Biology subjects

Ganss, K.

Publications and source records attributed to Ganss, K..

7 recordsLinked to original sources

bioRxiv2026

pH-dependent trapping of cationic amphiphilic drugs perturbs insulin granule homeostasis

Pancreatic {beta}-cells store insulin in acidic secretory granules (SGs), specialized organelles that also contain monoamine neurotransmitters such as serotonin. Many neuroactive drugs with monoaminergic activity are cationic amphiphilic drugs (CADs) that accumulate in acidic compartments by pH-dependent trapping. Yet, whether insulin SGs represent a site of CAD accumulation and if this affects their properties such as monoamine storage and pH remain unclear. Here, we show that Slc18a1/VMAT1 is required for vesicular monoamine uptake and maintenance of cellular serotonin levels in insulinoma INS-1 cells. In contrast, neuroactive CADs accumulate via pH-dependent trapping at luminal pH values characteristic of insulin SGs. CADs inhibit VMAT-mediated uptake of the fluorescent monoamine probe FFN206 and induce its efflux to the extracellular space without detectable changes in SG luminal pH. Conversely, natural VMAT substrates such as serotonin and dopamine increase SG pH in a VMAT-dependent manner. These findings identify insulin SGs as acidic organelles susceptible to CAD accumulation and uncover distinct mechanisms regulating secretory granule homeostasis.

cell biology↗

bioRxiv2026

Villin-SNAP25 interaction regulates insulin secretion downstream of ICA512 signaling

Pancreatic islet {beta}-cells step-up insulin production and secretion in response to the elevation of blood glucose levels. ICA512/PTPRN is a transmembrane cargo of the insulin secretory granules (SGs). Upon SG exocytosis, its Ca2+/calpain mediated cleavage at the plasma membrane generates an ICA512-cleaved cytosolic fragment (ICA512-CCF) which as a decoy phosphatase prolongs phospho-STAT5/3 activities, thereby enhancing the transcription of mRNAs for SG cargoes, including insulin and its own, to replenish SG stores. In addition, ICA512 positively regulates the expression of F-actin modifier villin. Villin, in turn, modulates the size of actin cages surrounding cortical SGs, hence regulating SG mobility and exocytosis. Here we show that villin controls the number of SG docking sites for exocytosis by directly interacting at low glucose with the t-SNARE SNAP-25, thus restricting in this condition SG access to fusion sites and insulin release. Replacement of ICA512-CCF N-terminal ubiquitin-acceptor lysine 609 (K609) with valine (V) stabilized ICA512-CCF in insulinoma cells and homozygous ICA512K609V mice and enhanced the levels of phospho-STAT5/3 and villin. In ICA512K609V female mice these molecular traits correlated with reduced body weight, improved insulin sensitivity, reduced basal insulin secretion and onset time for glucose stimulated insulin secretion. Taken together, these data demonstrates that SG exocytosis induced ICA512 retrograde signaling acts in concert with the novel SNARE complex regulator villin to plastically adapt {beta}-cell actin cytoskeleton and access to SG docking sites for optimal control of insulin secretion in response to variations in extracellular glucose levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/699220v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@37d835org.highwire.dtl.DTLVardef@5389f1org.highwire.dtl.DTLVardef@855d98org.highwire.dtl.DTLVardef@1c3c639_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Model of ICA512 and STAT3/5-dependent regulation of -cell function. At basal conditions, villin limits secretory granule (SG) docking by interacting with SNAP-25, restricting insulin release (1). Increased metabolic load triggers hyperglycemia-induced ICA512 processing and growth factor-mediated STAT3/5 phosphorylation (2-4), enhancing ISG mRNAs, granule biogenesis, and villin expression (5,6). Villin modulates SNARE protein availability, controlling SG docking and secretion. Together, ICA512 retrograde signaling and villin-mediated SNARE regulation dynamically remodel the -cell actin cytoskeleton, adjusting SG docking site access to ensure appropriate insulin secretion in response to fluctuating glucose levels. C_FIG

cell biology↗

bioRxiv2025

GGA2-depletion in beta cells by Enteroviruses causes Golgi acidification, premature activation of cathepsins and alters the MHC class I immunopeptidome

