Coupling during collective cell migration is controlled by a vinculin mechanochemical switch.

The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors.

Rho activation drives luminal collapse and eversion in epithelial acini.

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. Although some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternative pathway for altered polarity. Because mechanical alterations are common in human cancer, including an upregulation of RhoA-mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of ß1-integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model that balances tension at cell-cell interfaces revealed a fivefold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Progerin-expressing endothelial cells are unable to adapt to shear stress.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. Patients with HGPS experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study, we examined how progerin-expressing endothelial cells adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control endothelial cells to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in endothelial cells, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that endothelial cells either overexpressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). Endothelial cells overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing endothelial cells had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, the prealignment of cells to shear stress before progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of endothelial cells to properly adapt to shear stress.

Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network.

Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca2+-dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases.

The Elephant in the Cell: Nuclear Mechanics and Mechanobiology.

The 2021 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C) featured a workshop titled "The Elephant in the Room: Nuclear Mechanics and Mechanobiology." The goal of this workshop was to provide a perspective from experts in the field on the current understanding of nuclear mechanics and its role in mechanobiology. This paper reviews the major themes and questions discussed during the workshop, including historical context on the initial methods of measuring the mechanical properties of the nucleus and classifying the primary structures dictating nuclear mechanics, physical plasticity of the nucleus, the emerging role of the linker of nucleoskeleton and cytoskeleton (LINC) complex in coupling the nucleus to the cytoplasm and driving the behavior of individual cells and multicellular assemblies, and the computational models currently in use to investigate the mechanisms of gene expression and cell signaling. Ongoing questions and controversies, along with promising future directions, are also discussed.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration.

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.

Spatial Proliferation of Epithelial Cells Is Regulated by E-Cadherin Force.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. Prior experiments have shown that externally applied forces, such as stretch, result in increased proliferation in an E-cadherin force-dependent manner. In this study, the spatial regulation of cell proliferation in large epithelial colonies was examined. Surprisingly, cells at the center of the colony still had increased proliferation as compared to cells in confluent monolayers. E-cadherin forces were found to be elevated for both cells at the edge and center of these larger colonies when compared to confluent monolayers. To determine if high levels of E-cadherin force were necessary to induce proliferation at the center of the colony, a lower-force mutant of E-cadherin was developed. Cells with lower E-cadherin force had significantly reduced proliferation for cells at the center of the colony but minimal differences for cells at the edges of the colony. Similarly, increasing substrate stiffness was found to increase E-cadherin force and increase the proliferation rate across the colony. Taken together, these results show that forces through cell-cell junctions regulate proliferation across large groups of epithelial cells. In addition, an important finding of this study is that junction forces are dynamic and modulate cellular function even in the absence of externally applied loads.

Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.

Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 amino acid repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.

The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.

Flow-dependent cellular mechanotransduction in atherosclerosis.

Atherosclerosis depends on risk factors such as hyperlipidemia, smoking, hypertension and diabetes. Although these risk factors are relatively constant throughout the arterial circulation, atherosclerotic plaques occur at specific sites where flow patterns are disturbed, with lower overall magnitude and complex changes in speed and direction. Research over the past few decades has provided new insights into the cellular mechanisms of force transduction and how mechanical effects act in concert with conventional risk factors to mediate plaque formation and progression. This Commentary summarizes our current understanding of how mechanotransduction pathways synergize with conventional risk factors in atherosclerosis. We attempt to integrate cellular studies with animal and clinical data, and highlight major questions that need to be answered to develop more effective therapies.

A homeostatic role of nucleus-actin filament coupling in the regulation of cellular traction forces in fibroblasts.

