Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern. Video S1: Video depicting motion of single particles of SNAP-CD44-1 GFP labeled with two different dyes (coded in green, JF549-cpSNAP ligand, and red, JF646-SNAP ligand) acquired for 400 frames at 16ms exposure time. Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s1.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd56827777e574c0020c9975ac006d333b0fc5d8a3bdf4a58d9642095d14efdf4722b8e104ba117e006fc97f8265 Video S2: Video depicting a track of two differently labeled SNAP-CD44-GFP (coded in green, JF549-cpSNAP ligand, and red, JF646-SNAP ligand) showing co-localization of the protein over multiple frames. Download Original Video (.2 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s2.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd55827731dd00e5d8e270eb1a277ab80b2d6e07f4bf3a64e0a46b9c10da7ff92a5a65609c0acbad41f31b7cd99f Video S3: Video depicting motion of single particles of SNAP-CD44-GFP acquired at ˜60fps (3) and an example individual track (4). Download Original Video (1.4 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s3.,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b49252dfe02d1f87e44537fa094ec05070714f8be30f4b85a6204a2bce0d6e685dc5c0685a106b9e5908621039e5898952b0cec40822e73afff2dd2522c7dcdaafbf6dc390666d0fc2a2bb719d5cf570637e70f759e1830fac2efe6eef8327c323af1840ed Video S4: Video depicting motion of single particles of SNAP-CD44-GFP acquired at ˜60fps (3) and an example individual track (4). Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s4.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518512abb28b22067a1f9884d85997ddab96149b9477c71571c229bbd7b1ea6e1e798c3f82e72b1b4002e6517d9153759c9a032a9739c89529d759d2c5032763fe3f7fba7042c7f8b08b6e8e1fdc38b4326600e69ba8cc6d8657ded8991be4c48e038c31c8713035497a3e2f7cc05fb2d1ef75c840aa9077ac4ae63dba6ff0cea30e766ab940a0a704010cb0e3775ae4886049b8473be4b956a0d7a1 Video S5: Video depicting motion of single particles of SNAP-CD44TmICD-GFP acquired at ˜60fps. Download Original Video (4.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s5.,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b49252dfe02d1f87e44537fa094ec05070714f8be30f4b85a6204a2bce0d6e685dc5c0685a106b9e5908621039e5898954b0ce211bc15be0d1dd62af8ded514c9e5af927c71a0ef396c2fc1e5ada373c37ef836f320753db56a6dba92fcdcc4eec61b76fc7 Video S6: Video depicting motion of single particles of SNAP-CD44Tm-GFP acquired at ˜60fps. Download Original Video (4.6 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s6.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd518277bf465b1f0ff7119a74c08313cf581f53c42895d8a6145d39aec619edd173d56d5d60122c99e71eb8c6f1

Basement membranes (BMs) are specialized sheets of extracellular matrix that underlie epithelial and endothelial tissues. BMs regulate the traffic of cells and molecules between compartments, and participate in signaling, cell migration, and organogenesis. The dynamics of mammalian BMs, however, are poorly understood, largely due to a lack of models in which core BM components are endogenously labeled. Here, we describe the mTurquoise2-Col4a1 mouse in which we fluorescently tag collagen IV, the main component of BMs. Using an innovative planar-sagittal live imaging technique to visualize the BM of developing skin, we directly observe BM deformation during hair follicle budding and basal progenitor cell divisions. The BM’s inherent pliability enables dividing cells to remain attached to and deform the BM, rather than lose adhesion as generally thought. Using FRAP, we show BM collagen IV is extremely stable, even during periods of rapid epidermal growth. These findings demonstrate the utility of the mTurq2-Col4a1 mouse to shed new light on mammalian BM developmental dynamics.

Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH ₄ Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern.