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Insulin signaling is essential for glucose metabolism, and insulin decreases insulin receptor (InsR) levels in a dose-dependent and time-dependent manner. However, the regulatory mechanisms of InsR reduction upon insulin stimulation remain poorly understood. Here, we show that Eph receptor B4 (EphB4), a tyrosine kinase receptor that modulates cell adhesion and migration, can bind directly to InsR, and this interaction is markedly enhanced by insulin. Due to the adaptor protein 2 (Ap2) complex binding motif in EphB4, the interaction of EphB4 and InsR facilitates clathrin-mediated InsR endocytosis and degradation in lysosomes. Hepatic overexpression of EphB4 decreases InsR and increases hepatic and systemic insulin resistance in chow-fed mice, whereas genetic or pharmacological inhibition of EphB4 improve insulin resistance and glucose intolerance in obese mice. These observations elucidate a role for EphB4 in insulin signaling, suggesting that EphB4 might represent a therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including G alpha proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into beta-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.

International audience; Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables β3-integrin-mediated force generation independently of β1 integrin. β3-integrin-mediated forces were associated with a decrease in β3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in β3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

Plasma membrane damage and repair frequently happen in cells. A critical process underlying plasma membrane repair is to redirect repair factors, such as protein kinase C and the exocyst complex, from the polarized site to the damage site. However, the mechanism underlying the repair factor delivery to the damage site remains unknown. Here, we demonstrate that clathrin-mediated trafficking of repair factors is involved in plasma membrane/cell wall repair in budding yeast. Using laser-induced plasma membrane/cell wall damage assay, we identified phospholipid flippases, Lem3-Dnf1/Dnf2 and Cdc50-Drs2, as essential clathrin cargos for plasma membrane/cell wall repair. We found that flippase impairment significantly compromised the recruitment of exocyst Exo70 to the damage site. In contrast, the recruitment of protein kinase C (Pkc1) was only mildly compromised. Taken together, clathrin-mediated trafficking of the phospholipid flippases is critical for the recruitment of exocyst to the damage site. Mechanisms to redirect exocyst via the clathrin and flippase-mediated pathways may be a general feature of effective plasma membrane repair in polarized cells.

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is known to associate with AP180. An AP180 N-terminal homology domain (1–289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38–K39–K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages., Graphical abstract Image 1

As the main exchanger of electroneutral NaCl absorption, sodium-hydrogen exchanger isoform 3 (NHE3) circulates in the epithelial brush border (BB) and intracellular compartments in a multi-protein complex. The size of the NHE3 complex changes during rapid regulation events. Recycling regulation of NHE3 in epithelial cells can be roughly divided into three stages. First, when stimulated by Ca(2+), cGMP, and cAMP-dependent signaling pathways, NHE3 is converted from an immobile complex found at the apical microvilli (MV) into an easily internalized and mobile form that relocates to a compartment near the base of the MV. Second, NHE3 is internalized by clathrin and albumin-dependent pathways into cytoplasmic endosomal compartments, where the complex is reprocessed and reassembled. Finally, NHE3 is translocated from the recycling endosomes (REs) to the apex of epithelial cells, a process that can be stimulated by an increase in sodium-glucose cotransporter 1 (SGLT1) activity, epidermal growth factor receptor (EGFR) signaling, Ca(2+) signaling, and binding to βPix and SH3 and multiple ankyrin repeat domains 2 (Shank2) proteins. This review describes the molecular steps and protein interactions involved in the recycling movement of NHE3 from the apex of epithelial cells, into vesicles, where it is reprocessed and reassembled, and returned to its original location on the plasma membrane, where it exerts its physiological function.

Anther dehiscence is a crucial step for pollen grain release and male fertility. Filaments, which transport water, nutrients and hormones to the anthers, are important for anther dehiscence. In this study, we characterized the Arabidopsis microtubule organization 1 (MOR1) gene that involves in the filament functions and plays important roles in anther dehiscence. The Arabidopsis microtubule organization 1-1 (mor1-1) mutant exhibited an anther indehiscence phenotype at 24°C. Such the defect in anther dehiscence did not occur at a lower temperature (19°C). Further analysis indicated that both the cortical microtubule (CMT) organization and plasma membrane homeostasis were drastically impaired and disturbed in mor1-1 filament cells under the growth conditions of 24°C. Transmission electron microscopy (TEM) and FM4-64 up-take assays showed that endocytosis process in the mor1-1 filament cells were disrupted at 24°C. Furthermore, the cortical-associated RFP tagged clathrin light chain (CLC-RFP) foci were reduced in the mor1-1 filament cells. These results suggested that the MOR1-mediated CMT organization is important for clathrin-mediated endocytosis in the filament cells, and critical for anther dehiscence in thermosensitivity.

