Jerome Gilleron

Connexin a check-point component of cell apoptosis in normal and physiopathological conditions

Gap junction protein connexins (Cxs) play essential roles in cell homeostasis, growth, differentiation and death. Therefore, Cx dysfunction has been associated with many diseases and with tumor development. Cxs control cell apoptosis through different molecular mechanisms. First, gap junction channels classically facilitate the influx and flux of apoptotic signals between adjacent cells and hemichannels between the intracellular and extracellular environments. Second, recent studies demonstrate that Cx proteins, independently from their functional role through channels or hemichannels and in conjunction with their intracytoplasmic localization, may act as signaling effectors able to activate the canonical mitochondrial apoptotic pathway. In the present review, we dissected both functions of Cx in apoptosis, providing new avenues for apoptosis-mediated cancer therapy.

Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1–2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

Integration of Chemical and RNAi Multiparametric Profiles Identifies Triggers of Intracellular Mycobacterial Killing

Pharmacological modulators of host-microbial interactions can in principle be identified using high-content screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.

Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model.

Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durands validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12days. Exposure to low concentrations of chromium (10μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood-testis barrier dynamic is postulated.

SnapShot: Mammalian Rab Proteins in Endocytic Trafficking

RAB4A,B,C (CCV, EE, RE)Endocytic recycling ZFYVE20- VPS45, RABEP1, RUFY1, GRIPAP1, AKAP10, RAB11FIP1, CD2APRAB5A,B,C EE fusion, EE biogenesis EEA1, ZFYVE20- VPS45, APPL1/2, ANKFY1, RABEP1, RABEP2, PIK3R4, PIK3CB, OCRL, INPP5B, INPP4A, F8A1, VPS8 (yeast)RAB13 (EE, TJ) RE- to- PM transport, GLUT4 traffi ckingMICALL2, MICALL1RAB21 (EE) Integrin endocytosis, cytokinesisN.D.RAB22A EE- to- Golgi transport TBC1D2B, EEA1RAB31/RAB22B (EE, TGN, PG)TGN- to- EE transport, PG maturationTBC1D2B, OCRLRAB23 (PM, EE) PG- LY fusion, Hedgehog signaling, ciliogenesisN.D.RAB35 (PM, CCV, EE)Endocytic recycling, cytokine-sis, actin dynamicsOCRL, ACAP2, FSCN1RAB3A,B,C,D (SG, SV)Regulated exocytosis (SV cycle)RPH3A, RPH3AL, RIMS1/2, RAB3IP, SYN1, SYTL4/5RAB6A,B,C (Golgi)Golgi- to- PM, Golgi- to-endosome, intra- Golgi and ER- to- Golgi transport, cytokinesisGOLGB1, GCC2, OCRL, KIF20A, MYH9, MYH10, DENND5A, BICD1, CCDC64, TMF1, APBA3, VPS52RAB8A,B (RE, G4V)TGN- to- PM transport, GLUT4 and E- cadherin traffi cking, ciliogenesis, AJ assemblyOPTN, RPH3A, MYO5B, SYTL1, MICALL2, ODF2RAB10 (TGN, G4V)TGN- to- PM transport, GLUT4 traffi cking, PG maturationMICALL1, MYO5A/B/C, RIMS1RAB11A,B (TGN, RE)Endocytic recycling, RE- to-PM transport, cytokinesis, ciliogenesis, autophagyRAB11FIP1/FIP2/FIP3/FIP4/FIP5, EXOC6, RAB3IP, ZFYVE27*, MYO5B, TBC1D14RAB25 (RAB11C) (RE)RE- to- PM transport, integrin recyclingRAB11FIP1/FIP2/FIP3/FIP4/FIP5, RAB3IP, MYO5BRAB14 (EE, TGN) Golgi- to- EE transport KIF16B, ZFYVE20, RUFY1RAB15 (EE, RE) Endocytic recycling RABIF*, REP15RAB17 (RE, ML) Endocytic recycling, ciliogen-esis, melanosome traffi ckingN.D.RAB26 (SG) Regulated exocytosis RIMS1RAB33B (Golgi, ER- Golgi and intra- Golgi transport, autophagyGOLGA2, ATG16L1, RABEP1RAB37 (Golgi, SG)Mast cell degranulation, Wnt signaling, TNF- alpha secretionRIMS1, UNC13BRAB39A, B (Golgi, PG)Phagosomal acidifi cation, IL-1 secretionCASP1RAB7A,B (LE, LY, EE- to- LE and LE- to- Golgi transport, LY biogenesisRILP, VPS35 (retromer), VPS41 (HOPS complex), PIK3R4, FYCO1RAB9A,B (LE) Endosome- to- TGN transport PLIN3, GCC2, RHOBTB3, RABEPK, SGSM2, HPS4 (BLOC- 3 complex)RAB12 (RE, LY) Transport to LY N.D.RAB27A (ML, SG) Dynamics of LRO and SG RPH3A , RPH3AL MYO5AMLPH, SYTL1- 5, UNC13D, MYRIP, CORO1CRAB32 (TV, ML, Mito, APG)Autophagy, distribution of mitochondria, traffi cking of melanogenic enzyme to MLPRKAR2A, ANKRD7RAB34 (Golgi, PM, PG)Macropinocytosis, LY positioningRILP, UNC13BRAB36 (Golgi) Spatial distribution of LE and LY RILPRAB38 (TV, ML) Transport of tyrosinase to immature MLANKRD7RAB24 (APG?) - GABARAP

Identification of siRNA delivery enhancers by a chemical library screen

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

New cellular mechanisms of gap junction degradation and recycling

Connexins (Cxs), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore, alterations of Cx expression and degradation lead to the development of physiopathological disorders. Because of the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear.

Regulation of Liver Metabolism by the Endosomal GTPase Rab5

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierkes disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.