Mitosis, diffusible crosslinkers, and the ideal gas law.

During mitosis, molecular motors hydrolyze ATP to generate sliding forces between adjacent microtubules and form the bipolar mitotic spindle. Lansky et al. now show that the diffusible microtubule crosslinker Ase1p can generate sliding forces between adjacent microtubules, and it does so without ATP hydrolysis.

Optical Control of Microtubule Dynamics in Time and Space.

Small molecule inhibitors of microtubule dynamics are widely used as cell biology research tools and clinically as cancer chemotherapeutics. By slight modification to the chemical structure of a known microtubule inhibitor, combretastatin A-4, Borowiak et al. develop a photoswitchable derivative that can be turned "on" and "off" with low-intensity light to spatially and temporally control microtubule dynamics.

Regulation of the MEX-5 gradient by a spatially segregated kinase/phosphatase cycle.

Protein concentration gradients encode spatial information across cells and tissues and often depend on spatially localized protein synthesis. Here, we report that a different mechanism underlies the MEX-5 gradient. MEX-5 is an RNA-binding protein that becomes distributed in a cytoplasmic gradient along the anterior-to-posterior axis of the one-cell C. elegans embryo. We demonstrate that the MEX-5 gradient is a direct consequence of an underlying gradient in MEX-5 diffusivity. The MEX-5 diffusion gradient arises when the PAR-1 kinase stimulates the release of MEX-5 from slow-diffusive, RNA-containing complexes in the posterior cytoplasm. PAR-1 directly phosphorylates MEX-5 and is antagonized by the spatially uniform phosphatase PP2A. Mathematical modeling and in vivo observations demonstrate that spatially segregated phosphorylation and dephosphorylation reactions are sufficient to generate stable protein concentration gradients in the cytoplasm. The principles demonstrated here apply to any spatially segregated modification cycle that affects protein diffusion and do not require protein synthesis or degradation.

Rapid microtubule self-assembly kinetics.

Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.

Randomized Trial of Metformin, Ivermectin, and Fluvoxamine for Covid-19.

BACKGROUND: Early treatment to prevent severe coronavirus disease 2019 (Covid-19) is an important component of the comprehensive response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. METHODS: In this phase 3, double-blind, randomized, placebo-controlled trial, we used a 2-by-3 factorial design to test the effectiveness of three repurposed drugs - metformin, ivermectin, and fluvoxamine - in preventing serious SARS-CoV-2 infection in nonhospitalized adults who had been enrolled within 3 days after a confirmed diagnosis of infection and less than 7 days after the onset of symptoms. The patients were between the ages of 30 and 85 years, and all had either overweight or obesity. The primary composite end point was hypoxemia (≤93% oxygen saturation on home oximetry), emergency department visit, hospitalization, or death. All analyses used controls who had undergone concurrent randomization and were adjusted for SARS-CoV-2 vaccination and receipt of other trial medications. RESULTS: A total of 1431 patients underwent randomization; of these patients, 1323 were included in the primary analysis. The median age of the patients was 46 years; 56% were female (6% of whom were pregnant), and 52% had been vaccinated. The adjusted odds ratio for a primary event was 0.84 (95% confidence interval [CI], 0.66 to 1.09; P = 0.19) with metformin, 1.05 (95% CI, 0.76 to 1.45; P = 0.78) with ivermectin, and 0.94 (95% CI, 0.66 to 1.36; P = 0.75) with fluvoxamine. In prespecified secondary analyses, the adjusted odds ratio for emergency department visit, hospitalization, or death was 0.58 (95% CI, 0.35 to 0.94) with metformin, 1.39 (95% CI, 0.72 to 2.69) with ivermectin, and 1.17 (95% CI, 0.57 to 2.40) with fluvoxamine. The adjusted odds ratio for hospitalization or death was 0.47 (95% CI, 0.20 to 1.11) with metformin, 0.73 (95% CI, 0.19 to 2.77) with ivermectin, and 1.11 (95% CI, 0.33 to 3.76) with fluvoxamine. CONCLUSIONS: None of the three medications that were evaluated prevented the occurrence of hypoxemia, an emergency department visit, hospitalization, or death associated with Covid-19. (Funded by the Parsemus Foundation and others; COVID-OUT ClinicalTrials.gov number, NCT04510194.).

Dystrophin missense mutations alter focal adhesion tension and mechanotransduction.

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin-glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.

Favorable Antiviral Effect of Metformin on Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load in a Randomized, Placebo-Controlled Clinical Trial of Coronavirus Disease 2019.

BACKGROUND: Metformin has antiviral activity against RNA viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The mechanism appears to be suppression of protein translation via targeting the host mechanistic target of rapamycin pathway. In the COVID-OUT randomized trial for outpatient coronavirus disease 2019 (COVID-19), metformin reduced the odds of hospitalizations/death through 28 days by 58%, of emergency department visits/hospitalizations/death through 14 days by 42%, and of long COVID through 10 months by 42%. METHODS: COVID-OUT was a 2 × 3 randomized, placebo-controlled, double-blind trial that assessed metformin, fluvoxamine, and ivermectin; 999 participants self-collected anterior nasal swabs on day 1 (n = 945), day 5 (n = 871), and day 10 (n = 775). Viral load was quantified using reverse-transcription quantitative polymerase chain reaction. RESULTS: The mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95% confidence interval [CI], -1.05 to -.06; P = .027). Those who received metformin were less likely to have a detectable viral load than placebo at day 5 or day 10 (odds ratio [OR], 0.72; 95% CI, .55 to .94). Viral rebound, defined as a higher viral load at day 10 than day 5, was less frequent with metformin (3.28%) than placebo (5.95%; OR, 0.68; 95% CI, .36 to 1.29). The metformin effect was consistent across subgroups and increased over time. Neither ivermectin nor fluvoxamine showed effect over placebo. CONCLUSIONS: In this randomized, placebo-controlled trial of outpatient treatment of SARS-CoV-2, metformin significantly reduced SARS-CoV-2 viral load, which may explain the clinical benefits in this trial. Metformin is pleiotropic with other actions that are relevant to COVID-19 pathophysiology. CLINICAL TRIALS REGISTRATION: NCT04510194.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Optimal cell traction forces in a generalized motor-clutch model.

Cells exert forces on mechanically compliant environments to sense stiffness, migrate, and remodel tissue. Cells can sense environmental stiffness via myosin-generated pulling forces acting on F-actin, which is in turn mechanically coupled to the environment via adhesive proteins, akin to a clutch in a drivetrain. In this "motor-clutch" framework, the force transmitted depends on the complex interplay of motor, clutch, and environmental properties. Previous mean-field analysis of the motor-clutch model identified the conditions for optimal stiffness for maximal force transmission via a dimensionless number that combines motor-clutch parameters. However, in this and other previous mean-field analyses, the motor-clutch system is assumed to have balanced motors and clutches and did not consider force-dependent clutch reinforcement and catch bond behavior. Here, we generalize the motor-clutch analytical framework to include imbalanced motor-clutch regimes, with clutch reinforcement and catch bonding, and investigate optimality with respect to all parameters. We found that traction force is strongly influenced by clutch stiffness, and we discovered an optimal clutch stiffness that maximizes traction force, suggesting that cells could tune their clutch mechanical properties to perform a specific function. The results provide guidance for maximizing the accuracy of cell-generated force measurements via molecular tension sensors by designing their mechanosensitive linker peptide to be as stiff as possible. In addition, we found that, on rigid substrates, the mean-field analysis identifies optimal motor properties, suggesting that cells could regulate their myosin repertoire and activity to maximize force transmission. Finally, we found that clutch reinforcement shifts the optimum substrate stiffness to larger values, whereas the optimum substrate stiffness is insensitive to clutch catch bond properties. Overall, our work reveals novel features of the motor-clutch model that can affect the design of molecular tension sensors and provide a generalized analytical framework for predicting and controlling cell adhesion and migration in immunotherapy and cancer.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccination and Booster on Coronavirus Disease 2019 (COVID-19) Symptom Severity Over Time in the COVID-OUT Trial.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has decreasing protection from acquiring any infection with emergence of new variants; however, vaccination continues to protect against progression to severe coronavirus disease 2019 (COVID-19). The impact of vaccination status on symptoms over time is less clear. METHODS: Within a randomized trial on early outpatient COVID-19 therapy testing metformin, ivermectin, and/or fluvoxamine, participants recorded symptoms daily for 14 days. Participants were given a paper symptom diary allowing them to circle the severity of 14 symptoms as none (0), mild (1), moderate (2), or severe (3). This is a secondary analysis of clinical trial data on symptom severity over time using generalized estimating equations comparing those unvaccinated, SARS-CoV-2 vaccinated with primary vaccine series only, or vaccine-boosted. RESULTS: The parent clinical trial prospectively enrolled 1323 participants, of whom 1062 (80%) prospectively recorded some daily symptom data. Of these, 480 (45%) were unvaccinated, 530 (50%) were vaccinated with primary series only, and 52 (5%) vaccine-boosted. Overall symptom severity was least for the vaccine-boosted group and most severe for unvaccinated at baseline and over the 14 days (P < .001). Individual symptoms were least severe in the vaccine-boosted group including cough, chills, fever, nausea, fatigue, myalgia, headache, and diarrhea, as well as smell and taste abnormalities. Results were consistent over Delta and Omicron variant time periods. CONCLUSIONS: SARS-CoV-2 vaccine-boosted participants had the least severe symptoms during COVID-19, which abated the quickest over time. Clinical Trial Registration. NCT04510194.

