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Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network. Macropinocytosis permits the cellular uptake of fluids and nutrients via macropinosomes. Here, the authors show that Phafin2 is required for the formation of macropinosomes and permits their transit through dense actin networks. [ABSTRACT FROM AUTHOR]

The endosomal sorting complex required for transport (ESCRT) machinery is an assembly of protein subcomplexes (ESCRT I-III) that cooperate with the ATPase VPS4 to mediate scission of membrane necks from the inside. The ESCRT machinery has evolved as a multipurpose toolbox for mediating receptor sorting, membrane remodeling, and membrane scission, with ESCRT-III as the major membrane-remodeling component. Cellular membrane scission processes mediated by ESCRT-III include biogenesis of multivesicular endosomes, budding of enveloped viruses, cytokinetic abscission, neuron pruning, plasma membrane wound repair, nuclear pore quality control, nuclear envelope reformation, and nuclear envelope repair. We describe here the involvement of the ESCRT machinery in these processes and review current models for how ESCRT-III-containing multimeric filaments serve to mediate membrane remodeling and scission. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) makes abundant contacts with endosomes, and the numbers of contact sites increase as endosomes mature. It is already clear that such contact sites have diverse compositions and functions, but in this mini-review we will focus on two particular types of ER-endosome contact sites that regulate endosome positioning. Formation of ER-endosome contact sites that contain the cholesterol-binding protein oxysterol-binding protein-related protein 1L (ORP1L) is coordinated with loss of the minus-end-directed microtubule motor Dynein from endosomes. Conversely, formation of ER-endosome contact sites that contain the Kinesin-1-binding protein Protrudin results in transfer of the plus-end-directed microtubule motor Kinesin-1 from ER to endosomes. We discuss the possibility that formation of these two types of contact sites is coordinated as a 'gear-shift' mechanism for endosome motility, and we review evidence that Kinesin-1-mediated motility of late endosomes (LEs) to the cell periphery promotes outgrowth of neurites and other protrusions. [ABSTRACT FROM AUTHOR]

The main organelles of the secretory and endocytic pathways-the endoplasmic reticulum (ER) and endosomes, respectively-are connected through contact sites whose numbers increase as endosomes mature. One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase, whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein or mediate endosome fission. Cholesterol transfer and Ca2+ exchange have been proposed as additional functions of such sites. However, the compositions, activities and regulations of ER-endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth, forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE-ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER-LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth. [ABSTRACT FROM AUTHOR]

During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring. [ABSTRACT FROM AUTHOR]

Endocytosis and subsequent membrane traffic through endosomes are cellular processes that are integral to eukaryotic evolution, and numerous human diseases are associated with their dysfunction. Consequently, it is important to untangle the molecular machineries that regulate membrane dynamics and protein flow in the endocytic pathway. Central in this context is class III phosphatidylinositol 3-kinase, an evolutionarily conserved enzyme complex that phosphorylates phosphatidylinositol into phosphatidylinositol 3-phosphate. Phosphatidylinositol 3-phosphate recruits specific effector proteins, most of which contain FYVE or PX domains, to promote endocytosis, endosome fusion, endosome motility and endosome maturation, as well as cargo sorting to lysosomes, the biosynthetic pathway or the plasma membrane. Here we review the functions of key phosphatidylinositol 3-phosphate effectors in regulation of endocytic membrane dynamics and protein sorting. [ABSTRACT FROM AUTHOR]

Lysosomes fuse with the plasma membrane to help repair membrane lesions, but how they are positioned close to these lesions is not fully understood. Now, Encarnação et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511093) demonstrate that the lysosomal GTPase Rab3a and its effectors orchestrate lysosome positioning and plasma membrane repair. [ABSTRACT FROM AUTHOR]

Abstract: Clinical features characterizing Angelman syndrome, previously shown to be caused by disruption of UBE3A, were recently also described in neurologically disabled patients with mutations in SLC9A6, which encodes the Na+/H+ exchanger NHE6. In the present work we have focused on NHE6Δ255–256, the protein product of a specific 6-bp patient deletion in SLC9A6. To resolve the molecular mechanism causing the cellular dysfunction associated with this mutant, we have characterized its intracellular behaviour in comparison to wild type NHE6. Our study demonstrates that NHE6Δ255–256 is much less stable than the wild type protein. Whereas wild type NHE6 is transported to the plasma membrane and early endosomes and remains stable, NHE6Δ255–256 is degraded via two independent pathways mediated by proteasomes and lysosomes, respectively. Depletion of NHE6 had no detectable effect on endosomal pH, but co-depletion of NHE6 and the closely related NHE9 caused enhanced acidification of early endosomes. Our results suggest that NHE6 participates in regulation of endosomal pH and provides a cellular basis for understanding the loss of NHE6 function leading to a neurological phenotype resembling Angelman syndrome. [Copyright &y& Elsevier]

Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling. [ABSTRACT FROM AUTHOR]

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway. [Copyright &y& Elsevier]

The canonical Wnt/β-catenin signaling pathway regulates cell proliferation in development and adult tissue homeostasis. Dysregulated signaling contributes to human diseases, in particular cancer. Growing evidence suggests a role for clathrin and/or endocytosis in the regulation of this pathway, but conflicting results exist and demand a deeper mechanistic understanding. We investigated the consequences of clathrin depletion on Wnt/β-catenin signaling in cell lines and found a pronounced reduction in β-catenin protein levels, which affects the amount of nuclear β-catenin and β-catenin target gene expression. Although we found no evidence that clathrin affects β-catenin levels via endocytosis or multivesicular endosome formation, an inhibition of protein transport through the biosynthetic pathway led to reduced levels of a Wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), and cell adhesion molecules of the cadherin family, thereby affecting steady-state levels of β-catenin. We conclude that clathrin impacts on Wnt/β-catenin signaling by controlling exocytosis of transmembrane proteins, including cadherins and Wnt co-receptors that together control the membrane-bound and soluble pools of β-catenin. [ABSTRACT FROM AUTHOR]

Endosomes are known to undergo budding and fission reactions that separate regions destined for lysosomal degradation from carriers to be recycled to the plasma membrane. A recent paper (Rowland et al, ) shows that contact sites between endosomes and the endoplasmic reticulum ( ER) define the position and timing for fission. This uncovers an unanticipated role for the ER in controlling endosomal sorting and maturation. [ABSTRACT FROM AUTHOR]

Cancer cells break tissue barriers by use of small actin-rich membrane protrusions called invadopodia. Complete invadopodia maturation depends on protrusion outgrowth and the targeted delivery of the matrix metalloproteinase MT1-MMP via endosomal transport by mechanisms that are not known. Here, we show that the ER protein Protrudin orchestrates invadopodia maturation and function. Protrudin formed contact sites with MT1-MMP--positive endosomes that contained the RAB7-binding Kinesin-1 adaptor FYCO1, and depletion of RAB7, FYCO1, or Protrudin inhibited MT1-MMP--dependent extracellular matrix degradation and cancer cell invasion by preventing anterograde translocation and exocytosis of MT1- MMP. Moreover, when endosome translocation or exocytosis was inhibited by depletion of Protrudin or Synaptotagmin VII, respectively, invadopodia were unable to expand and elongate. Conversely, when Protrudin was overexpressed, noncancerous cells developed prominent invadopodia-like protrusions and showed increased matrix degradation and invasion. Thus, Protrudin-mediated ER--endosome contact sites promote cell invasion by facilitating translocation of MT1-MMP--laden endosomes to the plasma membrane, enabling both invadopodia outgrowth and MT1-MMP exocytosis. [ABSTRACT FROM AUTHOR]