Search

Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain- containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging., (© 2024 The Authors. Traffic published by John Wiley & Sons Ltd.)

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) in an inward/ER-directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis-localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS-CoV-2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions., (© 2024 The Authors. Traffic published by John Wiley & Sons Ltd.)

ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

To combat microbial infections, mammalian cells use a variety of innate immune response pathways to induce synthesis of anti-microbial proteins. The cGAS/STING pathway recognizes cytoplasmic viral or cellular DNA to elicit signals that lead to type I interferon and other cytokine synthesis. cGAMP, synthesized by DNA-activated cGAS, activates the ER-associated protein, STING, which oligomerizes and translocates to other intracellular membrane compartments to trigger different branches of signaling. We have reported that, in the ER, EGFR-mediated phosphorylation of Tyr245 of STING is required for its transit to the late endosomes, where it recruits and activates the transcription factor IRF3 required for IFN induction. In the current study, we inquired whether STING Tyr245 phosphorylation per se or STING's location in the late endosomes was critical for its ability to recruit IRF3 and induce IFN. Using pharmacological inhibitors or genetic ablation of proteins that are essential for specific steps of STING trafficking, we demonstrated that the presence of STING in the late endosomal membranes, even without Tyr245 phosphorylation, was sufficient for IRF3-mediated IFN induction., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

The epsin-related adaptor proteins Ent3p and Ent5p participate in budding of clathrin coated vesicles in transport between trans-Golgi network and endosomes in yeast. Transport of the arginine permease Can1p was analyzed, which recycles between plasma membrane and endosomes and can be targeted to the vacuole for degradation. ent3∆ cells accumulate Can1p-GFP in endosomes. Can1p-GFP is transported faster to the vacuole upon induction of degradation in ent5∆ cells than in wild type cells. The C-terminal domain of Ent5p was sufficient to restore recycling of the secretory SNARE GFP-Snc1p between plasma membrane and TGN in ent3∆ ent5∆ cells. The SNARE Tlg2p was identified as interaction partner of the Ent5p ENTH domain by in vitro binding assays and the interaction site on Ent5p was mapped. Tlg2p functions in transport from early endosomes to the trans-Golgi network and in homotypic fusion of these organelles. Tlg2p is partially shifted to denser fractions in sucrose density gradients of organelles from ent5∆ cells while distribution of Kex2p is unaffected demonstrating that Ent5p acts as cargo adaptor for Tlg2p in vivo. Taken together we show that Ent3p and Ent5p have different roles in transport and function as cargo adaptors for distinct SNAREs., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previously, we found that age-dependent accumulation of beta-amyloid is not sufficient to cause synaptic decline. Late-endocytic organelles (LEOs) may be driving synaptic decline as lysosomes (Lys) are a target of cellular aging and relevant for synapses. We found that LAMP1-positive LEOs increased in size and number and accumulated near synapses in aged neurons and brains. LEOs' distal accumulation might relate to the increased anterograde movement in aged neurons. Dissecting the LEOs, we found that late-endosomes accumulated while there are fewer terminal Lys in aged neurites, but not in the cell body. The most abundant LEOs were degradative Lys or endolysosomes (ELys), especially in neurites. ELys activity was reduced because of acidification defects, supported by the reduction in v-ATPase subunit V0a1 with aging. Increasing the acidification of aged ELys recovered degradation and reverted synaptic decline, while alkalinization or v-ATPase inhibition, mimicked age-dependent Lys and synapse dysfunction. We identify ELys deacidification as a neuronal mechanism of age-dependent synapse loss. Our findings suggest that future therapeutic strategies to address endolysosomal defects might be able to delay age-related synaptic decline., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Several functions of the human cell, such as sensing nutrients, cell movement and interaction with the surrounding environment, depend on a myriad of transmembrane proteins and their associated proteins and lipids (collectively termed "cargoes"). To successfully perform their tasks, cargo must be sorted and delivered to the right place, at the right time, and in the right amount. To achieve this, eukaryotic cells have evolved a highly organized sorting platform, the endosomal network. Here, a variety of specialized multiprotein complexes sort cargo into itineraries leading to either their degradation or their recycling to various organelles for further rounds of reuse. A key sorting complex is the Endosomal SNX-BAR Sorting Complex for Promoting Exit (ESCPE-1) that promotes the recycling of an array of cargos to the plasma membrane and/or the trans-Golgi network. ESCPE-1 recognizes a hydrophobic-based sorting motif in numerous cargoes and orchestrates their packaging into tubular carriers that pinch off from the endosome and travel to the target organelle. A wide range of pathogens mimic this sorting motif to hijack ESCPE-1 transport to promote their invasion and survival within infected cells. In other instances, ESCPE-1 exerts restrictive functions against pathogens by limiting their replication and infection. In this review, we discuss ESCPE-1 assembly and functions, with a particular focus on recent advances in the understanding of its role in membrane trafficking, cellular homeostasis and host-pathogen interaction., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic β-amyloid peptide (Aβ). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aβ in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aβ, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

