Mode Of Endocytosis Research Articles

SNX9 family mediates βarrestin-independent GPCR endocytosis

Agonist-stimulated GPCR endocytosis typically occurs via the multi-faceted adaptor proteins known as βarrestins. However, endocytosis of several GPCRs occurs independently of β-arrestins, suggesting an additional mode of GPCR endocytosis, but the mechanisms remain unknown. Here we provide evidence that sorting nexin 9 (SNX9), a previously described endocytic remodeling protein, functions as a novel cargo adaptor that promotes agonist-stimulated GPCR endocytosis. We show that SNX9 and SNX18, but not β-arrestins, are necessary for endocytosis of the chemokine receptor CXCR4. SNX9 is recruited to CXCR4 at the plasma membrane and interacts directly with the carboxyl-terminal tail of the receptor in a phosphorylation-dependent manner. We also provide evidence that some receptors do not require SNX9 and SNX18 nor β-arrestins for endocytosis, suggesting additional modes for GPCR endocytosis. These results provide novel insights into the mechanisms regulating GPCR trafficking and broaden our overall understanding of GPCR regulation.

EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes β-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

HOPS, CORVET and newly-identified Hybrid tethering complexes contribute differentially towards multiple modes of endocytosis

Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic fusion and vacuole protein sorting (HOPS) and class C core endosomal vacuole tethering (CORVET) interact with Rab-GTPases and SNARE proteins to regulate vesicle transport, fusion, and maturation in autophagy and endocytosis pathways. In this study, we identified two novel “Hybrid” tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced with the subunit from the other. Substrates taken up by receptor-mediated endocytosis or pinocytosis were transported by distinctive pathways, and the newly identified hybrid complexes contributed to pinocytosis in the presence of HOPS, whereas receptor-mediated endocytosis was exclusively dependent on HOPS. Our study provides new insights into the molecular mechanisms of the endocytic pathway and the function of the vacuolar protein sorting-associated (VPS) protein family.

SV2B defines a subpopulation of synaptic vesicles.

Synaptic vesicles can undergo several modes of exocytosis, endocytosis, and trafficking within individual synapses, and their fates may be linked to different vesicular protein compositions. Here, we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy. SV2B was almost completely absent from docked vesicles and a distinct cluster of vesicles found near the active zone. In contrast, SV2A was found in all domains of the synapse and was slightly enriched near the active zone. SV2B and SV2A were found on the membrane in the peri-active zone, suggesting the recycling from both clusters of vesicles. SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation. Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients. The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found more important for vesicle release.

Phosphatidylinositol 4-kinase IIα is a glycogen synthase kinase 3-regulated interaction hub for activity-dependent bulk endocytosis

Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3β (GSK3β) activity. We reveal the GSK3β substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3β site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3β-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis

Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo.

Four modes of endocytosis and subsequent synaptic vesicle (SV) recycling have been described at the presynapse to ensure the availability of SVs for synaptic release. However, it is unclear to what extend these modes operate under physiological activity patterns in vivo. The coat protein clathrin can regenerate SVs either directly from the plasma membrane (PM) via clathrin-mediated endocytosis (CME), or indirectly from synaptic endosomes by SV budding. Here, we examined the role of clathrin in SV recycling under physiological conditions by applying the clathrin inhibitor Pitstop-2 to the calyx of Held, a synapse optimized for high frequency synaptic transmission in the auditory brainstem, in vivo. The effects of clathrin-inhibition on SV recycling were investigated by serial sectioning scanning electron microscopy (S3EM) and 3D reconstructions of endocytic structures labeled by the endocytosis marker horseradish peroxidase (HRP). We observed large endosomal compartments as well as HRP-filled, black SVs (bSVs) that have been recently recycled. The application of Pitstop-2 led to reduced bSV but not large endosome density, increased volumes of large endosomes and shifts in the localization of both types of endocytic compartments within the synapse. These changes after perturbation of clathrin function suggest that clathrin plays a role in SV recycling from both, the PM and large endosomes, under physiological activity patterns, in vivo.

Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells.

