Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

International audienceBackground information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/ Wrch-1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N-and C-terminal domains. The N-terminal extension promotes binding to SH3 (Src homology 3)-domain-containing adaptors, whereas the C-terminal extension mediates membrane targeting through palmitoyl-ation of its non-conventional CAAX box. RhoU/Wrch-1 possesses transforming activity, which is negatively regulated by its N-terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c-Src-expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N-terminal extension and the palmitoyl-ation site were dispensable, whereas the C-terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked-down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C-terminal domains are critical for these functions

Double fluorescent knock-in mice to investigate endogenous mu-delta opioid heteromer subscellular distribution.

The heteromerization of Mu (MOP) and delta (DOP) opioid receptors has been extensively studied in heterologous systems. These studies demonstrated significant functional interaction of MOP and DOP evidenced by new pharmacological properties and intracellular signalling in transfected cells co-expressing the receptors. Due to the lack of appropriate tools for receptor visualization, such as specific antibodies, the pharmacological and functional properties of MOP-DOP heteromers in cells naturally expressing these receptors remains poorly understood. To address endogenous MOP-DOP heteromer trafficking and signalling in vivo and in primary neuronal cultures, we generated a double knock-in mouse line expressing functional fluorescent versions of DOP and MOP receptors. This mouse model has successfully been used to map the neuroanatomic distribution of the receptors and to identify brain regions in which the MOP-DOP heteromers are expressed. Here, we describe a method to quantitatively and automatically analyze changes in the subcellular distribution of MOP-DOP heteromers in primary hippocampal culture from this mouse model. This approach provides a unique tool to address specificities of endogenous MOP-DOP heteromer trafficking

How important are Rho GTPases in neurosecretion?

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18F-FDOPA PET/CT Uptake Parameters Correlate with Catecholamine Secretion in Human Pheochromocytomas

International audienceBackground: 18 F-FDOPA positron emission tomography/ computed tomography (PET/CT) is a sensitive nuclear imaging technology for the diagnosis of pheochromocytomas (PHEO). However, its utility in determining predictive factors for the secretion of catecholamines remains poorly studied. Methods: Thirty-nine histologically confirmed PHEO were included in this retrospective single-center study. Patients underwent 18 F-FDOPA PET/CT before surgery, with an evaluation of several uptake parameters (standardized uptake values [SUV max and SUV mean ] and the metabolic burden [MB] calculated as follows: MB = SUV mean × tumor volume) and measurement of plasma and/or urinary metanephrine (MN), normetanephrine (NM), and chromogranin A. Thirty-five patients were screened for germline mutations in the RET, SDHx, and VHL genes. Once resected, primary cultures of 5 PHEO were used for real-time measurement of catechol-amine release by carbon fiber amperometry. Results: The MB of the PHEO positively correlated with 24-h urinary excre-tion of NM (r = 0.64, p < 0.0001), MN (r = 0.49, p = 0.002), combined MN and NM (r = 0.75, p < 0.0001), and eventually plasma free levels of NM (r = 0.55, p = 0.006). In the mutated patients (3 SDHD, 2 SDHB, 3 NF1, 1 VHL, and 3 RET), a similar correlation was observed between MB and 24-h urinary combined MN and NM (r = 0.86, p = 0.0012). For the first time, we demonstrate a positive correlation between the PHEO-to-liver SUV max ratio and the mean number of secretory granule fusion events of the corresponding PHEO cells revealed by amperometric spikes (p = 0.01). Conclusion: While the 18 F-FDOPA PET/CT MB of PHEO strongly correlates with the concentration of MN, amperometric recordings suggest that 18 F-FDOPA uptake could be enhanced by overactivity of cat-echolamine exocytosis

Exocytosis and Endocytosis in Neuroendocrine Cells: Inseparable Membranes!

International audienceAlthough much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid toward understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotrans-mitters, neuropeptides, and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval

Phospholipid Scramblase-1-Induced Lipid Reorganization Regulates Compensatory Endocytosis in Neuroendocrine Cells

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1−/−mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis

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

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells