ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Rab3 mediates a pathway for endocytic sorting and plasma membrane recycling of ordered microdomains.

The composition of the plasma membrane (PM) must be tightly controlled despite constant, rapid endocytosis, which requires active, selective recycling of endocytosed membrane components. For many proteins, the mechanisms, pathways, and determinants of this PM recycling remain unknown. We report that association with ordered, lipid-driven membrane microdomains (known as rafts) is sufficient for PM localization of a subset of transmembrane proteins and that abrogation of raft association disrupts their trafficking and leads to degradation in lysosomes. Using orthogonal, genetically encoded probes with tunable raft partitioning, we screened for the trafficking machinery required for efficient recycling of engineered microdomain-associated cargo from endosomes to the PM. Using this screen, we identified the Rab3 family as an important mediator of PM localization of microdomain-associated proteins. Disruption of Rab3 reduced PM localization of raft probes and led to their accumulation in Rab7-positive endosomes, suggesting inefficient recycling. Abrogation of Rab3 function also mislocalized the endogenous raft-associated protein Linker for Activation of T cells (LAT), leading to its intracellular accumulation and reduced T cell activation. These findings reveal a key role for lipid-driven microdomains in endocytic traffic and suggest Rab3 as a mediator of microdomain recycling and PM composition.

Myosin V facilitates polarised E-cadherin secretion.

E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis.

Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism.

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport.

The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles.

The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation.

Mitophagy, a type of selective autophagy, specifically targets damaged mitochondria. The ULK complex regulates Parkin-mediated mitophagy, but the mechanism through which the ULK complex initiates mitophagosome formation remains unknown. The Rab7 GTPase (herein referring to Rab7a) is a key initiator of mitophagosome formation, and Ser-72 phosphorylation of Rab7 is important for this process. We have previously identified LRRK1 as a protein kinase responsible for Rab7 Ser-72 phosphorylation. In this study, we investigated the role of LRRK1 in mitophagy. We showed that LRRK1 functions downstream of ULK1 and ULK2 in Parkin-mediated mitophagy. Furthermore, we demonstrated that ectopic targeting of active LRRK1 to mitochondria is sufficient to induce the Ser-72 phosphorylation of Rab7, circumventing the requirement for ATG13, a component of the ULK complex. Thus, the ULK complex recruits LRRK1 to mitochondria by interacting with ATG13 to initiate mitophagosome formation. This study highlights the crucial role of the ULK complex-LRRK1 axis in the regulation of Parkin-mediated mitophagy.

Downregulation of O-GlcNAc transferase activity impairs basal autophagy and late endosome positioning under nutrient-rich conditions in human colon cells.

Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.

Acetyl-11-keto-beta-boswellic acid inhibits cell proliferation and growth of oral squamous cell carcinoma via RAB7B-mediated autophagy.

Natural products can overcome the limitations of conventional chemotherapy. Acetyl-11-keto-beta-boswellic acid (AKBA) as a natural product extracted from frankincense, exhibited chemotherapeutic activities in different cancers. However, whether AKBA exerts inhibiting effect of oral squamous cell carcinoma (OSCC) cells growth and the mechanism need to be explored. We attempted to investigate the therapeutic effects of AKBA against OSCC and explore the mechanism involved. Here we attempt to disclose the cell-killing effect of AKBA on OSCC cell lines and try to figure out the specifical pathway. The presence of increase autophagosome and the production of mitochondrial reactive oxygen species were confirmed after the application of AKBA on OSCC cells, and RAB7B inhibition enhanced autophagosome accumulation. Though the increase autophagosome was detected induced by AKBA, autophagic flux was inhibited as the failure fusion of autophagosome and lysosome. Cal27 xenografts were established to verify the role of anti-OSCC cells of AKBA in vivo. Based above findings, we speculate that natural product AKBA suppresses OSCC cells growth via RAB7B-mediated autophagy and may serve as a promising strategy for the therapy of OSCC.

Comparative proteomics reveals different protein expression in platelets in patients with alcoholic liver cirrhosis.