Cellular traction forces are contractile forces that depend on the material/substrate stiffness and play essential roles in sensing mechanical environments and regulating cell morphology and function. Traction forces are primarily generated by the actin cytoskeleton and transmitted to the substrate through focal adhesions. The cell nucleus is also believed to be involved in the regulation of this type of force; however, the role of the nucleus in cellular traction forces remains unclear. In this study, we explored the effects of nucleus-actin filament coupling on cellular traction forces in human dermal fibroblasts cultured on substrates with varying stiffness (5, 15, and 48 kPa). To investigate these effects, we transfected the cells with a dominant-negative Klarsicht/ANC-1/Syne homology (DN-KASH) protein that was designed to displace endogenous linker proteins and disrupt nucleus-actin cytoskeleton connections. The force that exists between the cytoskeleton and the nucleus (nuclear tension) was also evaluated with a fluorescence resonance energy transfer (FRET)-based tension sensor. We observed a biphasic change in cellular traction forces with a peak at 15 kPa, regardless of DN-KASH expression, that was inversely correlated with the nuclear tension. In addition, the relative magnitude and distribution of traction forces in nontreated wild-type cells were similar across different stiffness conditions, while DN-KASH-transfected cells exhibited a different distribution pattern that was impacted by the substrate stiffness. These results suggest that the nucleus-actin filament coupling play a homeostatic role by maintaining the relative magnitude of cellular traction forces in fibroblasts under different stiffness conditions.

Desmosomal Cadherin Tension Loss in Pemphigus Vulgaris Mediated by the Inhibition of Active RhoA at Cell-Cell Adhesions.

Binding of autoantibodies to keratinocyte surface antigens, primarily desmoglein 3 (Dsg3) of the desmosomal complex, leads to the dissociation of cell-cell adhesion in the blistering disorder pemphigus vulgaris (PV). After the initial disassembly of desmosomes, cell-cell adhesions actively remodel in association with the cytoskeleton and focal adhesions. Growing evidence highlights the role of adhesion mechanics and mechanotransduction at cell-cell adhesions in this remodeling process, as their active participation may direct autoimmune pathogenicity. However, a large part of the biophysical transformations after antibody binding remains underexplored. Specifically, it is unclear how tension in desmosomes and cell-cell adhesions changes in response to antibodies, and how the altered tensional states translate to cellular responses. Here, we showed a tension loss at Dsg3 using fluorescence resonance energy transfer (FRET)-based tension sensors, a tension loss at the entire cell-cell adhesion, and a potentially compensatory increase in junctional traction force at cell-extracellular matrix adhesions after PV antibody binding. Further, our data indicate that this tension loss is mediated by the inhibition of RhoA at cell-cell contacts, and the extent of RhoA inhibition may be crucial in determining the severity of pathogenicity among different PV antibodies. More importantly, this tension loss can be partially restored by altering actomyosin based cell contractility. Collectively, these findings provide previously unattainable details in our understanding of the mechanisms that govern cell-cell interactions under physiological and autoimmune conditions, which may open the window to entirely new therapeutics aimed at restoring physiological balance to tension dynamics that regulates the maintenance of cell-cell adhesion.

Coupling During Collective Cell Migration is Controlled by a Vinculin Mechanochemical Switch.

Collective cell migration (CCM) plays important roles in development, physiological, and pathological processes. A key feature of CCM is the dynamic mechanical coupling between cells, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to adapt to forces. Recent efforts have identified key proteins and implicated cellular-scale mechanical properties, but how key proteins give rise to these larger-scale mechanical processes is unclear. Using force-sensitive biosensors, cell migration assays, and molecular clutch models, we sought a molecular understanding of adhesion strengthening that could bridge this gap. We found that the mechanical linker protein vinculin bears substantial loads at AJs, FAs, and in the cytoplasm during epithelial sheet migration, and we identified a switch-like residue on vinculin that regulates its conformation and loading at the AJs during CCM. In vinculin KO-rescue, this switch jointly controlled the speed and coupling length-scale of CCM, which suggested changes in adhesion-based friction. To test this, we developed molecularly detailed friction clutch models of the FA and AJ. They show that open, loaded vinculin increases friction in adhesive structures, with larger affects observed in AJs. Thus, this work elucidates how load-bearing linker proteins can be regulated to alter mechanical properties of cells and enable rapid tuning of mechanical coupling in CCM.

Nuclear lamina strain states revealed by intermolecular force biosensor.

Nuclear lamins have been considered an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that the nanobody-based approach allows construction of biosensors for complex protein structures for mechanobiology studies.

