HSPA8 Is Identified as a Novel Regulator of Hypertensive Disorders in Pregnancy by Modulating the ß-Arrestin1/A1AR Axis.

Heat shock protein alpha 8 (HSPA8) was found to be downregulated in the placentas of patients with hypertensive disorders in pregnancy (HDP). We aim to explore the underlying role and mechanism of HSPA8 in HDP progression. Herein, HSPA8 mRNA expression in placentas and peripheral blood of patients with HDP and normal pregnant controls was measured with RT-qPCR. We found that HSPA8 expression was downregulated in placentas and peripheral blood of patients with HDP. HTR8/SVneo human trophoblast cells were transfected with pcDNA-HSPA8 or si-HSPA8. HSPA8 overexpression promoted cell proliferation, migration, and MMP-2 and MMP-9 protein levels, and inhibited apoptosis, while HSPA8 silencing showed the opposite results. Co-immunoprecipitation assay validated the binding between HSPA8 and ß-arrestin1, as well as ß-arrestin1 and A1AR proteins. HSPA8 bound with ß-arrestin1 protein and promoted ß-arrestin1 expression. ß-arrestin1 bound with A1AR protein and inhibited A1AR expression. Then, HTR8/SVneo cells were transfected with pcDNA-HSPA8 alone or together with si-ß-arrestin1, as well as transfected with pcDNA-ß-arrestin1 alone or together with pcDNA-A1AR. ß-arrestin1 silencing reversed the effects of HSPA8 overexpression on HTR8/SVneo cell functions. ß-arrestin1 overexpression promoted cell proliferation migration, and MMP-2 and MMP-9 protein levels, and inhibited apoptosis, while these effects were reversed by A1AR overexpression. Lentivirus HSPA8 overexpression vector (Lv-HSPA8) was injected into a preeclampsia (PE) rat model, which attenuated blood pressure and fetal detrimental changes in PE rats. In conclusion, HSPA8 promoted proliferation and migration and inhibited apoptosis in trophoblast cells, and attenuated the symptoms of PE rats by modulating the ß-arrestin1/A1AR axis. Our study provided a novel theoretical evidence and potential strategy for HDP treatment.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

AtHsc70-1 negatively regulates the basal heat tolerance in Arabidopsis thaliana through affecting the activity of HsfAs and Hsp101.

Heat shock protein 70 (Hsp70) chaperones are highly conserved and essential proteins with diverse cellular functions, including plant abiotic stress tolerance. Hsp70 proteins have been linked with basal heat tolerance in plants. Hsp101 likewise is an important chaperone protein that plays a critical role in heat tolerance in plants. We observed that Arabidopsis hsc70-1 mutant seedlings show elevated basal heat tolerance compared with wild-type. Over-expression of Hsc70-1 resulted in increased heat sensitivity. Hsp101 transcript and protein levels were increased during non-heat stress (HS) and post-HS conditions in hsc70-1 mutant seedlings. In contrast, Hsp101 was repressed in Hsc70-1 over-expressing plants after post-HS conditions. Hsc70-1 showed physical interaction with HsfA1d and HsfA1e protein in the cytosol under non-HS conditions. In transient reporter gene analysis, HsfA1d, HsfA1e and HsfA2 showed transcriptional response on the Hsp101 promoter. HsfA1d and HsfA2 transcripts were at higher levels in hsc70-1 mutant compared with wild-type. We provide genetic evidence that Hsc70-1 is a negative regulator affecting HsfA1d/A1e/A2 activators, which in turn regulate Hsp101 expression and basal thermotolerance.

HSPA8/HSC70 in Immune Disorders: A Molecular Rheostat that Adjusts Chaperone-Mediated Autophagy Substrates.

HSPA8/HSC70 is a molecular chaperone involved in a wide variety of cellular processes. It plays a crucial role in protein quality control, ensuring the correct folding and re-folding of selected proteins, and controlling the elimination of abnormally-folded conformers and of proteins daily produced in excess in our cells. HSPA8 is a crucial molecular regulator of chaperone-mediated autophagy, as a detector of substrates that will be processed by this specialized autophagy pathway. In this review, we shortly summarize its structure and overall functions, dissect its implication in immune disorders, and list the known pharmacological tools that modulate its functions. We also exemplify the interest of targeting HSPA8 to regulate pathological immune dysfunctions.