Directed cell migration towards softer environments.

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.

Chromosome congression by Kinesin-5 motor-mediated disassembly of longer kinetochore microtubules.

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.

Enhanced substrate stress relaxation promotes filopodia-mediated cell migration.

Cell migration on two-dimensional substrates is typically characterized by lamellipodia at the leading edge, mature focal adhesions and spread morphologies. These observations result from adherent cell migration studies on stiff, elastic substrates, because most cells do not migrate on soft, elastic substrates. However, many biological tissues are soft and viscoelastic, exhibiting stress relaxation over time in response to a deformation. Here, we have systematically investigated the impact of substrate stress relaxation on cell migration on soft substrates. We observed that cells migrate minimally on substrates with an elastic modulus of 2 kPa that are elastic or exhibit slow stress relaxation, but migrate robustly on 2-kPa substrates that exhibit fast stress relaxation. Strikingly, migrating cells were not spread out and did not extend lamellipodial protrusions, but were instead rounded, with filopodia protrusions extending at the leading edge, and exhibited small nascent adhesions. Computational models of cell migration based on a motor-clutch framework predict the observed impact of substrate stress relaxation on cell migration and filopodia dynamics. Our findings establish substrate stress relaxation as a key requirement for robust cell migration on soft substrates and uncover a mode of two-dimensional cell migration marked by round morphologies, filopodia protrusions and weak adhesions.

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.

mTOR inhibition in COVID-19: A commentary and review of efficacy in RNA viruses.

In this commentary, we shed light on the role of the mammalian target of rapamycin (mTOR) pathway in viral infections. The mTOR pathway has been demonstrated to be modulated in numerous RNA viruses. Frequently, inhibiting mTOR results in suppression of virus growth and replication. Recent evidence points towards modulation of mTOR in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We discuss the current literature on mTOR in SARS-CoV-2 and highlight evidence in support of a role for mTOR inhibitors in the treatment of coronavirus disease 2019.

Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote.

Protein concentration gradients organize cells and tissues and commonly form through diffusion away from a local source of protein. Interestingly, during the asymmetric division of the Caenorhabditis elegans zygote, the RNA-binding proteins MEX-5 and PIE-1 form opposing concentration gradients in the absence of a local source. In this study, we use near-total internal reflection fluorescence (TIRF) imaging and single-particle tracking to characterize the reaction/diffusion dynamics that maintain the MEX-5 and PIE-1 gradients. Our findings suggest that both proteins interconvert between fast-diffusing and slow-diffusing states on timescales that are much shorter (seconds) than the timescale of gradient formation (minutes). The kinetics of diffusion-state switching are strongly polarized along the anterior/posterior (A/P) axis by the PAR polarity system such that fast-diffusing MEX-5 and PIE-1 particles are approximately symmetrically distributed, whereas slow-diffusing particles are highly enriched in the anterior and posterior cytoplasm, respectively. Using mathematical modeling, we show that local differences in the kinetics of diffusion-state switching can rapidly generate stable concentration gradients over a broad range of spatial and temporal scales.

