A pathway for association of receptors, adaptors, and actin during endocytic internalization

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.     

Disruption of endocytic pathway regulatory genes activates autophagy in C. elegans

Autophagy and endocytic pathway are highly regulated catabolic processes. Both processes are crucial for cell growth, development, differentiation, disease and homeostasis and exhibit membrane rearrangement for their function. Autophagy and endocytic pathway represent branches of the lysosomal digestive system, autophagy being responsible for degradation of cytoplasmic components and endocytic pathway for degradation of exogenous substances. Here we report that autophagy is activated when endocytic pathway regulatory genes such as rab-5 and rabx-5 are disrupted. Defects in the ubiquitin binding domain of RABX-5 are critical in activating autophagy. We also observed that the elevated autophagy level does not contribute to lifespan extension of rabx-5 mutant. Our results suggest that autophagy may compensate for the endocytic pathway when regulatory genes for the endocytic pathway malfunction, providing a case of complementation between two functionally related cellular processes.     

Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic

Mucolipin-1 is a membrane protein encoded by the gene MCOLN1, mutations in which result in the lysosomal storage disorder mucolipidosis type IV (MLIV). Efficient lysosomal targeting of mucolipin-1 requires di-leucine motifs in both the N-terminal and the C-terminal cytosolic tails. We have shown that aberrant lactosylceramide trafficking in MLIV cells may be rescued by wild-type mucolipin-1 expression but not by mucolipin-1 mistargeted to the plasma membrane or by lysosome-localized mucolipin-1 mutated in its predicted ion pore-selectivity region. Our data demonstrate that the correct localization of mucolipin-1 and the integrity of its ion pore are essential for its physiological function in the late endocytic pathway.     

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. Here, we provide evidence for a clathrin-independent endocytic pathway in yeast. In cells lacking the clathrin-binding adaptor proteins Ent1, Ent2, Yap1801, and Yap1802, we identify a second endocytic pathway that depends on the GTPase Rho1, the downstream formin Bni1, and the Bni1 cofactors Bud6 and Spa2. This second pathway does not require components of the better-studied endocytic pathway, including clathrin and Arp2/3 complex activators. Thus, our results reveal the existence of a second pathway for endocytosis in yeast, which suggests similarities with the RhoA-dependent endocytic pathways of mammalian cells.     

A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann-Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.     

Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers

A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added.     

Yeast endocytosis

The presence of an endocytic pathway in cells from a wide range of species and the conservation of the proteins involved in this process throughout evolution suggest that endocytosis is of fundamental importance for the eukaryotic cell. However, some surprising recent results have shown that both Dictyostelium discoideum and Saccharomyces cerevisiae can live under laboratory conditions with substantially reduced levels of endocytosis. In this review, I concentrate on endocytosis in S. cerevisiae. Recent progress in the study of intermediates of the endocytic pathway and of mutants affecting the endocytic pathway make this organism an interesting model with which to study the mechanism and functions of endocytosis.     

HIP1: trafficking roles and regulation of tumorigenesis

During recent years, alterations in proteins of the endocytic pathway have been associated with tumors. Disrupted regulation of the endocytic pathway is a relatively unstudied mechanism of tumorigenesis, which can concomitantly disrupt several different signaling pathways to affect growth, differentiation and survival. Several endocytic proteins have been identified, either as part of tumor-associated translocations or to have the ability to transform cells. Here, we summarize the information known about huntingtin interacting protein 1 (HIP1), an endocytic protein with transforming properties that is involved in a cancer-causing translocation and which is overexpressed in a variety of human cancers. We describe the known normal functions of HIP1 in endocytosis and receptor trafficking, the evidence for its role as an oncoprotein and how HIP1 might be altered to promote tumorigenesis.     

Rare variants in the endocytic pathway are associated with Alzheimer’s disease,its related phenotypes,and functional consequences

Late-onset Alzheimer’s disease (LOAD) is the most common type of dementia causing irreversible brain damage to the elderly and presents a major public health challenge. Clinical research and genome-wide association studies have suggested a potential contribution of the endocytic pathway to AD, with an emphasis on common loci. However, the contribution of rare variants in this pathway to AD has not been thoroughly investigated. In this study, we focused on the effect of rare variants on AD by first applying a rare-variant gene-set burden analysis using genes in the endocytic pathway on over 3,000 individuals with European ancestry from three large whole-genome sequencing (WGS) studies. We identified significant associations of rare-variant burden within the endocytic pathway with AD, which were successfully replicated in independent datasets. We further demonstrated that this endocytic rare-variant enrichment is associated with neurofibrillary tangles (NFTs) and age-related phenotypes, increasing the risk of obtaining severer brain damage, earlier age-at-onset, and earlier age-of-death. Next, by aggregating rare variants within each gene, we sought to identify single endocytic genes associated with AD and NFTs. Careful examination using NFTs revealed one significantly associated gene, ANKRD13D. To identify functional associations, we integrated bulk RNA-Seq data from over 600 brain tissues and found two endocytic expression genes (eGenes), HLA-A and SLC26A7, that displayed significant influences on their gene expressions. Differential expressions between AD patients and controls of these three identified genes were further examined by incorporating scRNA-Seq data from 48 post-mortem brain samples and demonstrated distinct expression patterns across cell types. Taken together, our results demonstrated strong rare-variant effect in the endocytic pathway on AD risk and progression and functional effect of gene expression alteration in both bulk and single-cell resolution, which may bring more insight and serve as valuable resources for future AD genetic studies, clinical research, and therapeutic targeting.     

Signaling-mediated control of ubiquitin ligases in endocytosis

Ubiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation.     

Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking

Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.     

Interferon-γ–inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages

Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2–positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.     

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.     

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.     

Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation. Role of cholesterol and actin cytoskeleton

The endocytic compartment of eukaryotic cells is a complex intracellular structure involved in sorting, processing, and degradation of a great variety of internalized molecules. Recently, the uptake through caveolae has emerged as an alternative internalization pathway, which seems to be directly related with some signal transduction pathways. However, the mechanisms, molecules, and structures regulating the transport of caveolin from the cell surface into the endocytic compartment are largely unknown. In this study, normal quiescent fibroblasts (normal rat kidney (NRK)) were used to demonstrate that epidermal growth factor causes partial redistribution of caveolin from the cell surface into a cellubrevin early endocytic compartment. Treatment of NRK cells with cytochalasin D or latrunculin A inhibits this pathway and the concomitant activation of Mek and mitotic-activated protein (MAP) kinase; however, if cells were pre-treated with filipin, cytochalasin D does not inhibit the phosphorylation of MAP kinase induced by epidermal growth factor. From these results we conclude that in NRK cells the intact actin cytoskeleton is necessary for the EGF-mediated transport of caveolin from the cell surface into the early endocytic compartment and the activation of MAP kinase pathway.     

A role for adaptor protein-3 complex in the organization of the endocytic pathway in Dictyostelium

Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium.     

Signaling on the endocytic pathway

Endocytosis regulates many cellular signaling processes by controlling the number of functional receptors available at the cell surface. Conversely, some signaling processes regulate the endocytic pathway. Furthermore, various cellular signaling events appear to occur on endosome membranes. The endocytic pathway, by providing a set of dynamic and biochemically specialized endomembrane structures that physically communicate with the plasma membrane, is increasingly viewed as a highly flexible scaffold for mediating precise spatiotemporal control and transport of diverse biological signals. General principles of endosome-based signaling are beginning to emerge but, in many cases, the physiological significance of signaling on the endocytic pathway remains poorly understood.