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation. This article is protected by copyright. All rights reserved.

The role of Golgi/endosome-localized clathrin adapters in the maintenance of steady-state cell surface epidermal growth factor receptor (EGFR) is not well known. Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation. Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes. Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation. Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4, and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model. Moreover, AP-1 was expressed in endosomes at higher levels in some cancer tissues. Collectively, these results suggest that AP-1 and GGA2 function in recycling endosomes to retrieve endocytosed EGFR, thereby sustaining its cell surface expression and, consequently, cancer cell growth.

Ligand-independent EGFR endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid signaling towards the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and PKA activity. This PA/PDE4/PKA pathway induces clathrin-dependent and clathrin-independent endocytosis followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this PA/PDE4/PKA pathway using biosensors of PA, cAMP and PKA in living cells. Propranolol used to inhibit PA hydrolysis triggers this pathway, which then activates p38 and ERK1/2 downstream PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increased the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is likely due to EGFR phosphorylation, as detected in Thr1047/1047 and Ser669 residues. However, EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin and Rab11, while a small proportion distributes to Alix-endosomes. This indicates a recycling arrest, which includes includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer. This article is protected by copyright. All rights reserved.

Adaptin proteins (APs) play a crucial role in intracellular cell trafficking. The ‘classical’ role of APs is carried out by AP1‒3, which bind to clathrin, cargo, and accessory proteins. Accordingly, AP1–3 are crucial for both vesicle formation and sorting. All APs consist of four subunits that are indispensable for their functions. In fact, based on studies using cells, model organism knockdown/knock-out, and human variants, each subunit plays crucial roles and contributes to the specificity of each AP. These studies also revealed that the sorting and intracellular trafficking function of AP can exert varying effects on pathology by controlling features such as cell development, signal transduction related to the apoptosis and proliferation pathways in cancer cells, organelle integrity, receptor presentation, and viral infection. Although the roles and functions of AP1‒3 are relatively well studied, the functions of the less abundant and more recently identified APs, AP4 and AP5, are still to be investigated. Further studies on these APs may enable a better understanding and targeting of specific diseases. APs known or suggested locations and functions., GRAPHICAL ABSTRACT

The clathrin-associated adaptor protein complex AP-1 stops STING signaling at the Golgi.

β-arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of β-arrestins is ligand-dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical ‘mode’ for GPCR-mediated triggering of the endocytic activity is presently known– displacement of the β-arrestin C-terminus (CT) to expose CCP-binding determinants that are masked in the inactive state. Here we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the β-arrestin CT and, instead, requires the cytosolic base of the β-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind β-arrestin transiently (‘class A’) primarily triggering the CLB-dependent mode and GPCRs that bind more stably (‘class B’) triggering both the CT and CLB-dependent modes in combination. Moreover, we show that each mode has opposing effects on the net signaling output of receptors– with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how β-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.

During clathrin-mediated endocytosis, a flat plasma membrane is shaped into an invagination that undergoes scission to form a vesicle. In mammalian cells, the force that drives the transition from invagination to vesicle is primarily provided by the GTPase dynamin that acts in concert with crescent-shaped BAR domain proteins. In yeast cells, the mechanism of endocytic scission is unclear. The yeast BAR domain protein complex Rvs161/167 (Rvs) nevertheless plays an important role in this process: deletion of Rvs dramatically reduces scission efficiency. A mechanistic understanding of the influence of Rvs on scission, however, remains incomplete. We used quantitative live-cell imaging and genetic manipulation to understand the recruitment and function of Rvs and other late-stage proteins at yeast endocytic sites. We found that arrival of Rvs at endocytic sites is timed by interaction of its BAR domain with specific membrane curvature. A second domain of Rvs167-the SH3 domain-affects localization efficiency of Rvs. We show that Myo3, one of the two type-I myosins in

Endocytosis is an essential mechanism for cellular uptake in many human tissues. A range of endocytic mechanisms occur including clathrin-dependent and -independent mechanisms. However, the role of endocytosis in the placenta and the spatial localisation of individual mechanisms is not well understood. The two principal cell layers that comprise the placental barrier to maternal-fetal transfer are the syncytiotrophoblast and fetal capillary endothelium. Endocytic uptake into the syncytiotrophoblast has been demonstrated for physiological maternal molecules such as transferrin-bound iron and low density lipoprotein (LDL) and may play an important role in the uptake of several other micronutrients, serum proteins, and therapeutics at both major placental cell barriers. These mechanisms may also mediate placental uptake of some viruses and nanoparticles. This review introduces the mechanisms of cargo-specific endocytosis and what is known about their localisation in the placenta, focussing predominantly on the syncytiotrophoblast. A fuller understanding of placental endocytosis is necessary to explain both fetal nutrition and the properties of the placental barrier. Characterising placental endocytic mechanisms and their regulation may allow us to identify their role in pregnancy pathologies and provide new avenues for therapeutic intervention.