Hsc70 Interacts with ß4GalT5 to Regulate the Growth of Gliomas.

Heat shock cognate protein 70 (Hsc70) is a key mediator for the maintenance of intracellular proteins and regulates cellular activities. And it is elevated in various tumor tissues including glioma, which is closely related to the malignancy and poor prognosis of the tumors. However, the effects of Hsc70 on gliomas and its regulatory mechanism have not yet been elucidated. In the present study, we found that Hsc70 was overexpressed in glioma tissues and cultured glioma cells. Furthermore, Hsc70 expression exhibited positive correlation with the grades of gliomas. Knockdown of Hsc70 could effectively inhibit cell proliferation and increase cell apoptosis. Furthermore, we identified that ß4GalT5 was a critical target for Hsc70-mediated anti-glioma effects. Blocking ß4GalT5 activity could effectively reverse the anti-tumor effect of Hsc70. Taken together, these data indicate that Hsc70 regulates ß4GalT5 levels, and possibly plays a role in cell proliferation and apoptosis of glioma.

Functional analysis of HSPA1A and HSPA8 in parturition.

Many factors are involved in parturition, such as apoptosis, inflammatory mediators, and hormones. Previous studies indicated that HSP70 directly or indirectly regulates apoptosis, inflammatory immune response and hormone stimulus. To gain new insights into molecular mechanism underlying HSP70 for regulating parturition, we overexpressed and knocked down two representative members of HSP70 (HSPA1A and HSPA8) through transfection of their recombinant plasmid and si-RNA separately in WISH (human amniotic epithelial) cells. The expression changes of several pathways' marker genes were investigated by Western blotting and quantitative real-time PCR (qRT-PCR). Results showed extreme expression changes in the genes of IL-8 and ESR2. HSP70 was found to stimulate estrogen response by regulating ESR2 through ERK1/2 after treating WISH cells with the special phosphorylation inhibitor of ERK1/2 and analyzing the changes of E2 concentration by ELISA. HSP70 was also observed to contribute to preterm birth after administering the special inhibitor of HSP70-PFT-µ with LPS-induced preterm birth mouse model. Overall, HSP70 induces parturition through stimulating immune inflammatory and estrogen response. The balanced HSP70 expression could ensure a smooth parturition, while the imbalanced expression may cause a pathological state like preterm.

Hsc70 facilitates TGF-ß-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-ß-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-ß signaling, was also suppressed in the early phase of TGF-ß stimulation. Upon stimulation with TGF-ß, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-ß-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-ß-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-ß-induced Smad signaling in NRK-49F cells.

Heat shock protein A8 stabilizes the bull sperm plasma membrane during cryopreservation: Effects of breed, protein concentration, and mode of use.

Heat shock protein A8 (HSPA8) is a highly conserved member of the Hsp70 family, which is expressed in oviductal cells, translocated into oviductal fluid, and becomes attached to the sperm surface during sperm transport. Previous research has shown that HSPA8 supports mammalian sperm viability during in vitro incubation at both 5 °C and body temperature. The present series of experiments was designed to explore the possibility that bovine recombinant HSPA8 might therefore protect bull spermatozoa during cryopreservation through its beneficial effects on the sperm plasma membrane. Soy-based cryopreservation media were used in these experiments. The effects of HSPA8 addition before freezing were examined at concentrations ranging from 0.2 to 6.4 µg/mL, whereas the effects of postthaw HSPA8 addition were tested between 0.2 and 12.8 µg/mL. When bull spermatozoa (from beef and dairy breeds) were frozen in the presence of HSPA8, beneficial but complex effects on postthaw viability were observed. Low HSPA8 concentrations (0.2 and 0.4 µg/mL) resulted in significantly reduced postthaw sperm viability, but concentrations above 0.8 µg/mL improved plasma membrane integrity. If HSPA8 was added to spermatozoa after thawing, outcomes were also biphasic and beneficial effects on viability were only seen if the HSPA8 concentration exceeded 3.2 µg/mL. Beneficial effects were significantly more apparent with beef rather than dairy breeds. When HSPA8 was used in combination with cholesterol-loaded cyclodextrin, spermatozoa from the beef breeds showed significantly lower apoptotic effects. This was not observed with the dairy breeds.