Enteroviruses (EVs), such as Coxsackievirus B5 (CVB5), are linked to pancreatic beta cell autoimmunity in type 1 diabetes (T1D). CVB5 infection of beta cells blocks their cap-dependent translation but spares cap-independent translation of insulin secretory granule (SG) cargoes, including major T1D autoantigens. Despite this, insulin SG stores are depleted. We show that CVB5 protease 2A rapidly depletes Golgi-associated sorting factor GGA2 in beta cells due to its short half-life. Immunostaining of pancreas sections from recent-onset T1D donors confirmed reduced GGA2 in beta cells expressing EV capsid protein VP1. Both GGA2 depletion and CVB5 2A expression impair SG biogenesis at the TGN without affecting MHC class I trafficking. They also disrupt the sorting of vacuolar ATPase and cathepsins, leading to TGN acidification and premature lysosomal hydrolase activation, which typically generate peptides presented in HLA-II alleles. Notably, the unique immunopeptidome of EV-infected, GGA2-depleted ECN90 beta-like cells is mainly presented via HLA-B alleles with an average isoelectric point (pI) of [~]5, compared to a pI of [~]7 of the prevailing HLA-I A allele immunopeptidome of non-infected cells. We propose that delayed sorting at the acidified TGN promotes cathepsin-mediated processing of in-transit secretory proteins, including SG cargoes, thereby generating novel peptides that may replace HLA class I antigens due to the acidic environment. This novel cross-presentation mechanism could have implications for T1D pathogenesis and antigen presentation in general.

cell biology↗

bioRxiv2024

Post-transcriptional regulation of insulin mRNA storage by G3BP1/2+ condensates in beta cells

Hyperglycemia upregulates insulin translation in pancreatic beta cells. Several RNA- binding proteins involved in this process have been identified, including G3BP1, a stress granule marker downregulated in islets of subjects with type 2 diabetes. We show that in mouse insulinoma MIN6-K8 cells exposed to fasting glucose levels G3BP1 and its paralog G3BP2 colocalize to cytosolic condensates with eIF3b and Ins1/2 mRNA. Upon glucose stimulation, the condensates dissolve and G3BP1/2, eIF3b, and insulin mRNAs redistribute throughout the cytosol. Intriguingly, G3BP1+ condensates in MIN6-K8 cells differ from sodium arsenate-induced stress granules in regards to eIF2 and AMPK phosphorylation. Knockout of G3BP1 or G3BP2 prevented condensate assembly, but only G3BP1 deletion decreased the levels of Ins1/2 mRNA and proinsulin and impaired polysome formation. Like glucose, other insulin secretagogues such as Exendin-4 and palmitate, but not high KCl, prompted the dissolution of G3BP1+ condensates. G3BP1+/Ins mRNA+ condensates were also present in mouse and human beta cells from normoglycemic donors. Hence, G3BP1+ condensates represent a glucose-regulated compartment for the physiological storage and protection of insulin mRNA in resting beta cells.

