Dynamin-2 mediates clathrin-dependent endocytosis for amyloid-ß internalization in brain microvascular endothelial cells.

Dynamin is recognized as a crucial regulator for membrane fission and has three isoforms in mammals. But the expression patterns of dynamin isoforms and their roles in non-neuronal cells are incompletely understood. In this study, the expression profiles of dynamin isoforms and their roles in endocytosis was investigated in brain endothelial cells. We found that Dyn2 was expressed at highest levels, whereas the expression of Dyn1 and Dyn3 were far less than Dyn2. Live-cell imaging was used to investigate the effects of siRNA-mediated knockdown of individual dynamin isoforms on transferrin uptake, and we found that Dyn2, but not Dyn1 or Dyn3, is required for the endocytosis in brain endothelial cells. Results of dextran uptake assay showed that dynamin isoforms are not involved in the clathrin-independent fluid-phase internalization of brain endothelial cells, suggesting the specificity of the role of Dyn2 in clathrin-dependent endocytosis. Immunofluorescence and electron microscopy analysis showed that Dyn2 co-localizes with clathrin and acts at the late stage of vesicle fission in the process of endocytosis. Further results showed that Dyn2 is necessary for the basolateral-to-apical internalization of amyloid-ß into brain endothelial cells. We concluded that Dyn2, but not Dyn1 or Dyn3, mediates the clathrin-dependent endocytosis for amyloid-ß internalization particularly from basolateral to apical side into brain endothelial cells.

A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells.

Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with Dyn2 but inactivated in non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3ß) kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual- and triple-channel total internal reflection fluorescence microscopy, we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2 but not Dyn1 exhibits a pronounced burst of assembly, presumably into supramolecular collar-like structures that drive membrane scission and clathrin-coated vesicle (CCV) formation. Activation of Dyn1 by acute inhibition of GSK3ß results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation, and decreased CCP lifetimes but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME that occur well upstream of fission, even when present at low, substoichiometric levels relative to Dyn2. Under physiological conditions, Dyn1 is activated downstream of epidermal growth factor receptor (EGFR) signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoform-specific regulatory role for Dyn1 in CME.

Mechanoregulation of clathrin-mediated endocytosis.

We characterized the tension response of clathrin-mediated endocytosis by using various cell manipulation methodologies. Elevated tension in a cell hinders clathrin-mediated endocytosis through inhibition of de novo coat initiation, elongation of clathrin coat lifetimes and reduction of high-magnitude growth rates. Actin machinery supplies an inward pulling force necessary for internalization of clathrin coats under high tension. These findings suggest that the physical cues cells receive from their microenvironment are major determinants of clathrin-mediated endocytic activity.

Single-Particle Tracking Reveals the Sequential Entry Process of the Bunyavirus Severe Fever with Thrombocytopenia Syndrome Virus.

The Bunyavirales is one of the largest groups of RNA viruses, which encompasses many strains that are highly pathogenic to animals and humans. Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans, with a high fatality rate of up to 30%. To date, the entry process of bunyavirus infection remains obscure. Here, using quantum dot (QD)-based single-particle tracking and multicolor imaging, the dynamic molecular process of SFTSV entry and penetration is systematically dissected. The results show that internalization of SFTSV into host cells is initiated by recruiting clathrin onto the cell membrane for the formation of clathrin-coated pits and further pinching off from the plasma membrane to form discrete vesicles. These vesicular carriers further deliver virions to Rab5+ early endosomes, and then to Rab7+ late endosomes. The intracellular transport of virion-carrying endocytic vesicles is dependent first on actin filaments at the cell periphery, and then on microtubules toward the cell interior. The final fusion events occur at ≈15-60 min post-entry, and are triggered by the acidic environment at ≈pH5.6 within the late endosomes. These results reveal the multistep SFTSV entry process and the dynamic virus-host interactions involved.

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function.

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance.

Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology.

Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.

A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly.

The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

Ultrastructural correlates of antidiuretic response of rat kidney.

We studied ultrastructural features of epithelial cells of the inner medullary collecting tubules in Brattleboro and Wistar under the action of desmopressin (dDAVP, 5 µg/100 g body weight for 2 days). Intracellular reorganization of transepithelial barrier for osmotic water transport depended on the capacity of rats to the synthesis of endogenous vasopressin.

The function of endocytosis in podocytes.

PURPOSE OF REVIEW: In this review, we discuss the role of endocytosis, a fundamental process internalizing molecules from the plasma membrane, and its critical importance in podocyte biology. RECENT FINDINGS: Endocytic clathrin and nonclathrin-coated pits have been visualized in podocytes using electron microscopy, but the functional biological relevance has not been well defined. Recent evidence suggests that loss of key clathrin endocytic regulatory apparatus, such as dynamin, synaptojanin 1 or endophilin, in genetic mouse models of disease results in severe proteinuria and foot process effacement. In addition, several genes implicated in human nephrotic syndrome directly or indirectly associate with these endocytic proteins, thus creating a protein network that is linked in actin dynamics, signalling and endocytosis. SUMMARY: This review summarizes our current understanding of membrane trafficking specifically in podocytes, thus giving further novel insights into the molecular mechanisms and pathogenesis of nephrotic syndrome.

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit.

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)-RME-6 fusion protein primarily localizes to clathrin-coated pits, physically interacts with alpha-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants, transport from the plasma membrane to endosomes is defective, and small 110-nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin-coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes.

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell's sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.

Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution.

Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the "pseudo 3D" morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

CAR-associated vesicular transport of an adenovirus in motor neuron axons.

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover.

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor beta (TGF-beta) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-beta receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-beta signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-beta receptors into distinct endocytic compartments regulates Smad activation and receptor turnover.

Direct supercritical angle localization microscopy for nanometer 3D superresolution.

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

Loss of myosin VI no insert isoform (NoI) induces a defect in clathrin-mediated endocytosis and leads to caveolar endocytosis of transferrin receptor.

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell's waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell's waltzer fibroblasts.

EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.

EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.