Membrane-enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with the Bin/Amphiphysin/Rvs (N-BAR) domain protein, AMPH-1. Here we show, using a free-solution single-particle technique known as burst analysis spectroscopy (BAS), that AMPH-1 alone creates small, tubular-vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH-1 and is slowed in the presence of RME-1. Unexpectedly, AMPH-1-induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP-stimulated membrane fission activity seen for AMPH-1 is recapitulated by the heterodimeric N-BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP-stimulated membrane fission is a general property of this important class of N-BAR proteins., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The activity of the matrix metalloproteinase (MMP) MT1-MMP is strictly regulated by expression and cellular location. In macrophages LPS activation leads to the up-regulation of MT1-MMP and this need to be at the cell surface for them to degrade the dense extracellular matrix (ECM) components to create a path to migrate into injured and infected tissues. Fixed and live imaging shows newly made MT1-MMP is packaged into vesicles that traffic to and fuse with LBPA + LAMP1 + late endosomes en route to the surface. The R-SNARE VAMP4, found on Golgi-derived vesicles that traffic to late endosomes, forms a trans-SNARE complex with the Q-SNARE complex Stx6/Stx7/Vti1b. The Stx6/Stx7/Vti1b complex has been shown to be up-regulated in lipopolysaccharide (LPS)-activated cells to increase trafficking of key cytokines through the classical pathway and now we show here it is up-regulation also plays a role in the late endosomal pathway of MT1-MMP trafficking. Depletion of any of the SNAREs in this complex reduces surface MT1-MMP and gelatin degradation. Conversely, overexpression of the Stx6/Stx7/Vti1b components increases surface MT1-MMP levels. This suggests that Stx6/Stx7/Vti1b is a key Q-SNARE complex in macrophages during an immune response and in partnership with VAMP4 it regulates transport of newly made MT1-MMP., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Intercellular organelle transfer has been documented in several cell types and has been proposed to be important for cell-cell communication and cellular repair. However, the mechanisms by which organelle transfer occurs are uncertain. Recent studies indicate that the gap junction protein, connexin 43 (Cx43), is required for mitochondrial transfer but its specific role is unknown. Using three-dimensional electron microscopy and immunogold labeling of Cx43, this report shows that whole organelles including mitochondria and endosomes are incorporated into double-membrane vesicles, called connexosomes or annular gap junctions, that form as a result of gap junction internalization. These findings demonstrate a novel mechanism for intercellular organelle transfer mediated by Cx43 gap junctions., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Retromer core complex is an endosomal scaffold that plays a critical role in orchestrating protein trafficking within the endosomal system. Here we characterized the effect of the Parkinson's disease-linked Vps35 D620N in the endo-lysosomal system using Vps35 D620N rescue cell models. Vps35 D620N fully rescues the lysosomal and autophagy defects caused by retromer knock-out. Analogous to Vps35 knock out cells, the endosome-to-trans-Golgi network transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) is impaired in Vps35 D620N rescue cells because of a reduced capacity to form endosome transport carriers. Cells expressing the Vps35 D620N variant have altered endosomal morphology, resulting in smaller, rounder structures with less tubule-like branches. At the molecular level retromer incorporating Vps35 D620N variant has a decreased binding to retromer associated proteins wiskott-aldrich syndrome protein and SCAR homologue (WASH) and SNX3 which are known to associate with retromer to form the endosome transport carriers. Hence, the partial defects on retrograde protein trafficking carriers in the presence of Vps35 D620N represents an altered cellular state able to cause Parkinson's disease., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Modern cell biology is now rich with data acquired at the whole genome and proteome level. We can add value to this data through integration and application of specialist knowledge. To illustrate, we will focus on the SNARE and RAB proteins; key regulators of intracellular fusion specificity and organelle identity. We examine published mass spectrometry data to gain an estimate of protein copy number and organelle distribution in HeLa cells for each family member. We also survey recent global CRISPR/Cas9 screens for essential genes from these families. We highlight instances of co-essentiality with other genes across a large panel of cell lines that allows for the identification of functionally coherent clusters. Examples of such correlations include RAB10 with the SNARE protein Syntaxin4 (STX4) and RAB7/RAB21 with the WASH and the CCC (COMMD/CCDC22/CCDC93) complexes, both of which are linked to endosomal recycling pathways., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome-to-Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP-activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P 2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P 2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability., (© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.)