LAP1 is a crucial protein for the maintenance of the nuclear envelope structure and cell cycle progression.

Cell division in eukaryotes requires the disassembly of the nuclear envelope (NE) at the beginning of mitosis and its reassembly at the end of mitosis. These processes are complex and involve coordinated steps where NE proteins have a crucial role. Lamina-associated polypeptide 1 (LAP1) is an inner nuclear membrane protein that has been associated with cell cycle events. In support of this role, LAP1 has been implicated in the regulation of the NE reassembly and assembly of the mitotic spindle during mitosis. In this study, we demonstrated that LAP1 intracellular levels vary during the cell cycle in SH-SY5Y cells, and that LAP1 is highly phosphorylated during mitosis. It is also clear that LAP1 co-localized with acetylated α-tubulin in the mitotic spindle and with γ-tubulin in centrosomes (main microtubule organizing center) in mitotic cells. Moreover, LAP1 knockdown resulted in decreased number of mitotic cells and decreased levels of acetylated α-tubulin (marker of microtubules stability) and lamin B1. Additionally, it was possible to determine that LAP1 is important for centrosome positioning near the NE. These findings place LAP1 at a key position to participate in the maintenance of the NE structure and progression of the cell cycle.

Crosstalk Between Macroautophagy and Chaperone-Mediated Autophagy: Implications for the Treatment of Neurological Diseases.

Macroautophagy and chaperone-mediated autophagy (CMA) are two important subtypes of autophagy that play a critical role in cellular quality control under physiological and pathological conditions. Despite the marked differences between these two autophagic pathways, macroautophagy and CMA are intimately connected with each other during the autophagy-lysosomal degradation process, in particular, in the setting of neurological illness. Macroautophagy serves as a backup mechanism to removal of malfunctioning proteins (i.e., aberrant α-synuclein) from the cytoplasm when CMA is compromised, and vice versa. The molecular mechanisms underlying the conversation between macroautophagy and CMA are being clarified. Herein, we survey current overviews concentrating on the complex interactions between macroautophagy and CMA, and present therapeutic potentials through utilization and manipulation of macroautophagy-CMA crosstalk in the treatment of neurological diseases.

Regulation of FSP27 protein stability by AMPK and HSC70.

Fat-specific protein 27 (FSP27) plays a pivotal role in controlling the formation of large lipid droplet and energy metabolism. The cellular levels of FSP27 are tightly regulated through the proteasomal ubiquitin-mediated degradation. However, the upstream signals that trigger FSP27 degradation and the underlying mechanism(s) have yet to be identified. Here we show that AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide) or phenformin induced the ubiquitination of FSP27 and promoted its degradation in 3T3-L1 adipocytes. The levels of FSP27 protein could be maintained by either knocking down AMPKα1 or blocking proteasomal pathway. Moreover, AICAR treatment induced multilocularization of LDs in 3T3-L1 adipocytes, reminiscent of the morphological changes in cells depleted of FSP27. Furthermore, mass spectrometry-based proteomic analysis identified heat shock cognate 70 (HSC70) as a novel binding protein of FSP27. The specific interaction was confirmed by co-immunoprecipitation of both ectopically expressed and endogenous proteins. Importantly, knockdown of HSC70 by small interference RNA resulted in increased half-life of FSP27 in cells treated with a protein synthesis inhibitor cycloheximide (CHX) or AICAR. However, silencing of the E3 ubiquitin ligase CHIP (COOH terminus of HSC70-interacting protein) failed to alter the stability of FSP27 protein under both conditions. Taken together, our data indicate that AMPK is a negative regulator of FSP27 stability through the proteasomal ubiquitin-dependent protein catabolic process. Promotion of FSP27 degradation may be an important factor responsible for the beneficial effect of AMPK activators on energy metabolism.

The critical roles of HSC70 in physiological and pathological processes.