Background The cellular prion protein (PrPC) is a membrane-bound, multifunctional protein mainly expressed in neuronal tissues. Recent studies indicate that the native trafficking of PrPC can be misused to internalize misfolded amyloid beta and α-synuclein (aSyn) oligomers. Objectives We define PrPC's role in internalizing misfolded aSyn in α-synucleinopathies and identify further involved proteins. Methods We performed comprehensive behavioral studies on four transgenic mouse models (ThySyn and ThySynPrP00, TgM83 and TgMPrP00) at different ages. We developed PrPC-(over)-expressing cell models (cell line and primary cortical neurons), used confocal laser microscopy to perform colocalization studies, applied mass spectrometry to identify interactomes, and determined disassociation constants using surface plasmon resonance (SPR) spectroscopy. Results Behavioral deficits (memory, anxiety, locomotion, etc.), reduced lifespans, and higher oligomeric aSyn levels were observed in PrPC-expressing mice (ThySyn and TgM83), but not in homologous Prnp ablated mice (ThySynPrP00 and TgMPrP00). PrPC colocalized with and facilitated aSyn (oligomeric and monomeric) internalization in our cell-based models. Glimepiride treatment of PrPC-overexpressing cells reduced aSyn internalization in a dose-dependent manner. SPR analysis showed that the binding affinity of PrPC to monomeric aSyn was lower than to oligomeric aSyn. Mass spectrometry-based proteomic studies identified clathrin in the immunoprecipitates of PrPC and aSyn. SPR was used to show that clathrin binds to recombinant PrP, but not aSyn. Experimental disruption of clathrin-coated vesicles significantly decreased aSyn internalization. Conclusion PrPC's native trafficking can be misused to internalize misfolded aSyn through a clathrin-based mechanism, which may facilitate the spreading of pathological aSyn. Disruption of aSyn-PrPC binding is, therefore, an appealing therapeutic target in α-synucleinopathies.

Specificity and sensitivity in biochemical reactions can be achieved through regulation of equilibrium binding affinity or through proofreading mechanisms that allow for the dissociation of unwanted intermediates. In this essay, we aim to provide our perspectives on how the concept of kinetic proofreading might apply in the context of cargo sorting in clathrin-mediated endocytosis.

The contents of numerous membrane lipids change upon ageing. However, it is unknown whether and how any of these changes are causally linked to lifespan regulation. Acyl chains contribute to the functional specificity of membrane lipids. In this study, working with C. elegans, we identified an acyl chain-specific sphingolipid, C22 glucosylceramide, as a longevity metabolite. Germline deficiency, a conserved lifespan-extending paradigm, induces somatic expression of the fatty acid elongase ELO-3, and behenic acid (22:0) generated by ELO-3 is incorporated into glucosylceramide for lifespan regulation. Mechanistically, C22 glucosylceramide is required for the membrane localization of clathrin, a protein that regulates membrane budding. The reduction in C22 glucosylceramide impairs the clathrin-dependent autophagic lysosome reformation, which subsequently leads to TOR activation and longevity suppression. These findings reveal a mechanistic link between membrane lipids and ageing and suggest a model of lifespan regulation by fatty acid-mediated membrane configuration., The membrane lipids change with ageing and function as regulatory molecules, but the underlying mechanisms are incompletely understood. Here, the authors identify C22 glucosylceramide as a regulator of the longevity transcription factor SKN-1, and show that C22 glucosylceramide regulates lifespan by controlling lysosome homeostasis and subsequent TOR activation.

Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE.

The COVID-19 pandemic that started in 2019 has left in its wake colossal devastation. While several countries continue to struggle with the second, third, and even the fourth waves of the pandemic and government agencies grapple with the difficult task of predicting how best to deal with emergent strains, vaccines have become available and are being deployed worldwide. This pandemic has only reinforced the redeeming quality of scientific research. Numerous research initiatives have culminated in providing knowledge on the viral genome, proteome, and protein structures that offer potentially druggable pathways for therapy. But a crucial ingredient to the success of the scientific endeavor has been the ability for communities of scientists to seamlessly disseminate data and thereby bring in fresh perspective. The sum total of all of these developments is that the world has witnessed a striking and unprecedent rapidity in scientific progress in tacking the pandemic. Its 2021 and we write this editorial with a sense of hope and promise of return to normal life. We would sincerely like to thank the commendable efforts of our contributing authors, who have come up with insightful reviews under circumstances that were anything but inspiring.

Clathrin-dependent endocytosis and exocytosis are coordinated to maintain cell homeostasis in Arabidopsis roots., Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.

