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Despite recent experimental progress in characterizing cell migration mechanics, our understanding of the mechanisms governing rapid cell movement remains limited. To effectively limit tumor growth, antitumoral T cells need to rapidly migrate to find and kill cancer cells. To investigate the upper limits of cell speed, we developed a new hybrid stochastic-mean field model of bleb-based cell motility. We first examined the potential for adhesion-free bleb-based migration and show that cells migrate inefficiently in the absence of adhesion-based forces, i.e., cell swimming. While no cortical contractility oscillations are needed for cells to swim in viscoelastic media, high-to-low cortical contractility oscillations are necessary for cell swimming in viscous media. This involves a high cortical contractility phase with multiple bleb nucleation events, followed by an intracellular pressure buildup recovery phase at low cortical tensions, resulting in modest net cell motion. However, our model suggests that cells can employ a hybrid bleb- and adhesion-based migration mechanism for rapid cell motility and identifies conditions for optimality. The model provides a momentum-conserving mechanism underlying rapid single-cell migration and identifies factors as design criteria for engineering T cell therapies to improve movement in mechanically complex environments.

Cell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. CD44 primarily binds to hyaluronic acid (HA) and integrins primarily bind to extracellular matrix (ECM) proteins such as collagen. However, the role of CD44 under integrin-mediated conditions and vice versa is not well known. Here, we performed traction force microscopy (TFM) on U251 cells seeded on collagen I-coated polyacrylamide gels to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. Furthermore, untreated WT and CD44-blocked WT exhibited comparable results. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted a higher traction force than untreated WT cells. Our data suggest that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other's ability to generate traction force on its cognate substrate., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Purpose: Glioblastoma (GBM) is an aggressive malignant brain tumor with 2 year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models., Methods: Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration., Results: We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells., Conclusions: Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch., Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00799-x., Competing Interests: Competing interestsThe authors, SMA, MK, and DJO have no conflicts of interest to declare., (© The Author(s) 2024.)

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2-year survival rates of 6.7% [1], [2]. One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue[3], [4]. To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Through imaging cell-vasculature interactions and utilizing drugs, antibodies, and genetic modifications to target motors and clutches, we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo , and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch.

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., Competing Interests: Declaration of interests P.P.P. is a member of the Scientific Advisory Board for Parthenon Therapeutics., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Computational models of integrin-based adhesion complexes have revealed important insights into the mechanisms by which cells establish connections with their external environment. However, how changes in conformation and function of individual adhesion proteins regulate the dynamics of whole adhesion complexes remains largely elusive. This is because of the large separation in time and length scales between the dynamics of individual adhesion proteins (nanoseconds and nanometers) and the emergent dynamics of the whole adhesion complex (seconds and micrometers), and the limitations of molecular simulation approaches in extracting accurate free energies, conformational transitions, reaction mechanisms, and kinetic rates, that can inform mechanisms at the larger scales. In this review, we discuss models of integrin-based adhesion complexes and highlight their main findings regarding: (i) the conformational transitions of integrins at the molecular and macromolecular scales and (ii) the molecular clutch mechanism at the mesoscale. Lastly, we present unanswered questions in the field of modeling adhesions and propose new ideas for future exciting modeling opportunities., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis., (© 2023. The Author(s).)