BACKGROUND: The role of platelets in disease progression as well as the function of platelets as part of the haemostatic and immunological system in patients with liver cirrhosis is only incompletely understood. This is partly due to difficulties in assessing platelet function. Proteome analyses of platelets have been used to further investigate the role of platelets in other diseases. AIM: To assess possible changes in the platelet proteome during different stages of alcohol induced liver cirrhosis compared to healthy donors. PATIENTS AND METHODS: A 45 ml blood sample was drawn from 18 participants aged 18-80 years evenly divided into three groups of healthy donors, patients with less advanced alcohol induced liver cirrhosis (Child-Pugh < 7) and patients with advanced liver cirrhosis (Child-Pugh > 10). The blood was processed to isolate platelets and perform subsequent two-dimensional gel-electrophoresis using a SYPRO™ Ruby dye. After computational analysation significantly in- or decreased protein spots (defined as a two-fold abundance change between different study cohorts and ANOVA < 0.05) were identified via liquid chromatography-mass spectrometry (LCMS) and searching against human protein databases. RESULTS: The comparative analysis identified four platelet proteins with progressively decreased protein expression in patients with liver cirrhosis. More specifically Ras-related protein Rab-7a (Rab-7a), Ran-specific binding protein 1 (RANBP1), Rho GDP-dissociation inhibitor 1 (RhoGDI1), and 14-3-3 gamma. CONCLUSION: There is significant change in protein expression in the platelet proteome throughout the disease progression of alcohol induced liver cirrhosis. The identified proteins are possibly involved in haemostatic and immunoregulatory function of platelets.

Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells.

BACKGROUND: Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. METHODS: Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. RESULTS: We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. CONCLUSIONS: Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts.

We previously reported that the a3 subunit of proton-pumping vacuolar-type ATPase (V-ATPase) interacts with Rab7 and its guanine nucleotide exchange factor, Mon1a-Ccz1, and recruits them to secretory lysosomes in osteoclasts, which is essential for anterograde trafficking of secretory lysosomes. The a3 subunit interacts with Mon1a-Ccz1 through its cytosolic N-terminal domain. Here, we examined the roles of this domain in the interaction with Rab7 and trafficking of secretory lysosomes. Immunoprecipitation experiments showed that a3 interacted with Rab7 through its cytosolic domain, similar to the interaction with Mon1a-Ccz1. We connected this domain with a lysosome localization signal and expressed it in a3-knockout (a3KO) osteoclasts. Although the signal connected to the cytosolic domain was mainly detected in lysosomes, impaired lysosome trafficking in a3KO osteoclasts was not rescued. These results indicate that the cytosolic domain of a3 can interact with trafficking regulators, but is insufficient to induce secretory lysosome trafficking. The C-terminal domain of a3 and other subunits of V-ATPase are likely required to form a fully functional complex for secretory lysosome trafficking.

Protrudin-mediated ER-endosome contact sites promote phagocytosis.

During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis.

Effect of MEHP on testosterone synthesis via Sirt1/Foxo1/Rab7 signaling pathway inhibition of lipophagy in TM3 cells.

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure. MEHP exposure inhibited lipophagy and the Sirt1/Foxo1/Rab7 signaling pathway in TM3 cells, causing aberrant accumulation of intracellular lipid droplets. Addition of the Sirt1 agonist SRT1720 and Rab7 agonist ML-098 alleviated the inhibition of lipophagy and increased free cholesterol and testosterone contents in TM3 cells. SRT1720 alleviated the inhibitory effect of MEHP on the Sirt1/Foxo1/Rab7 signaling pathway, whereas ML-098 only alleviated the inhibition of Rab7 protein expression by MEHP and had no effect on Sirt1 and Foxo1 protein expression. This suggests that MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway, ultimately leading to a further decrease in cellular testosterone secretion. This study improves our current understanding of the toxicity and molecular mechanisms of action of MEHP and provides new insights into the reproductive effects of phthalic acid esters.

Nerve Growth Factor Signaling Modulates the Expression of Glutaminase in Dorsal Root Ganglion Neurons during Peripheral Inflammation.

Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

Toll-Dorsal signaling regulates the spatiotemporal dynamics of yolk granule tubulation during Drosophila cleavage.

The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. We find that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which we show is controlled by the Toll-Dorsal signaling pathway. Our work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process.

Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy.

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimer's disease (AD) in PD patients.

Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion.

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.