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane-type-1 matrix metalloproteinase (MT1-MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1-MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1-MMP. Upon lipopolysaccharide (LPS) activation, MT1-MMP synthesis dramatically increases 10-fold at the surface by 15 hours. MT1-MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R-SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q-SNARE complex Stx4/SNAP23 to regulate MT1-MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1-MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1-MMP transport through this pathway could reduce macrophage migration and the resulting inflammation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution., (© 2019 The Authors Traffic Published by John Wiley & Sons Ltd.)

The endosomal system plays an essential role in cell homeostasis by controlling cellular signaling, nutrient sensing, cell polarity and cell migration. However, its place in the regulation of tissue, organ and whole body physiology is less well understood. Recent studies have revealed an important role for the endosomal system in regulating glucose and lipid homeostasis, with implications for metabolic disorders such as type 2 diabetes, hypercholesterolemia and non-alcoholic fatty liver disease. By taking insights from in vitro studies of endocytosis and exploring their effects on metabolism, we can begin to connect the fields of endosomal transport and metabolic homeostasis. In this review, we explore current understanding of how the endosomal system influences the systemic regulation of glucose and lipid metabolism in mice and humans. We highlight exciting new insights that help translate findings from single cells to a wider physiological level and open up new directions for endosomal research., (© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.)

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT-mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer-like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed "Retriever," this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called "Commander" to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus-end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward-directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP-170, as well as the association of CLIP-170 to MT plus-ends. In response to HGF, entry of Met into Rab4-positive endosomes results in Golgi-localized γ-ear-containing Arf-binding protein 3 (GGA3) and CLIP-170 recruitment to an activated Met RTK complex. We conclude that CLIP-170 co-ordinates the recycling and the transport of Met-positive endocytic vesicles to plus-ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration., (© 2018 The Authors. Traffic published by John Wiley & Sons Ltd.)

Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra- and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target-specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis. Sequential application of the different subprotocols allows isolation of morphological and functional intact organelles from one pool of cells. The enrichment is based on a selective labelling using receptor ligands or antibodies together with superparamagnetic microbeads followed by separation in a patented matrix-free high-gradient magnetic purification device. This unique magnetic chamber is based on a focusing system outside of the empty separation column, generating an up to 3 T high-gradient magnetic field focused at the wall of the column., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