Hydrogels are important platform materials for in vitro cellular studies. Mechanistic studies on durotaxis, the directional movement of a cell affected by a spatial gradient of stiffness of the underlying substrate, requires materials such as polyacrylamide, polyethylene glycol, or PDMS, in which the stiffness can be controlled in a spatiotemporal manner. Here, we describe the synthesis of an o-nitrobenzyl-based photocleavable cross-linker and its incorporation into a polyacrylamide hydrogel to render it photoresponsive. Precise control over the physical properties of the gel allows observation of glioblastoma durotaxis under surface stiffness conditions relevant to the actual brain environment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Long Covid is an emerging chronic illness potentially affecting millions, sometimes preventing the ability to work or participate in normal daily activities. COVID-OUT was an investigator-initiated, multi-site, phase 3, randomized, quadruple-blinded placebo-controlled clinical trial (NCT04510194). The design simultaneously assessed three oral medications (metformin, ivermectin, fluvoxamine) using two by three parallel treatment factorial assignment to efficiently share placebo controls and assessed Long Covid outcomes for 10 months to understand whether early outpatient treatment of SARS-CoV-2 with metformin, ivermectin, or fluvoxamine prevents Long Covid., Methods: This was a decentralized, remotely delivered trial in the US of 1,125 adults age 30 to 85 with overweight or obesity, fewer than 7 days of symptoms, and enrolled within three days of a documented SARS-CoV-2 infection. Immediate release metformin titrated over 6 days to 1,500mg per day 14 days total; ivermectin 430mcg/kg/day for 3 days; fluvoxamine, 50mg on day one then 50mg twice daily through 14 days. Medical-provider diagnosis of Long Covid, reported by participant by day 300 after randomization was a pre-specified secondary outcome; the primary outcome of the trial was severe Covid by day 14., Result: The median age was 45 years (IQR 37 to 54), 56% female of whom 7% were pregnant. Two percent identified as Native American; 3.7% as Asian; 7.4% as Black/African American; 82.8% as white; and 12.7% as Hispanic/Latino. The median BMI was 29.8 kg/m 2 (IQR 27 to 34); 51% had a BMI >30kg/m 2 . Overall, 8.4% reported having received a diagnosis of Long Covid from a medical provider: 6.3% in the metformin group and 10.6% in the metformin control; 8.0% in the ivermectin group and 8.1% in the ivermectin control; and 10.1% in the fluvoxamine group and 7.5% in the fluvoxamine control. The Hazard Ratio (HR) for Long Covid in the metformin group versus control was 0.58 (95% CI 0.38 to 0.88); 0.99 (95% CI 0.592 to 1.643) in the ivermectin group; and 1.36 in the fluvoxamine group (95% CI 0.785 to 2.385)., Conclusions: There was a 42% relative decrease in the incidence of Long Covid in the metformin group compared to its blinded control in a secondary outcome of this randomized phase 3 trial.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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.)., (Copyright © 2022 Massachusetts Medical Society.)

Background: Data conflict on whether vaccination decreases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load. The objective of this analysis was to compare baseline viral load and symptoms between vaccinated and unvaccinated adults enrolled in a randomized trial of outpatient coronavirus disease 2019 (COVID-19) treatment., Methods: Baseline data from the first 433 sequential participants enrolling into the COVID-OUT trial were analyzed. Adults aged 30-85 with a body mass index (BMI) ≥25 kg/m 2 were eligible within 3 days of a positive SARS-CoV-2 test and <7 days of symptoms. Log 10 polymerase chain reaction viral loads were normalized to human RNase P by vaccination status, by time from vaccination, and by symptoms., Results: Two hundred seventy-four participants with known vaccination status contributed optional nasal swabs for viral load measurement: median age, 46 years; median (interquartile range) BMI 31.2 (27.4-36.4) kg/m 2 . Overall, 159 (58%) were women, and 217 (80%) were White. The mean relative log 10 viral load for those vaccinated <6 months from the date of enrollment was 0.11 (95% CI, -0.48 to 0.71), which was significantly lower than the unvaccinated group ( P  = .01). Those vaccinated ≥6 months before enrollment did not differ from the unvaccinated with respect to viral load (mean, 0.99; 95% CI, -0.41 to 2.40; P  = .85). The vaccinated group had fewer moderate/severe symptoms of subjective fever, chills, myalgias, nausea, and diarrhea (all P  < .05)., Conclusions: These data suggest that vaccination within 6 months of infection is associated with a lower viral load, and vaccination was associated with a lower likelihood of having systemic symptoms., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Characterizing the motile properties of glioblastoma tumor cells could provide a useful way to predict the spread of tumors and to tailor the therapeutic approach. Radiomics has emerged as a diagnostic tool in the classification of tumor grade, stage, and prognosis. The purpose of this work is to examine the potential of radiomics to predict the motility of glioblastoma cells. Tissue specimens were obtained from 31 patients undergoing surgical resection of glioblastoma. Mean tumor cell motility was calculated from time-lapse videos of specimen cells. Manual segmentation was used to define the border of the enhancing tumor T1-weighted MR images, and 107 radiomics features were extracted from the normalized image volumes. Model parameter coefficients were estimated using the adaptive lasso technique validated with leave-one-out cross validation (LOOCV) and permutation tests. The R-squared value for the predictive model was 0.60 with p -values for each individual parameter estimate less than 0.0001. Permutation test models trained with scrambled motility failed to produce a model that out-performed the model trained on the true data. The results of this work suggest that it is possible for a quantitative MRI feature-based regression model to non-invasively predict the cellular motility of glioblastomas.