Predicting Confined 1D Cell Migration from Parameters Calibrated to a 2D Motor-Clutch Model.

Biological tissues contain micrometer-scale gaps and pores, including those found within extracellular matrix fiber networks, between tightly packed cells, and between blood vessels or nerve bundles and their associated basement membranes. These spaces restrict cell motion to a single-spatial dimension (1D), a feature that is not captured in traditional in vitro cell migration assays performed on flat, unconfined two-dimensional (2D) substrates. Mechanical confinement can variably influence cell migration behaviors, and it is presently unclear whether the mechanisms used for migration in 2D unconfined environments are relevant in 1D confined environments. Here, we assessed whether a cell migration simulator and associated parameters previously measured for cells on 2D unconfined compliant hydrogels could predict 1D confined cell migration in microfluidic channels. We manufactured microfluidic devices with narrow channels (60-µm2 rectangular cross-sectional area) and tracked human glioma cells that spontaneously migrated within channels. Cell velocities (vexp = 0.51 ± 0.02 µm min-1) were comparable to brain tumor expansion rates measured in the clinic. Using motor-clutch model parameters estimated from cells on unconfined 2D planar hydrogel substrates, simulations predicted similar migration velocities (vsim = 0.37 ± 0.04 µm min-1) and also predicted the effects of drugs targeting the motor-clutch system or cytoskeletal assembly. These results are consistent with glioma cells utilizing a motor-clutch system to migrate in confined environments.

Glioma Cell Migration Dynamics in Brain Tissue Assessed by Multimodal Optical Imaging.

Glioblastoma is a primary malignant brain tumor characterized by highly infiltrative glioma cells. Vasculature and white matter tracts are considered to be the preferred and fastest routes for glioma invasion through brain tissue. In this study, we systematically quantified the routes and motility of the U251 human glioblastoma cell line in mouse brain slices by multimodal imaging. Specifically, we used polarization-sensitive optical coherence tomography to delineate nerve fiber tracts while confocal fluorescence microscopy was used to image cell migration and brain vasculature. Somewhat surprisingly, we found that in mouse brain slices, U251 glioma cells do not follow white matter tracts but rather preferentially migrate along vasculature in both gray and white matter. In addition, U251 cell motility is ∼2-fold higher in gray matter than in white matter (91 vs. 43 µm2/h), with a substantial fraction (44%) of cells in both regions invading without close association with vasculature. Interestingly, within both regions, the rates of migration for the perivascular and televascular routes of invasion were indistinguishable. Furthermore, by imaging of local vasculature deformation dynamics during cell migration, we found that U251 cells are capable of exerting traction forces that locally pull on their environment, suggesting the applicability of a "motor-clutch"-based model for migration in vivo. Overall, by quantitatively analyzing the migration dynamics along the diverse pathways followed by invading U251 glioma cells as observed by our multimodal imaging approach, our studies suggest that effective antiinvasive strategies will need to simultaneously limit parallel routes of both perivascular and televascular invasion through both gray and white matter.

Multiscale Computational Modeling of Tubulin-Tubulin Lateral Interaction.

Microtubules are multistranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via "dynamic instability," in which the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e., what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length- and timescales, from atoms to cellular behavior. In this study, we describe a multiscale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermokinetic modeling of microtubules. Here, we investigated the underlying interaction energy when tubulin dimers associate laterally by performing all-atom MD simulations. We found that the lateral potential energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP and is estimated to be ≅ -11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers confirms that the lateral bond is weak on its own, with a mean lifetime of ∼0.1 µs, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral-bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore, the estimated lateral-bond strength (ΔGlat0≅ -5 kBT) is well-aligned with earlier estimates based on thermokinetic modeling and light microscopy measurements. Thus, we have computationally connected atomistic-level structural information, obtained by cryo-electron microscopy, to cellular-scale microtubule assembly dynamics using a combination of MD, BD, and thermokinetic models to bridge from Ångstroms to micrometers and from femtoseconds to minutes.