G protein-coupled receptors (GPCRs) physically connect extracellular information with intracellular signal propagation. Membrane trafficking plays a supportive role by "bookending" signaling events: movement through the secretory pathway delivers GPCRs to the cell surface where receptors can sample the extracellular environment, while endocytosis and endolysosomal membrane trafficking provide a versatile system to titrate cellular signaling potential and maintain homeostatic control. Recent evidence suggests that, in addition to these important effects, GPCR trafficking actively shapes the cellular signaling response by altering the location and timing of specific receptor-mediated signaling reactions. Here, we review key experimental evidence underlying this expanding view, focused on GPCR signaling mediated through activation of heterotrimeric G proteins located in the cytoplasm. We then discuss lingering and emerging questions regarding the interface between GPCR signaling and trafficking., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high-throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non-redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image-based tool to dissect guard cell responses and outline a genetic framework of stomatal closure., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein-coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo-lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo-lysosomal sorting and beyond., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Amyloid accumulation in the brain is the major pathological hallmark of Alzheimer disease (AD). Amyloid beta (Aβ) is cleared by the endosomal-autophagy-lysosomal system, which is impaired in AD pathogenesis by an unknown mechanism. Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been demonstrated to decrease the level of Aβ in APP/PS1 mouse brain and to protect neurons by inhibiting the activation of microglia in vitro. The present study showed that PF11 was capable of increasing the uptake and degradation of oligomeric Aβ in cultured microglia. Oligomeric Aβ (oAβ) interrupted the autophagy-lysosomal degradative system by regulating the nuclear translocation of transcription factor EB (TFEB), a master factor in lysosomal biogenesis. Conversion of Rab5 to Rab7, which is important for the mechanism of cargo progression from early to late endosomes, was also interrupted by high-concentration oAβ. Notably, in the PF11-treated microglial cells, a dramatic increase of the lysosome-associated proteins and enzyme expression were observed, along with the intracellular pH steady state, indicating the improvement of lysosomal function. In addition, PF11 induced TFEB nuclear translocation in microglia treated with high-concentration oAβ. Furthermore, PF11 was able to restore Rab conversion, suggesting an effective role of PF11 in the maturation of endosomes. These data provide evidence that PF11 can reverse the dysfunction of the endosomal-lysosomal system induced by high-concentration oAβ in microglia, and this might be the main mechanism by which PF11 facilitates oAβ clearance. Accordingly, we propose that PF11 should be considered as a potential agent for treating AD., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The tubular endolysosomal network is a quality control system that ensures the proper delivery of internalized receptors to specific subcellular destinations in order to maintain cellular homeostasis. Although retromer was originally described in yeast as a regulator of endosome-to-Golgi receptor recycling, mammalian retromer has emerged as a central player in endosome-to-plasma membrane recycling of a variety of receptors. Over the past decade, information regarding the mechanism by which retromer facilitates receptor trafficking has emerged, as has the identification of numerous retromer-associated molecules including the WASH complex, sorting nexins (SNXs) and TBC1d5. Moreover, the recent demonstration that several SNXs can directly interact with retromer cargo to facilitate endosome-to-Golgi retrieval has provided new insight into how these receptors are trafficked in cells. The mechanism by which SNX17 cargoes are recycled out of the endosomal system was demonstrated to involve a retromer-like complex termed the retriever, which is recruited to WASH positive endosomes through an interaction with the COMMD/CCDC22/CCDC93 (CCC) complex. Lastly, the mechanisms by which bacterial and viral pathogens highjack this complex sorting machinery in order to escape the endolysosomal system or remain hidden within the cells are beginning to emerge. In this review, we will highlight recent studies that have begun to unravel the intricacies by which the retromer and associated molecules contribute to receptor trafficking and how deregulation at this sorting domain can contribute to disease or facilitate pathogen infection., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long-lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo-lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo-lysosomal degradative pathway-loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endocytosis is a crucial process in eukaryotic cells. The GTPases Rab 5, 21 and 22 that mediate endocytosis are ancient eukaryotic features and all available evidence suggests retained conserved function. In animals and fungi, these GTPases are regulated in part by proteins possessing Vps9 domains. However, the diversity, evolution and functions of Vps9 proteins beyond animals or fungi are poorly explored. Here we report a comprehensive analysis of the Vps9 family of GTPase regulators, combining molecular evolutionary data with functional characterization in the non-opisthokont model organism Trypanosoma brucei. At least 3 subfamilies, Alsin, Varp and Rabex5 + GAPVD1, are found across eukaryotes, suggesting that all are ancient features of regulation of endocytic Rab protein function. There are examples of lineage-specific Vps9 subfamily member expansions and novel domain combinations, suggesting diversity in precise regulatory mechanisms between individual lineages. Characterization of the Rabex5 + GAPVD1 and Alsin orthologues in T. brucei demonstrates that both proteins are involved in endocytosis, and that simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21, indicating conservation of function. These data demonstrate that, for the Vps9-domain family at least, modulation of Rab function is mediated by evolutionarily conserved protein-protein interactions., (© 2018 The Authors. Traffic published by John Wiley & Sons Ltd.)