The heat stress cognate 70 is one of the major cytoplasmic chaperones to supply a multitude of the housekeeping chaperoning functions. In addition to its high constitutive expression, recent studies have demonstrated that it is also inducible. Another exciting discovery is that the regulation of heat stress cognate 70 plays important roles in the aging process and aging-related diseases. Besides the chaperone functions, heat stress cognate 70 is involved in the inflammatory signal pathways via extracellular interaction with TLR2/TLR4. Furthermore, studies have validated the ability of extracellular heat stress cognate 70 to regulate cancer cell proliferation and sperm storage. The discovery of heat stress cognate 70 secretion, in normal and cancer cells undergoing stress, presents novel therapeutic strategies.

Decreased expression of Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) correlated with prognosis of Hepatocellular carcinoma.

Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is an ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70 to recruit Hsc70 into complexes of diverse function. SGTB was identified as an isoform of SGT with 60% amino acid sequence homology. To investigate the expression of SGTB in hepatocellular carcinoma (HCC) and determine its correlation with tumor progression and prognosis, we evaluated the expression levels of SGTB in HCCs and corresponding adjacent non-tumor liver tissues. We also assessed the association between their expression and clinicopathologic parameters. The expression of SGTB was absent or low in HCCs while it was notable in paracancerous tissues from 108 patients by western blotting and immunochemistry (P < 0.05). Among the 108 HCCs, low expression of SGTB was associated with gender, histological grade (P<0.001) and HBsAg expression (P=0.002). Univariate analysis showed that the low SGTB expression was associated with poor prognosis (P<0.001). Thus, decreased expression of SGTB may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.

HSPA8/HSC70 chaperone protein: structure, function, and chemical targeting.

HSPA8/HSC70 protein is a fascinating chaperone protein. It represents a constitutively expressed, cognate protein of the HSP70 family, which is central in many cellular processes. In particular, its regulatory role in autophagy is decisive. We focused this review on HSC70 structure-function considerations and based on this, we put a particular emphasis on HSC70 targeting by small molecules and peptides in order to develop intervention strategies that deviate some of HSC70 properties for therapeutic purposes. Generating active biomolecules regulating autophagy via its effect on HSC70 can effectively be designed only if we understand the fine relationships between HSC70 structure and functions.

Structural insight into plant programmed cell death mediated by BAG proteins in Arabidopsis thaliana.

The recently identified plant Bcl-2-associated athanogene (BAG) family plays an extensive role in plant programmed cell death (PCD) processes ranging from growth and development to stress responses and even cell death. In the Arabidopsis thaliana BAG (AtBAG) protein family, four members (AtBAG1-4) have a domain organization similar to that of mammalian BAG proteins. Here, crystal structures of the BAG domains (BDs) of AtBAG1-4 have been determined; they have high homology and adopt a structure comprising three short parallel α-helices, similar to some mammalian BAG proteins. The crystal structure of a complex of the AtBAG1 ubiquitin-like domain and BAG domain (UBD) with the Hsc70 nucleotide-binding domain (NBD) was also determined. The binding of the AtBAG1 BD to the Hsc70 NBD induces conformational change of the Hsc70 NBD to the open state and reduces the affinity of the NBD for ADP. In vivo studies showed that bag2-1 mutant plants are larger than wild-type plants when growing under normal conditions, indicating that the AtBAG proteins might regulate plant PCD and confer tolerance to stresses in plants. These structural and functional analyses indicate that the AtBAG proteins function as nucleotide-exchange factors for Hsp70/Hsc70 in A. thaliana and that the mechanism of regulation of chaperone-mediated protein folding is conserved in plants.

Implication of Hsc70, PDI and integrin αvß3 involvement during entry of the murine rotavirus ECwt into small-intestinal villi of suckling mice.

In the present study, a homologous rotavirus, ECwt, infecting small intestinal villi isolated from ICR and BALB/c mice were used as a model for identifying cell-surface molecules involved in rotavirus entry. Small-intestinal villi were treated with anti-Hsc70, anti-PDI, anti-integrin ß3 or anti-ERp57 antibodies or their corresponding F(ab')2 fragments before inoculation with rotavirus ECwt, RRV or Wa. Pretreatment of villi decreased virus infectivity by about 50-100 % depending of the rotavirus strain, antibody structure and detection assay used. Similar results were obtained by treating viral inocula with purified proteins Hsc70, PDI or integrin ß3 before inoculation of untreated villi. Rotavirus infection of villi proved to be sensitive to membrane-impermeant thiol/disulfide inhibitors such as DTNB and bacitracin, suggesting the involvement of a redox reaction in infection. The present results suggest that PDI, Hsc70 and integrin ß3 are used by both homologous and heterologous rotaviruses during infection of isolated mouse villi.

Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry.

Dengue virus (DENV) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. It is transmitted to humans by Aedes mosquitoes. The first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. There are several molecules that participate in DENV infection of mosquitoes, but only a few have been identified. In this work, we co-purified 4 proteins from C6/36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins (HSP70/HSc70), Binding immunoglobulin protein (BiP), Thioredoxin/protein disulphide isomerase (PDI), and 44 kDa Endoplasmic reticulum resident protein (ERp44) via matrix-assisted laser desorption/ionisation time of flight (Maldi-ToF) analysis. Using immunofluorescence and flow cytometry assays, we observed re-localisation of HSP70/HSc70 and, to a lesser extent, BiP to the plasma membrane under stress conditions, such as during DENV infection. By performing binding and infection assays independently, we found that all 4 proteins participate in both processes, but to differing extents: HSP70/HSc70 is the most critical component, while ERp44 is less important. Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding, which suggests that DENV entry to C6/36 cells is mediated by these proteins at the same step and not sequentially.

Comprehensive review on the HSC70 functions, interactions with related molecules and involvement in clinical diseases and therapeutic potential.

Heat shock cognate protein 70 (HSC70) is a constitutively expressed molecular chaperone which belongs to the heat shock protein 70 (HSP70) family. HSC70 shares some of the structural and functional similarity with HSP70. HSC70 also has different properties compared with HSP70 and other heat shock family members. HSC70 performs its full functions by the cooperation of co-chaperones. It interacts with many other molecules as well and regulates various cellular functions. It is also involved in various diseases and may become a biomarker for diagnosis and potential therapeutic targets for design, discovery, and development of novel drugs to treat various diseases. In this article, we provide a comprehensive review on HSC70 from the literatures including the basic general information such as classification, structure and cellular location, genetics and function, as well as its protein association and interaction with other proteins. In addition, we also discussed the relationship of HSC70 and related clinical diseases such as cancer, cardiovascular, neurological, hepatic and many other diseases and possible therapeutic potential and highlight the progress and prospects of research in this field. Understanding the functions of HSC70 and its interaction with other molecules will help us to reveal other novel properties of this protein. Scientists may be able to utilize this protein as a biomarker and therapeutic target to make significant advancement in scientific research and clinical setting in the future.

Loss of nuclear pro-IL-16 facilitates cell cycle progression in human cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of non-Hodgkin lymphomas that affect the skin. The pathogenesis of these conditions is poorly understood. For example, the signaling mechanisms contributing to the dysregulated growth of the neoplastic T cells are not well defined. Here, we demonstrate that loss of nuclear localization of pro-IL-16 facilitates CTCL cell proliferation by causing a decrease in expression of the cyclin dependent-kinase inhibitor p27Kip1. The decrease in p27Kip1 expression was directly attributable to an increase in expression of S-phase kinase-associated protein 2 (Skp2). Regulation of Skp2 is in part attributed to the nuclear presence of the scaffold protein pro-IL-16. T cells isolated from 11 patients with advanced CTCL, but not those from healthy controls or patients with T cell acute lymphocytic leukemia (T-ALL), demonstrated reduction in nuclear pro-IL-16 levels. Sequence analysis identified the presence of mutations in the 5' end of the PDZ1 region of pro-IL-16, a domain required for association of pro-IL-16 with the nuclear chaperone HSC70 (also known as HSPA8). HSC70 knockdown led to loss of nuclear translocation by pro-IL-16 and subsequent increases in Skp2 levels and decreases in p27Kip1 levels, which ultimately enhanced T cell proliferation. Thus, our data indicate that advanced CTCL cell growth is facilitated, at least in part, by mutations in the scaffold protein pro-IL-16, which directly regulates Skp2 synthesis.

Heat shock cognate protein 70 regulates gephyrin clustering.

Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.