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.
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•LRRK2 interacts with the endosomal v-SNAREs VAMP4 and VAMP7•VAMP4 and VAMP7 mediate the secretion of the Parkinson’s disease biomarker VGF•LRRK2 regulates the accumulation vs. secretion of VGF
Filippini et al. report the direct interaction of leucine-rich repeat kinase 2 (LRRK2) with the endosomal vesicular SNAREs VAMP4 and VAMP7. LRRK2, VAMP4, and VAMP7 regulate the secretion of the Parkinson’s disease biomarker VGF. They provide evidence for the role of LRRK2 in regulating unconventional secretion in the disease.
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).
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•Primary neuron hippocampal culture for morphological analysis after starvation•In vitro neuronal starvation by culture media dilution•Fixation and immuno staining for morphological analysis•Image acquisition and determination of axonal length after 24 h starvation
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.
Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. ...LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.
Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying ...Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.
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.
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•Nutrient restriction and autophagy regulate axonal growth and neuronal polarity•VAMP7 mediates nutrient restriction/autophagy-dependent neurite growth•VAMP7 mediates the secretion of ER-phagy factor Reticulon 3
VAMP7 is involved in exocytosis-mediated neurite growth and degradative autophagy. Using secretomics, Wojnacki et al. show that VAMP7 mediates the release of tubular ER-phagy receptor Reticulon 3, a secretory pathway greatly enhanced in autophagy-null neuronal cells. ER-phagy and unconventional secretion regulate neurite growth and polarization during nutrient deprivation.
...Kindlin-1 induced the activation of β1-integrin as shown by increased levels of the active form of the protein specifically detected by the 9EG7 antibody on the cell surface (Figure 1C,D). ...Quantitative Real Time- Polymerase Chain Reaction;; EEA1: early endosomal antigen 1, FC: fold change We then verified that altered expression of other integrins and regulators was not the reason for the changes in cell adhesion and spreading. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed similar ITGB1 mRNA levels in Kind1 and control cells, confirming that the difference was not due to a Kindlin-1-mediated transcriptional regulation (Supplementary Figure S1D). Time-lapse imaging showed that the persistence (displacement D-over-traveled distance T directionality ratio) of individual cells was reduced in Kind1-cells (P = 0.007), accompanied by a 3.8-fold increase in the velocity of cell migration (P < 0.001) (Supplementary Figure S2C-E). ...Kind1-cells had an increased ability to migrate in multiple directions while traveling a similar distance in a 3D environment.
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.
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•VAMP7 is important for mechano-adaptation to the rigidity of cellular environment•Substrate rigidity regulates VAMP7 exocytosis•LRRK1 and VARP compete for VAMP7 binding•LRRK1 and VARP generate a tug-of-war for the transport of VAMP7
Cell Biology; Functional Aspects of Cell Biology; Biomechanics
Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target ...soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N‐ethylmaleimide‐sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI‐VAMP/VAMP7 and cellubrevin/VAMP3, two v‐SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI‐VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.
SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction ...between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.
Background: SNAREs mediate membrane fusion and need to be addressed to specific intracellular compartments.
Results: We show that the Q-SNARE SNAP-47 regulates the localization and function of a subset of v-SNAREs.
Conclusion: SNAP-47 localizes mainly in the early secretory pathway where it regulates the transport of selected v-SNAREs.
Significance: SNAP-47 may guide selected v-SNAREs through the early secretory pathway.
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