Vesicle fusion at preestablished plasma membrane release sites releases transmitters and hormones to mediate fundamental functions like neuronal network activities and fight-or-flight responses. This half-a-century-old concept—fusion at well-established release sites in excitable cells—needs to be modified to include the sequential compound fusion reported here—vesicle fusion at previously fused Ω-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we real-time visualized sequential compound fusion pore openings and content releases in generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic ranges in excitable cells. We also visualized subsequent compound fusion pore closure, a new mode of endocytosis termed compound kiss-and-run that enhances vesicle recycling capacity. These results suggest modifying current exo-endocytosis concepts by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion and kiss-and-run take place to enhance exo-endocytosis capacity and dynamic ranges.

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome-cytopharynx complex of Trypanosoma cruzi.

Eating is fundamental and from this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment. As part of the world's aquatic ecosystems, the expansive family of heterotrophic protozoans uses self-generated currents to funnel prokaryotic prey into an ancient, yet highly enigmatic, oral apparatus known as the cytostome-cytopharynx complex prior to digestion. Despite its near ubiquitous presence in protozoans, little is known mechanistically about how this feeding organelle functions. Intriguingly, one class of these flagellated phagotrophic predators known as the kinetoplastids gave rise to a lineage of obligate parasitic protozoa, the trypanosomatids, that can infect a wide variety of organisms ranging from plants to humans. One parasitic species of humans, Trypanosoma cruzi, has retained this ancestral organelle much like its free-living relatives and continues to use it as its primary mode of endocytosis. In this review, we will highlight foundational observations made regarding the cytostome-cytopharynx complex and examine some of the most pressing questions regarding the mechanistic basis for its function. We propose that T.cruzi has the potential to serve as an excellent model system to dissect the enigmatic process of protozoal phagotrophy and thus enhance our overall understanding of fundamental eukaryotic biology.

FMRP Sustains Presynaptic Function via Control of Activity-Dependent Bulk Endocytosis.

Synaptic vesicle (SV) recycling is essential for the maintenance of neurotransmission, with a number of neurodevelopmental disorders linked to defects in this process. Fragile X syndrome (FXS) results from a loss of fragile X mental retardation protein (FMRP) encoded by the FMR1 gene. Hyperexcitability of neuronal circuits is a key feature of FXS, therefore we investigated whether SV recycling was affected by the absence of FMRP during increased neuronal activity. We revealed that primary neuronal cultures from male Fmr1 knock-out (KO) rats display a specific defect in activity-dependent bulk endocytosis (ADBE). ADBE is dominant during intense neuronal activity, and this defect resulted in an inability of Fmr1 KO neurons to sustain SV recycling during trains of high-frequency stimulation. Using a molecular replacement strategy, we also revealed that a human FMRP mutant that cannot bind BK channels failed to correct ADBE dysfunction in KO neurons, however this dysfunction was corrected by BK channel agonists. Therefore, FMRP performs a key role in sustaining neurotransmitter release via selective control of ADBE, suggesting intervention via this endocytosis mode may correct the hyperexcitability observed in FXS.SIGNIFICANCE STATEMENT Loss of fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), however whether its loss has a direct role in neurotransmitter release remains a matter of debate. We demonstrate that neurons lacking FMRP display a specific defect in a mechanism that sustains neurotransmitter release during intense neuronal firing, called activity-dependent bulk endocytosis (ADBE). This discovery provides key insights into mechanisms of brain communication that occur because of loss of FMRP function. Importantly it also reveals ADBE as a potential therapeutic target to correct the circuit hyperexcitability observed in FXS.

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.

Transsynaptic N-Cadherin Adhesion Complexes Control Presynaptic Vesicle and Bulk Endocytosis at Physiological Temperature.

