Biology subjects

Stephens, S.

Publications and source records attributed to Stephens, S..

5 recordsLinked to original sources

bioRxiv2022

Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts

Osteoclasts are giant bone-digesting cells that harbour specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclasts resorptive apparatus. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the ruffled border. Thus, Slc37a2 is a physiological component of the osteoclasts unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

cell biology↗

bioRxiv2021

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules

Insulin is a key regulator of human metabolism, and its dysfunction leads to diseases such as type 2 diabetes. It remains unknown how proinsulin is targeted from the trans-Golgi network (TGN) to secretory storage granules as no cargo receptor has been identified. Chromogranin proteins (CGs) are central regulators of granule biosynthesis, and it was proposed that their aggregation is critical for this process. However, the molecular mechanism by which these molecules facilitate sorting at the TGN is poorly understood. Here, we show that CGs undergo liquid-liquid phase separation (LLPS) at low pH independently of divalent cations, such as calcium. Liquid CG condensates, but not aggregates, recruit and sort proinsulin and other granule-destined cargo molecules towards secretory granules. Cargo selectivity is independent of sequence or structural elements but is based on the size and concentration of the client molecules at the TGN. Finally, electrostatic interactions and the N-terminal intrinsically disordered domain of chromogranin B facilitate LLPS and are critical for granule formation. We propose that phase-separated CGs act as a "cargo sponge" within the TGN lumen, gathering soluble client proteins into the condensate independently of specific sequence or structural elements, facilitating receptor-independent sorting. These findings challenge the canonical TGN sorting models and provide insights into granule biosynthesis in insulin-secreting {beta}-cells. One sentence summaryLiquid Chromogranin condensates recruit cargo molecules at the TGN for their delivery to secretory storage granules.

cell biology↗

bioRxiv2021

RABL6A promotes pancreatic neuroendocrine tumor angiogenesis and progression in vivo

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet {beta} cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

cancer biology↗

bioRxiv2020

Perilipin2 down-regulation in beta cells impairs insulin secretion under nutritional stress and damages mitochondria

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in {beta} cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects {beta} cell function under nutritional stress, PLIN2 was down-regulated in mouse {beta} cells, INS1 cells, and human islet cells. {beta} cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in {beta} cells have an important role in preserving insulin secretion, {beta} cell metabolism and mitochondrial function under nutritional stress.

molecular biology↗

bioRxiv2019

Identification of a small molecule that stimulates human beta-cell proliferation and insulin secretion, and protects against cytotoxic stress in rat insulinoma cells

A key event in the development of both major forms of diabetes is the loss of functional pancreatic islet {beta}-cell mass. Strategies aimed at enhancing {beta}-cell regeneration have long been pursued, but methods for reliably inducing human {beta}-cell proliferation with full retention of key functions such as glucose-stimulated insulin secretion (GSIS) are still very limited. We have previously reported that overexpression of the homeobox transcription factor Nkx6.1 stimulates {beta}-cell proliferation, while also enhancing GSIS and providing protection against {beta}-cell cytotoxicity through induction of the VGF prohormone. We developed an Nkx6.1 pathway screen by stably transfecting 832/13 rat insulinoma cells with a VGF promoter-luciferase reporter construct, using the resultant cell line to screen a 630,000 compound chemical library. We isolated three compounds with consistent effects to stimulate human islet cell proliferation. Further studies of the most potent of these compounds, GNF-9228, revealed that it selectively activates human {beta}-cell relative to -cell proliferation and has no effect on {delta}-cell replication. In addition, pre-treatment, but not short term exposure of human islets to GNF-9228 enhances GSIS. GNF-9228 also protects 832/13 insulinoma cells against ER stress- and inflammatory cytokine-induced cytotoxicity. In contrast to recently emergent Dyrk1a inhibitors that stimulate human islet cell proliferation, GNF-9228 does not activate NFAT translocation. These studies have led to identification of a small molecule with pleiotropic positive effects on islet biology, including stimulation of human {beta}-cell proliferation and insulin secretion, and protection against multiple agents of cytotoxic stress.

cell biology↗