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP-containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane-disrupting and cell-penetrating activities., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome-like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant-like features suggest T. gondii endocytic trafficking involves transit through early and late endosome-like compartments (ELCs) and potentially the trans-Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin-related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post-invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host-derived proteins, suggesting that DrpB is not required for parasite endocytosis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Increasing evidence has pointed to that dysregulation of the endo-lysosomal system is an early cellular phenotype of pathogenesis for Alzheimer's disease (AD). Rab5, a small GTPase, plays a critical role in mediating these processes. Abnormal overactivation of Rab5 has been observed in post-mortem brain samples of Alzheimer's patients as well as brain samples of mouse models of AD. Recent genome-wide association studies of AD have identified RIN3 (Ras and Rab interactor 3) as a novel risk factor for the disease. RIN3 that functions as a guanine nucleotide exchange factor for Rab5 may serve as an important activator for Rab5 in AD pathogenesis. In this review, we present recent research highlights on the possible roles of dysregulation of Rab5-mediated endocytic pathways in contributing to early pathogenesis of AD., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1; Video S2., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The retromer cargo-selective complex (CSC) comprising Vps35, Vps29 and Vps26 mediates the endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR). Or does it? Recently published data have questioned the validity of this long-established theory. Here, the evidence for and against a role for the retromer CSC in CIMPR endosome-to-Golgi retrieval is examined in the light of the new data that the SNX-BAR dimer is actually responsible for CIMPR retrieval., (© 2017 The Authors. Traffic published by John Wiley & Sons Ltd.)

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The protein complex composed of the kinase PIKfyve, the phosphatase FIG4 and the scaffolding protein VAC14 regulates the metabolism of phosphatidylinositol 3,5-bisphosphate, which serves as both a signaling lipid and the major precursor for phosphatidylinositol 5-phosphate. This complex is involved in the homeostasis of late endocytic compartments, but its precise role in maintaining the dynamic equilibrium of late endosomes, endolysosomes and lysosomes remains to be determined. Here, we report that inhibition of PIKfyve activity impairs terminal lysosome reformation from acidic and hydrolase-active, but enlarged endolysosomes. Our live-cell imaging and electron tomography data show that PIKfyve activity regulates extensive membrane remodeling that initiates reformation of lysosomes from endolysosomes. Altogether, our findings show that PIKfyve activity is required to maintain the dynamic equilibrium of late endocytic compartments by regulating the reformation of terminal storage lysosomes., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We analyzed plant-derived α1,4-fucosyltransferase (FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc-green fluorescent protein (GFP) or tomato LeFucTc-GFP restored Lewis-a formation in a fuctc mutant, confirming functionality in the trans-Golgi. AtFucTc-GFP partly accumulated at the nuclear envelope (NE) not observed for other homologs or truncated AtFucTc lacking the N-terminus or catalytic domain. Analysis of At/LeFucTc-GFP swap constructs with exchanged cytosolic, transmembrane and stalk (CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino-acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N-terminal GFP copies did, indicating localization at the inner nuclear membrane (INM). Tunicamycin treatment of AtFucTc-GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N-glycosylation. Yet neither expression in protoplasts of Arabidopsis N-glycosylation mutants nor elimination of the N-glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum (ER)-to-Golgi transport by co-expression of Sar1(H74L) trapped tunicamycin-released AtFucTc-GFP in the ER, however, without NE localization. Since recovery after tunicamycin-washout required de novo-protein synthesis, our analyses suggest that AtFucTc localizes to the NE/INM due to interaction with an unknown (glyco)protein., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)