Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host.

In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide-sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Role of SNAREs in Unconventional Secretion-Focus on the VAMP7-Dependent Secretion.

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus have been collectively termed as Unconventional protein secretion (UPS). UPS can comprise of cargo without a signal peptide or proteins which escape the Golgi in spite of entering the ER. UPS has been classified further depending on the mode of transport. Type I and Type II unconventional secretion are non-vesicular and non-SNARE protein dependent whereas Type III and Type IV dependent on vesicles and on SNARE proteins. In this review, we focus on the Type III UPS which involves the import of cytoplasmic proteins in membrane carriers of autophagosomal/endosomal origin and release in the extracellular space following SNARE-dependent intracellular membrane fusion. We discuss the role of vesicular SNAREs with a strong focus on VAMP7, a vesicular SNARE involved in exosome, lysosome and autophagy mediated secretion. We further extend our discussion to the role of unconventional secretion in health and disease with emphasis on cancer and neurodegeneration.

Contributions of Andrée Tixier-Vidal (1923-2021) to modern cell biology.

This article illustrates the main stages of the scientific career of Dr Andrée Tixier-Vidal, a pioneer in cell biology research in France. She made important discoveries in the field of hormone secretion and neuronal morphogenesis. She played a key role in developing pituitary and neuronal cultures and using electron microscopy to study cellular structures. Her scientific influence continues to irradiate through her students and collaborators.

A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.

Protocol to study starvation-induced autophagy in developing rat neurons.

Autophagy is being involved in an increasing number of cellular pathways. It now appears that autophagy stimulation and inhibition have complex effects in neurons. Here, we present a simple yet powerful protocol to induce autophagy in primary neurons in culture by partial nutrient deprivation, in neurons with or without transfection of plasmids encoding the Longin domain of VAMP7 or a nanobody directed against VAMP7. Although limited to cells in culture, this protocol can facilitate the study of autophagy in neurons. For complete details on the use and execution of this protocol, please refer to Wojnacki et al. (2020).

ER-PM Contact Sites - SNARING Actors in Emerging Functions.

The compartmentalisation achieved by confining cytoplasm into membrane-enclosed organelles in eukaryotic cells is essential for maintaining vital functions including ATP production, synthetic and degradative pathways. While intracellular organelles are highly specialised in these functions, the restricting membranes also impede exchange of molecules responsible for the synchronised and responsive cellular activities. The initial identification of contact sites between the ER and plasma membrane (PM) provided a potential candidate structure for communication between organelles without mixing by fusion. Over the past decades, research has revealed a far broader picture of the events. Membrane contact sites (MCSs) have been recognized as increasingly important actors in cell differentiation, plasticity and maintenance, and, upon dysfunction, responsible for pathological conditions such as cancer and neurodegenerative diseases. Present in multiple organelles and cell types, MCSs promote transport of lipids and Ca2+ homoeostasis, with a range of associated protein families. Interestingly, each MCS displays a unique molecular signature, adapted to organelle functions. This review will explore the literature describing the molecular components and interactions taking place at ER-PM contact sites, their functions, and implications in eukaryotic cells, particularly neurons, with emphasis on lipid transfer proteins and emerging function of SNAREs.

Introducing secretory reticulophagy/ER-phagy (SERP), a VAMP7-dependent pathway involved in neurite growth.

Together with the proteasome, macroautophagy is a main pathway for the degradation of intracellular elements. Endoplasmic reticulum (ER)-autophagy i.e. reticulophagy/ER-phagy leads to the encapsulation of pieces of the ER in forming autophagosomes. This is generally followed by fusion with lysosomes and degradation of these ER components by lysosomal hydrolases. Recent work by our group shows that ER elements could also be incorporated into late endosomes and later be released by a secretory mechanism which we will herein refer to as secretory reticulophagy/ER-phagy (SERP). In the absence of macroautophagy, such as by knocking out Atg5, SERP is more efficient, leading to an increased secretion of MAP1LC3B-II and LC3-interacting region (LIR)-containing proteins of the ER, reticulons and atlastins. In this scenario, neurites grow longer and neuronal polarity is altered. In the absence of SERP, such as by knocking out Vamp7, secretion of MAP1LC3B-II, ER-LIR containing proteins and neurite growth are severely inhibited. We argue that SERP might be a main secretory mechanism bypassing the Golgi apparatus, and that it is particularly active and important in neurite growth.

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth.

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.

Role of the Sec22b-E-Syt complex in neurite growth and ramification.

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here, we show that Sec22b interacts with members of the extended synaptotagmin (E-Syt) family of ER lipid transfer proteins (LTPs), and this interaction depends on the longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, and expression of the Sec22b longin domain and a Sec22b mutant with an extended linker between the SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with LTPs, such as E-Syts.This article has an associated First Person interview with the first author of the paper.