At mammalian glutamatergic synapses, most basic elements of synaptic transmission have been shown to be modulated by specific transsynaptic adhesion complexes. However, although crucial for synapse homeostasis, a physiological regulation of synaptic vesicle endocytosis by adhesion molecules has not been firmly established. The homophilic adhesion protein N-cadherin is localized at the peri-active zone, where the highly temperature-dependent endocytosis of vesicles occurs. Here, we demonstrate an important modulatory role of N-cadherin in endocytosis at near physiological temperature by synaptophysin-pHluorin imaging. Different modes of endocytosis including bulk endocytosis were dependent on N-cadherin expression and function. N-cadherin modulation might be mediated by actin filaments because actin polymerization ameliorated the knockout-induced endocytosis defect. Using super-resolution imaging, we found strong recruitment of N-cadherin to glutamatergic synapses upon massive vesicle release, which might in turn enhance vesicle endocytosis. This provides a novel, adhesion protein-mediated mechanism for efficient coupling of exo- and endocytosis.

Minibrain kinase and calcineurin coordinate activity-dependent bulk endocytosis through synaptojanin.

Neurons use multiple modes of endocytosis, including clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), during mild and intense neuronal activity, respectively, to maintain stable neurotransmission. While molecular players modulating CME are well characterized, factors regulating ADBE and mechanisms coordinating CME and ADBE activations remain poorly understood. Here we report that Minibrain/DYRK1A (Mnb), a kinase mutated in autism and up-regulated in Down's syndrome, plays a novel role in suppressing ADBE. We demonstrate that Mnb, together with calcineurin, delicately coordinates CME and ADBE by controlling the phosphoinositol phosphatase activity of synaptojanin (Synj) during varying synaptic demands. Functional domain analyses reveal that Synj's 5'-phosphoinositol phosphatase activity suppresses ADBE, while SAC1 activity is required for efficient ADBE. Consequently, Parkinson's disease mutation in Synj's SAC1 domain impairs ADBE. These data identify Mnb and Synj as novel regulators of ADBE and further indicate that CME and ADBE are differentially governed by Synj's dual phosphatase domains.

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy.

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

Endocytosis and the Participation of Glycosaminoglycans Are Important to the Mechanism of Cell Death Induced by β-Hairpin Antimicrobial Peptides.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for β-hairpin peptides.

Spatial arrangements of spherical nanoparticles on lipid vesicles.

We report results of a numerical investigation of the modes of adhesion of two spherical nanoparticles (NPs) on lipid vesicles based on molecular dynamics simulations, in conjunction with the weighted histogram analysis method, of an implicit-solvent model of self-assembled membranes. Our investigation shows that the NPs exhibit a sequence of three modes of adhesion. For low adhesive interactions, the adhering NPs are apart from each other. As the adhesive interaction is increased, the NPs dimerize into in-plane dimers. As the adhesive interaction is further increased for relatively large vesicles, the NPs dimerize into tubular dimers. However, for small vesicles, the tubular dimer state is not observed. For higher values of the adhesive interaction, four endocytosis modes are observed, depending on the initial locations of the NPs on the vesicle and the relative size of the NPs with respect to that of the vesicle. For relatively large vesicles, the NPs are endocytosed individually or as a dimer. For relatively small vesicles, only one NP is endocytosed if the initial distance between the NPs is large, while the second NP remains adhered to the outer leaflet of the vesicle. However, if the initial distance between the NPs is small, one NP is endocytosed, while the other is internalized in the vesicle through a pore.

Visualizing Membrane Transformation in Mediating Endocytosis and Generating Various Endocytic Modes in Live Cells

Visualizing membrane transformation of both endocytosis and exocytosis in live neuroendocrine cells, we found that endocytic transitions and kiss-and-run (fusion-pore-opening-and-closure) mediate various modes of endocytosis that may differed in speeds, amount and vesicle sizes.

Monitoring Activity-Dependent Bulk Endocytosis in Primary Neuronal Culture Using Large Fluorescent Dextrans.

The efficient recycling of synaptic vesicles (SVs) during neuronal activity is central for sustaining brain function. During intense neuronal activity, the dominant mechanism of SV retrieval is activity-dependent bulk endocytosis (ADBE). Here, we describe a method to monitor ADBE in isolation from other SV endocytosis modes, via the uptake of large fluorescent fluid-phase markers in primary neuronal culture. Furthermore, we outline how to monitor ADBE using this approach across a field of neurons or in individual neurons.

The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms.

Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For example, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these diverse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of individual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and individual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.

Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.