Oligophrenin-1: the link between calcium-regulated exocytosis and compensatory endocytosis in neuroendocrine cells

In neuroendocrine cells, hormones and neuropeptides are released from large-dense core vesicles (secretory granules) by calcium-regulated exocytosis. Following exocytosis, compensatory uptake of membrane is required to maintain membrane homeostasis and allow recycling of secretory vesicle membranes. How these cells initiate and regulate this compensatory endocytosis remains poorly understood. Our recent data suggests that oligophrenin-1 (OPHN1) is a link coupling calcium-regulated exocytosis to compensatory endocytosis of secretory granules in the adrenal chromaffin cells (Houy et al., 2015, J Neurosci. 2015, 35:11045-55). Here, we highlight the major evidence and discuss how OPHN1 could couple these two processes

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.journal articleresearch support, non-u.s. gov't2014 Oct 152014 08 20importe

The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected].

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.journal articleresearch support, non-u.s. gov't20132013 09 06importe

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

A fine balance of synaptophysin levels underlies efficient retrieval of synaptobrevin II to synaptic vesicles

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance

GTPase-activating protein oligophrenin 1 is a new partner of multifunctional adapter protein intersectin 1

Intersectin 1 (ITSN1) is a multifunctional adaptor protein which is involved in endocytosis, exocytosis and cellular signaling and it is also associated with such pathologies as Down syndrome and Alzheimer’s disease. The aim of this study was to identify new ITSN1 protein partners which are implicated in membrane trafficking. Methods. In silico analysis by Scansite online resource had identified a GTPase activating protein oligophrenin 1 (OPHN1) as a potential partner of ITSN1 SH3A domain. GST pull-down and immunoprecipitation were used to prove complex formation between ITSN1 and OPHN1. Subcellular protein localization was determined by immunofluorescence and confocal microscopy. Results. We have shown that brain-specific and ubiquitously expressed SH3A domain isoforms of ITSN1 interact with OPHN1. ITSN1 and OPHN1 form complexes in both resting and stimulated to exocytosis PC12 cell line. Conclusions. GTPase activating protein OPHN1 and adaptor protein ITSN1 interact in PC12 cell line independently of exocytosis stimulation

Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation