Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

Low-dose fluvoxamine modulates endocytic trafficking of SARS-CoV-2 spike protein: a potential mechanism for anti-COVID-19 protection by antidepressants

Commonly prescribed antidepressants may be associated with protection against severe COVID-19, with one drug (fluvoxamine) currently undergoing a Phase 3 clinical trial. The mechanism of their action, however, remains unknown. Here, I investigated the effect of fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

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.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

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.

SNX17 regulates antigen internalization and phagosomal maturation by dendritic cells

Cross-presentation is the process whereby antigenic peptides derived from exogenous antigens are associated to MHC class I molecules triggering the activation of CD8+ T lymphocytes. The endocytic route of dendritic cells (DCs) is strongly specialized to achieve antigen cross-presentation efficiently, which is crucial to initiate cytotoxic immune responses against many pathogens (i.e. Toxoplasma gondii) and tumors. Nevertheless, the endosomal molecular effectors involved in this process are not completely understood. In particular, the role of sorting nexin (SNX) proteins in cross-presentation has never been addressed. In this work, we identify the endosomal protein SNX17 as a key regulator of antigen internalization and cross-presentation by DCs. Our results demonstrate that SNX17 expression in DCs is essential to guarantee a normal cross-presentation of soluble, particulate and T. gondii-associated antigens. The silencing of SNX17 expression in DCs significantly affected the uptake of exogenous antigens by fluid-phase endocytosis and phagocytosis, but not by receptor-mediated endocytosis. Moreover, the knock-down of SNX17 impaired T. gondii invasion, CD11b integrin recycling and hampered the organization of the actin cytoskeleton. Finally, we show that SNX17 controls the proper maturation of DC phagosomes. Our findings provide compelling evidence that SNX17 plays a central role in the modulation of DC endocytic network, which is crucial for competent antigen internalization and cross-presentation.

Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain (TD), which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis.SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis

BackgroundThe kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown.MethodsTo investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach.ResultsUptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology.ConclusionsStriking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.