Cell lines are the mainstay in understanding the biology of COVID-19 infection but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue would allow for the ex vivo study of the interindividual heterogeneity of host response to SARS-CoV-2, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infected with HCoV-OC43 and SARS-CoV-2 and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6, CXCL8, and IFNB1 in response to SARS-CoV-2. Treatment of tissues with dexamethasone and the experimental drug N-hydroxycytidine suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a system for the ex vivo study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Microtubule "dynamic instability," the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the β subunit of the αβ-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~ 4 k B T) and an outward bending preference (~ 1.5 k B T) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment., (© 2021. The Author(s).)

Plus-end tracking proteins (+TIPs) associate with the growing end of microtubules and mediate important cellular functions. The majority of +TIPs are directed to the plus-end through a family of end-binding proteins (EBs), which preferentially bind the stabilizing cap of GTP-tubulin present during microtubule growth. One outstanding question is whether there may exist other microtubule-associated proteins (MAPs) that preferentially bind specific nucleotide states of tubulin. Here, we report that the neuronal MAP tau preferentially binds GDP-tubulin ( K D  = 0.26 μM) over GMPCPP-tubulin ( K D  = 1.1 μM) in vitro, as well as GTP-tubulin at the tips of growing microtubules, causing tau binding to lag behind the plus-end both in vitro and in live cells. Thus, tau is a microtubule tip avoiding protein, establishing the framework for a possible new class of tip avoiding MAPs. We speculate that disease-relevant tau mutations may exert their phenotype by their failure to properly recognize GDP-tubulin., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

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-μm 2 rectangular cross-sectional area) and tracked human glioma cells that spontaneously migrated within channels. Cell velocities (v exp  = 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 (v sim  = 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., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Semaphorins, specifically type IV, are important regulators of axonal guidance and have been increasingly implicated in poor prognoses in a number of different solid cancers. In conjunction with their cognate PLXNB family receptors, type IV members have been increasingly shown to mediate oncogenic functions necessary for tumor development and malignant spread. In this study, we investigated the role of semaphorin 4C (SEMA4C) in osteosarcoma growth, progression, and metastasis. We investigated the expression and localization of SEMA4C in primary osteosarcoma patient tissues and its tumorigenic functions in these malignancies. We demonstrate that overexpression of SEMA4C promotes properties of cellular transformation, while RNAi knockdown of SEMA4C promotes adhesion and reduces cellular proliferation, colony formation, migration, wound healing, tumor growth, and lung metastasis. These phenotypic changes were accompanied by reductions in activated AKT signaling, G1 cell cycle delay, and decreases in expression of mesenchymal marker genes SNAI1, SNAI2, and TWIST1. Lastly, monoclonal antibody blockade of SEMA4C in vitro mirrored that of the genetic studies. Together, our results indicate a multi-dimensional oncogenic role for SEMA4C in metastatic osteosarcoma and more importantly that SEMA4C has actionable clinical potential.

Mechanotransduction research focuses on understanding how cells sense and respond to mechanical stimuli by converting mechanical signals into biochemical and biological responses. Cells have been shown to respond to mechanical stimuli through specialized biological machinery such as adhesion complexes. Research in the last two decades helped in identifying key components of cellular mechanotransduction. In recent years, integrated approaches, which are highlighted here, are emerging to provide new insights into the mechanistic and theoretical underpinnings of mechanotransduction. In particular, mathematical modeling has helped elucidate the mechanism underlining ligand spacing and distribution sensing, as well as sensing viscoelastic properties of the extracellular matrix. In addition, molecular tension sensors have helped dissect the forces involved in mechanotransduction at high spatial and temporal resolutions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 μm 2 /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., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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 k B T. 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 (ΔG lat 0 ≅ -5 k B T) 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., (Copyright © 2019. Published by Elsevier Inc.)

