the molecular mechanism of regulation of HSP-43 by CeHDs,we firstly used site-directed mutagenesis to find the protein doamains where HSP-43 bind to the CeHD.We discovered that HSP-43 had multiple sites physically interacting with CeHD proteins,and truncation of the N-or C-terminal would detach HSP-43 from hemidesmosomes and result in obvious aggregation in the cytoplasm of epidermal cells.We examined the thermotolerance of the transgenic strains harbouring the deleted versions of the N-terminal and N/C-terminal HSP-43.As expected,the HSP-43 truncated proteins,which were not completely located on the CeHD could not effectively rescue the thermotolerance caused by hsp-43(ok647)mutation,suggesting that the CeHD localization of HSP-43 was essential for its heat shock protection.In order to better answer why HSP-43 is located on the CeHDs,we proposed two hypotheses:(1)in normal unchallenged conditions,too many unbound sHSPs in the epidermis could be harmful to the cells and may interfere with the normal organismal function,(2)In the non-stressed state,the unbound sHSPs in the epidermal cells cannot maintain its stability and could be easily degraded by proteasomes.Experimental results,showed that overexpressing truncated HSP-43 which partially localized in the cytosol hinders normal development and growth of worms.In addition,the protein level of HSP-16.48 that was not located in the CeHD was lower than that of HSP-43 driven by the same promoter,indicating that both hypotheses are possible.In summary,we believe that the expression level of most sHSPs is very low under normal circumstances;high levels of expression will be induced only after heat shock.However,CeHD,a stable cell adhesion structure,serves as a chaperone depot to store a large amount of HSP-43 to prevent them from being released into the cytoplasm and affecting the normal function of the cells.When facing high temperature stimuli,CeHD will quickly release HSP-43 to associate with misfolded proteins,thus achieving the rapid thermotolerance of worms to heat stress.Compared to the transcriptional control of most sHSPs,this approach may be a faster protection strategy for cells to cope with sudden heat stress.Our previous studies have found that the CeHDs is a signaling center participating in biological processes much more than cell adhesion,so our findings will help people understand more about the new functions of CeHDs and the modulatory mechanisms for sHSPs.