Modulation of E-Cadherin Function through the AmotL2 Isoforms Promotes Ameboid Cell Invasion.

The spread of tumor cells and the formation of distant metastasis remain the main causes of mortality in cancer patients. However, the mechanisms governing the release of cells from micro-environmental constraints remain unclear. E-cadherin negatively controls the invasion of epithelial cells by maintaining cell-cell contacts. Furthermore, the inactivation of E-cadherin triggers invasion in vitro. However, the role of E-cadherin is complex, as metastasizing cells maintain E-cadherin expression, which appears to have a positive role in the survival of tumor cells. In this report, we present a novel mechanism delineating how E-cadherin function is modulated to promote invasion. We have previously shown that E-cadherin is associated with p100AmotL2, which is required for radial actin formation and the transmission of mechanical force. Here, we present evidence that p60AmotL2, which is expressed in invading tumor cells, binds to the p100AmotL2 isoform and uncouples the mechanical constraint of radial actin filaments. We show for the first time that the coupling of E-cadherin to the actin cytoskeleton via p100AmotL2 is directly connected to the nuclear membrane. The expression of p60AmotL2 inactivates this connection and alters the properties of the nuclear lamina, potentiating the invasion of cells into micropores of the extracellular matrix. In summary, we propose that the balance of the two AmotL2 isoforms is important in the modulation of E-cadherin function and that an imbalance of this axis promotes ameboid cell invasion.

Differential Pathomechanisms of Desmoglein 1 Transmembrane Domain Mutations in Skin Disease.

Dominant and recessive mutations in the desmosomal cadherin, desmoglein (DSG) 1, cause the skin diseases palmoplantar keratoderma (PPK) and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, respectively. In this study, we compare two dominant missense mutations in the DSG1 transmembrane domain (TMD), G557R and G562R, causing PPK (DSG1PPK-TMD) and SAM syndrome (DSG1SAM-TMD), respectively, to determine the differing pathomechanisms of these mutants. Expressing the DSG1TMD mutants in a DSG-null background, we use cellular and biochemical assays to reveal the differences in the mechanistic behavior of each mutant. Super-resolution microscopy and functional assays showed a failure by both mutants to assemble desmosomes due to reduced membrane trafficking and lipid raft targeting. DSG1SAM-TMD maintained normal expression levels and turnover relative to wildtype DSG1, but DSG1PPK-TMD lacked stability, leading to increased turnover through lysosomal and proteasomal pathways and reduced expression levels. These results differentiate the underlying pathomechanisms of these disorders, suggesting that DSG1SAM-TMD acts dominant negatively, whereas DSG1PPK-TMD is a loss-of-function mutation causing the milder PPK disease phenotype. These mutants portray the importance of the DSG TMD in desmosome function and suggest that a greater understanding of the desmosomal cadherin TMDs will further our understanding of the role that desmosomes play in epidermal pathophysiology.

α-Catenin-dependent vinculin recruitment to adherens junctions is antagonistic to focal adhesions.

Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have demonstrated that mechanical perturbations of cells result in enhanced recruitment of vinculin to FAs and/or AJs. Likewise, many other studies have shown "cross-talk" between FAs and AJs. Vinculin itself has been suggested to be a probable regulator of this adhesion cross-talk. In this study we used MDCK as a model system of epithelia, developing cell lines in which vinculin recruitment was reduced or enhanced at AJs. Careful analysis of these cells revealed that perturbing vinculin recruitment to AJs resulted in a reduction of detectable FAs. Interestingly the cross-talk between these two structures was not due to a limited pool of vinculin, as increasing expression of vinculin did not rescue FA formation. Instead, we demonstrate that vinculin translocation between AJs and FAs is necessary for actin cytoskeleton rearrangements that occur during cell migration, which is necessary for large, well-formed FAs. Last, we show using a wound assay that collective cell migration is similarly hindered when vinculin recruitment is reduced or enhanced at AJs, highlighting that vinculin translocation between each compartment is necessary for efficient collective migration.

Chromatin condensation regulates endothelial cell adaptation to shear stress.

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS.

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture.

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drug-treatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.