Silver nanoparticles (AgNPs) have been reported as stressors for the bivalves' immune system at different regulatory levels, impacting the detection step and receptors, and other mediators, as well as effector molecules. However, studies on how AgNPs impact the transmission of signals from receptors and whether they have an effect on mediators and transcription factors are still scarce. This study aims to investigate the effect of 12 hours of in vivo exposure to 100 µg/L of AgNPs on the gene expression of the cytosolic adaptor Myeloid, the differentiation protein 88 (MgMyD88-b), and the interferon regulatory factor (Me4-IRF) in the gills and digestive gland of Mytilus galloprovincialis, before and after blocking two major uptake pathways of nanoparticles (clathrin- and caveolae-mediated endocytosis). The results illustrate a tissue-specific gene expression of the MgMyD88-b and the Me4-IRF in the gills and digestive gland of M. galloprovincialis. In the gills, AgNPs did not significantly impact the expression of the two genes. However, blocking the caveolae-mediated endocytosis decreased the expression of Me4-IRF. However, inhibition of clathrin-mediated endocytosis in the digestive gland recorded a significant decrease in the expression of MgMyD88-b. Overall, the inhibition of the AgNPs’ uptake routes have highlighted their potential interference with the immune response through the studied mediators’ genes, which need to be studied further in future investigations.

The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC, to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles. However, measurement of uptake speed in the presence of the inhibitors suggested that clathrin-independent processes contribute more extensively to endocytosis at the basal pole than the apical pole. Treatment with an inhibitor of myosin VI, 2,4,6-triiodophenol, significantly delayed both the apicobasal and the basoapical fluorescence signals. However, treatment with an inhibitor of kinesin, monastrol, or of dynein, ciliobrevin D, significantly delayed the signals only in the basoapical direction. The myosin-VI inhibitor, but neither the kinesin nor dynein inhibitors, significantly delayed the signals to the subsurface cisternae. That is, myosin VI carries vesicles in both longitudinal directions as well as radially to the sub-surface cisternae, whereas kinesin and dynein participate primarily in basoapical trafficking. This fundamental information is essential for elucidating recycling mechanisms of specific proteins involved in establishing, controlling, and maintaining the electromechanical action of OHCs and, therefore, vital for understanding auditory perception.

Background Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. Results Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. Conclusions Taken together, this provides the first comprehensive nanometric view of Giardia’s endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases—including actin nucleation promoting factors—and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

Fission of transport vesicles from endosomes is a crucial step in the recycling of lipids and receptors to the plasma membrane, but this process remains poorly understood. Although key components of the fission machinery, including the actin cytoskeleton and the ATPase Eps15 homology domain protein 1 (EHD1), have been implicated in endosomal fission, how this process is coordinately regulated is not known. We have identified the actin regulatory protein Coronin2A (CORO2A) as a novel EHD1 interaction partner. CORO2A localizes to stress fibers and actin microfilaments but also can be observed in partial overlap with EHD1 on endosomal structures. siRNA knockdown of CORO2A led to enlarged lamellae-like actin-rich protrusions, consistent with a role of other Coronin family proteins in attenuating actin-branching. Moreover, CORO2A depletion also caused a marked decrease in the internalization of clathrin-dependent cargo but had little impact on the uptake of clathrin-independent cargo, highlighting key differences in the role of branched actin for different modes of endocytosis. However, CORO2A was required for recycling of clathrin-independent cargo, and its depletion led to enlarged endosomes, supporting a role for CORO2A in the fission of endosomal vesicles. Our data support a novel role for CORO2A in coordinating endosomal fission and recycling with EHD1. [Media: see text].

Deciphering the endocytosis mechanisms of nanoparticle entry in cells is crucial to understand the fate of nanoparticles and the biological activity of the transported cargo. Such studies require the use of reporter agents such as fluorescent markers. Previously, we have reported the synthesis of self-fluorescent HAp nanoparticles as efficient nucleic acid delivery agents in prokaryotic and eukaryotic cells. Here, we show the application of biocompatible self-fluorescent nano delivery vehicle based on HAp and CPP- octa-arginine as an efficient system to study the mechanisms of intracellular fate of a gene delivery agent. The pathway of octa-arginine functionalized HAp NP (R8HNP) and HAp NP uptake in R1 ESCs was elucidated using confocal microscopy with the help of endocytic inhibitors. The NPs mainly enter R1 ESCs by clathrin mediated and macropinocytosis pathways. It was established that the NPs escape endosomal degradation by proton sponge effect owing to their ability to buffer the pH and trigger osmotic rupture. The functionalization of CPP, effectively enhanced the internalization and endosomal escape in R1 ESCs. The detailed results of these studies are outlined in this manuscript.