Post-synaptic Release of the Neuronal Tissue-Type Plasminogen Activator (tPA).

The neuronal serine protease tissue-type Plasminogen Activator (tPA) is an important player of the neuronal survival and of the synaptic plasticity. Thus, a better understanding the mechanisms regulating the neuronal trafficking of tPA is required to further understand how tPA can influence brain functions. Using confocal imaging including living cells and high-resolution cell imaging combined with an innovating labeling of tPA, we demonstrate that the neuronal tPA is contained in endosomal vesicles positives for Rabs and in exosomal vesicles positives for synaptobrevin-2 (VAMP2) in dendrites and axons. tPA-containing vesicles differ in their dynamics with the dendritic tPA containing-vesicles less mobile than the axonal tPA-containing vesicles, these laters displaying mainly a retrograde trafficking. Interestingly spontaneous exocytosis of tPA containing-vesicles occurs largely in dendrites.

MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience.

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright, a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono- and two-photon microscopy. These probes are compatible with long-term live-cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells, allowing identification of neurons in acute brain tissue sections and neuromuscular junctions without any use of transfection or transgenic animals. In addition, MemBright probes were used in super-resolution imaging to unravel the neck of dendritic spines. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques. VIDEO ABSTRACT.

ARAP1 Bridges Actin Dynamics and AP-3-Dependent Membrane Traffic in Bone-Digesting Osteoclasts.

Bone-resorbing osteoclasts play a central role in bone remodeling and its pathology. To digest bone, osteoclasts re-organize both F-actin, to assemble podosomes/sealing zones, and membrane traffic, to form bone-facing ruffled borders enriched in lysosomal membrane proteins. It remains elusive how these processes are coordinated. Here, we show that ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain-containing protein 1) fulfills this function. At podosomes/sealing zones, ARAP1 is part of a protein complex where its RhoGAP domain regulates actin dynamics. At endosomes, ARAP1 interacts with AP-3 adaptor complexes where its Arf-GAP domain regulates the Arf1-dependent AP-3 binding to membranes and, consequently lysosomal membrane protein transport to ruffled borders. Accordingly, ARAP1 or AP-3 depletion in osteoclasts alters their capacity to digest bone in vitro. and AP-3δ-deficient mocha mice, a model of the Hermansky-Pudlak storage pool syndrome, develop osteoporosis. Thus, ARAP1 bridges F-actin and membrane dynamics in osteoclasts for proper bone homeostasis.

Biomechanical Control of Lysosomal Secretion Via the VAMP7 Hub: A Tug-of-War between VARP and LRRK1.

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we show that cells deficient in the secretory lysosome v-SNARE VAMP7 are impaired in adaptation to substrate rigidity. Conversely, VAMP7-mediated secretion is stimulated by more rigid substrate and this regulation depends on the Longin domain of VAMP7. We further find that the Longin domain binds the kinase and retrograde trafficking adaptor LRRK1 and that LRRK1 negatively regulates VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controls the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. LRRK1 and VARP interact with VAMP7 in a competitive manner. We propose a mechanism whereby biomechanical constraints regulate VAMP7-dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral pool of secretory lysosomes.

Reciprocal link between cell biomechanics and exocytosis.

A cell is able to sense the biomechanical properties of the environment such as the rigidity of the extracellular matrix and adapt its tension via regulation of plasma membrane and underlying actomyosin meshwork properties. The cell's ability to adapt to the changing biomechanical environment is important for cellular homeostasis and also cell dynamics such as cell growth and motility. Membrane trafficking has emerged as an important mechanism to regulate cell biomechanics. In this review, we summarize the current understanding of the role of cell mechanics in exocytosis, and reciprocally, the role of exocytosis in regulating cell mechanics. We also discuss how cell mechanics and membrane trafficking, particularly exocytosis, can work together to regulate cell polarity and motility.

A new actin-binding domain glues autophagy together.

Autophagy breaks down nonessential cellular components to replenish macromolecular building blocks during starvation. Nevertheless, the downstream events regulating vesicle trafficking during this essential cellular process are not yet fully defined. Xu et al. combined approaches of crystallography, biochemistry, and cell biology to show that the guanine nucleotide exchange factor DENND3 contains an actin-binding site they call "PHenn domain" in a region previously thought to be unstructured. PHenn domain binding to microfilaments is necessary for DENND3's participation in autophagy, providing a new link between autophagic stimulation and actin microfilaments. The findings by Xu et al. shed important new light on how membrane trafficking participates in critical steps of autophagy in relationship with actin microfilaments.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.