molecular biology↗

bioRxiv2021

Insulin mRNA is stored in RNA granules in resting beta cells

The glucose-stimulated biosynthesis of insulin in pancreatic islet beta cells is post-transcriptionally regulated. Several RNA-binding proteins (RBPs) that regulate Insulin mRNA stability and translation also bind mRNAs coding for other insulin secretory granule (ISG) proteins. However, an overview of these interactions and their glucose-induced remodelling is still missing. Here we identify two distinct sets of RBPs that were preferentially pulled down with the 5-UTRs of mouse Ins1, Ins2, spliced Ins2, Ica512/Ptprn and Pc2/Pcsk2 mRNAs from extracts of either resting or stimulated mouse insulinoma MIN6 cells. Among RBPs binding to all tested transcripts in resting conditions was hnRNP A2/B1. Hnrnpa2b1 KO MIN6 cells contained lower levels of Ins1 mRNA, proinsulin and insulin, and had reduced insulin secretion. In resting cells, both hnRNP A2/B1 and Insulin mRNAs localized to stress granules, which dissolved upon glucose stimulation. Insulin mRNA-positive RNA granules were also found in human pancreatic beta cells in situ. Our results suggest that resting beta cells store mRNAs for insulin secretory granule proteins in stress granules through specific RNA protein interactions. Glucose stimulation remodels these interactions, releasing the transcripts, and another set of RBPs coordinates their translation.

cell biology↗

bioRxiv2020

Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. The competence of secretory granules for exocytosis depends on spatio-temporal variables such as proximity to the plasma membrane and age, with newly-generated granules being preferentially released. Despite recent advances, we still lack a comprehensive view about the molecular composition of insulin granules and how this may change over the lifetime of the organelles. Here we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtained intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time along with the physical properties of the membrane.

cell biology↗

bioRxiv2017

Mir-132 controls beta cell proliferation and survival in mouse model through the PTEN/AKT/FOXO3 signaling

Aim and hypothesismicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains a challenge. In this study we aimed to identify miRNAs and their downstream targets involved in regeneration of islet beta cells following partial pancreatectomy in mice.\n\nMethodsRNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in control of beta cell regeneration. Altered expression of selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were seleced through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA and protein expression levels and signaling upon miR-132 knockdown or overexpression in MIN6 cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice.\n\nResultsPartial pancreatectomy significantly increased the number of BrdU+/insulin+ positive islet cells. Microarray profiling revealed 14 miRNAs, including miR-132 and -141, to be significantly upregulated in LCM islets of partially pancreatectomized compared to LCM islets of control mice. In the same comparison miR-760 was the only miRNA found to be downregulated. Changed expression of these miRNAs in islets of partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we chose to focus our attention on miR-132. Downregulation of miR-132 in MIN6 cells reduced proliferation while enhancing the expression of proapoptic genes, which was instead reduced in miR-132 overexpression MIN6 cells. Microarray profiling, RT-PCR and immunoblotting of miR-132 overexpressing MIN6 cells revealed their downregulated expression of Pten, with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb as well as inactivation of pro-apoptotic Foxo3 via its phosphorylation. Finally, we show that regeneration of beta cells following partial pancreatectomy was reduced in miR-132-/- mice compared to control mice.\n\nConclusions/InterpretationsOur study provides compelling evidence for upregulation of miR-132 being critical for regeneration of mouse islet beta cells in vivo through downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.\n\nResearch in ContextWhat is already known?\n\nO_LISeveral miRNAs, including miR-132, are known to regulate beta cell function and mass in several mouse models of diabetes db/db, ob/ob and high fat-diet.\nC_LI\n\nWhat is the key question?\n\nO_LIWhich are miRNAs implicated in control of beta cell regeneration upon partial pancreatectomy and how?\nC_LI\n\nWhat are the new findings?\n\nO_LImiR-132 is critical to promote regeneration of mouse beta cells in vivo following partial pancreatectomy\nC_LIO_LIIn vitro studies in mouse MIN6 cells indicate that miR-132 fosters beta cell proliferation by down-regulating the expression of phosphatase Pten, thereby tilting the balance between anti-apoptotic factor Akt and pro-apoptotic factor Foxo3 activities towards proliferation through regulation of their phosphorylation.\nC_LI\n\nHow might this impact on clinical practice in the foreseeable future?\n\nO_LIThese findings strengthen the rationale for targeting the expression of miR-132 to increase beta cell mass in vivo (type 2 diabetes) or ex-vivo (islet transplantation in type 1 diabetes) for the treatment of diabetes.\nC_LI

cell biology↗