Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that the sphingomyelin (SM)-sequestered cholesterol, but not the accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol-independent. Whereas, the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein which activates actin nucleation, is recruited to IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. Importance: IAV infects the cells by harnessing cellular endocytic machineries. Better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies, and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol-independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results would provide new insights into IAV infection and pathway/cargo-specific involvement of cholesterol pool(s).

Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

SH3Ps recruit auxilin-like vesicle uncoating factors into clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is an essential process of cellular cargo uptake operating in all eukaryotes. In animal and yeast, CME involves BAR-SH3 domain proteins, endophilins and amphiphysins, which function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains BAR-SH3 domain proteins SH3P1-3, but their role is poorly understood. We identify SH3P1-3 as functional homologues of endophilin/amphiphysin. SH3P1-3 bind to discrete foci at the plasma membrane (PM), and colocalization indicates late recruitment of SH3P2 to a subset of clathrin-coated pits. PM recruitment pattern of SH3P2 is nearly identical to its interactor, a putative vesicle uncoating factor AUXILIN-LIKE1, and SH3P1-3 are required for most of AUXILIN-LIKE1 PM binding. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors, rather than the uncoating phosphatases synaptojanins. Furthermore, we identify an unexpected redundancy between SH3P1-3 and a plant-specific endocytic adaptor, TPLATE complex, showing a contribution of SH3P1-3 to gross CME.

Developmental patterning function of GNOM ARF-GEF mediated from the plasma membrane

The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.

Free diffusion of PI(4,5)P2 in the plasma membrane in the presence of high density effector protein complexes

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. It engages effector proteins that regulate diverse traffic, transport, signaling and cytoskeletal processes that define PM structure and function. How a single class of lipid molecules independently regulate so many parallel processes remains an open question. We tested the hypothesis that spatially segregated pools of PI(4,5)P2 are associated with, and thus reserved for regulation of, different functional complexes in the PM. The mobility of PI(4,5)P2 in the membrane was measured using lipid biosensors by single particle tracking photoactivation localization microscopy (sptPALM). We found that PI(4,5)P2, and several other classes of inner PM lipids, diffuse rapidly at approximately 0.3 microns squared per second with largely Brownian motion, although they spend approximately a third of their time diffusing much more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes, such endoplasmic-reticulum:PM contact sites, clathrin-coated structures, and several actin cytoskeletal elements including focal adhesions, did not cause a change in PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons were observed to produce a slowing of PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2-engaging effector proteins, such as clathrin coated pits and focal adhesions, do not corral free PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating parallel PM functions.

Dynamin conditional knockout fibroblasts: Tamoxifen inducible Knockout method v1

This cell line was described and characterized in the following paper: Ferguson, S.M., Raimondi, A., Paradise, S., Shen, H., Mesaki, K., Ferguson, A., Destaing, O., Ko, G., Takasaki, J., Cremona, O., O’Toole, E., De Camilli P. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Developmental Cell 17, 811-822, 2009. PMID: 20059951]. This procedure describes tamoxifeninducible KO method using this cell line.

Clathrin-mediated endocytosis-independent function of VAN3 ARF GTPase Activating Protein at the plasma membrane

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. The Arabidopsis thaliana ARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally in van3-1 knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.

SUMOylation of the Kv4.2 Ternary Complex Increases Surface Expression and Current Amplitude by Reducing Internalization in HEK 293 Cells

Kv4 α-subunits exist as ternary complexes (TC) with potassium channel interacting proteins (KChIP) and dipeptidyl peptidase-like proteins (DPLP); multiple ancillary proteins also interact with the α-subunits throughout the channel’s lifetime. Dynamic regulation of Kv4.2 protein interactions adapts the transient potassium current, IA, mediated by Kv4 α-subunits. Small ubiquitin-like modifier (SUMO) is an 11 kD peptide post-translationally added to lysine (K) residues to regulate protein–protein interactions. We previously demonstrated that when expressed in human embryonic kidney (HEK) cells, Kv4.2 can be SUMOylated at two K residues, K437 and K579. SUMOylation at K437 increased surface expression of electrically silent channels while SUMOylation at K579 reduced IA maximal conductance (Gmax) without altering surface expression. KChIP and DPLP subunits are known to modify the pattern of Kv4.2 post-translational decorations and/or their effects. In this study, co-expressing Kv4.2 with KChIP2a and DPP10c altered the effects of enhanced Kv4.2 SUMOylation. First, the effect of enhanced SUMOylation was the same for a TC containing either the wild-type Kv4.2 or the mutant K437R Kv4.2, suggesting that either the experimental manipulation no longer enhanced K437 SUMOylation or K437 SUMOylation no longer influenced Kv4.2 surface expression. Second, instead of decreasing IA Gmax, enhanced SUMOylation at K579 now produced a significant ∼37–70% increase in IA maximum conductance (Gmax) and a significant ∼30–50% increase in Kv4.2g surface expression that was accompanied by a 65% reduction in TC internalization. Blocking clathrin-mediated endocytosis (CME) in HEK cells expressing the Kv4.2 TC mimicked and occluded the effect of SUMO on IA Gmax; however, the amount of Kv4.2 associated with the major adaptor for constitutive CME, adaptor protein 2 (AP2), was not SUMO dependent. Thus, SUMOylation reduced Kv4.2 internalization by acting downstream of Kv4.2 recruitment into clathrin-coated pits. In sum, the two major findings of this study are: SUMOylation of Kv4.2 at K579 regulates TC internalization most likely by promoting channel recycling. Additionally, there is a reciprocity between Kv4.2 SUMOylation and the Kv4.2 interactome such that SUMOylation regulates the interactome and the interactome influences the pattern and effect of SUMOylation.

Phosphorylation of AMPA receptor subunit GluA1 regulates clathrin-mediated receptor internalization

Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic up-scaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 Serine 845 (S845) predominantly plays a role in receptor internalization than forward trafficking during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor, AP2, which recruits cargo proteins into endocytic clathrin coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation by two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization, and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.

Label-retention expansion microscopy

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.