Microtubule-targeting agents (MTAs) are widely used chemotherapy drugs capable of disrupting microtubule-dependent cellular functions, such as division and migration. We show that two clinically approved MTAs, paclitaxel and vinblastine, each suppress stiffness-sensitive migration and polarization characteristic of human glioma cells on compliant hydrogels. MTAs influence microtubule dynamics and cell traction forces by nearly opposite mechanisms, the latter of which can be explained by a combination of changes in myosin motor and adhesion clutch number. Our results support a microtubule-dependent signaling-based model for controlling traction forces through a motor-clutch mechanism, rather than microtubules directly relieving tension within F-actin and adhesions. Computational simulations of cell migration suggest that increasing protrusion number also impairs stiffness-sensitive migration, consistent with experimental MTA effects. These results provide a theoretical basis for the role of microtubules and mechanisms of MTAs in controlling cell migration., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding how cells migrate in fibrous environments is important in wound healing, immune function, and cancer progression. A key question is how fiber orientation and network geometry influence cell movement. Here we describe a quantitative, modeling-based approach toward identifying the mechanisms by which cells migrate in fibrous geometries having well controlled orientation. Specifically, U251 glioblastoma cells were seeded onto non-electrospinning Spinneret based tunable engineering parameters fiber substrates that consist of networks of suspended 400 nm diameter nanofibers. Cells were classified based on the local fiber geometry and cell migration dynamics observed by light microscopy. Cells were found in three distinct geometries: adhering two a single fiber, adhering to two parallel fibers, and adhering to a network of orthogonal fibers. Cells adhering to a single fiber or two parallel fibers can only move in one dimension along the fiber axis, whereas cells on a network of orthogonal fibers can move in two dimensions. We found that cells move faster and more persistently in 1D geometries than in 2D, with cell migration being faster on parallel fibers than on single fibers. To explain these behaviors mechanistically, we simulated cell migration in the three different geometries using a motor-clutch based model for cell traction forces. Using nearly identical parameter sets for each of the three cases, we found that the simulated cells naturally replicated the reduced migration in 2D relative to 1D geometries. In addition, the modestly faster 1D migration on parallel fibers relative to single fibers was captured using a correspondingly modest increase in the number of clutches to reflect increased surface area of adhesion on parallel fibers. Overall, the integrated modeling and experimental analysis shows that cell migration in response to varying fibrous geometries can be explained by a simple mechanical readout of geometry via a motor-clutch mechanism.

Cell migration is the physical movement of cells and is responsible for the extensive cellular invasion and metastasis that occur in high-grade tumors. Motivated by decades of direct observation of cell migration via light microscopy, theoretical models have emerged to capture various aspects of the fundamental physical phenomena underlying cell migration. Yet, the motility mechanisms actually used by tumor cells during invasion are still poorly understood, as is the role of cellular interactions with the extracellular environment. In this chapter, we review key physical principles of cytoskeletal self-assembly and force generation, membrane tension, biological adhesion, hydrostatic and osmotic pressures, and their integration in mathematical models of cell migration. With the goal of modeling-driven cancer therapy, we provide examples to guide oncologists and physical scientists in developing next-generation models to predict disease progression and treatment.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome., (© 2017 Mekhdjian, Kai, Rubashkin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Microenvironmental mechanics play an important role in determining the morphology, traction, migration, proliferation, and differentiation of cells. A stochastic motor-clutch model has been proposed to describe this stiffness sensitivity. In this work, we present a master equation-based ordinary differential equation (ODE) description of the motor-clutch model, from which we derive an analytical expression to for a cell's optimum stiffness (i.e. the stiffness at which the traction force is maximal). This analytical expression provides insight into the requirements for stiffness sensing by establishing fundamental relationships between the key controlling cell-specific parameters. We find that the fundamental controlling parameters are the numbers of motors and clutches (constrained to be nearly equal), and the time scale of the on-off kinetics of the clutches (constrained to favor clutch binding over clutch unbinding). Both the ODE solution and the analytical expression show good agreement with Monte Carlo motor-clutch output, and reduce computation time by several orders of magnitude, which potentially enables long time scale behaviors (hours-days) to be studied computationally in an efficient manner. The ODE solution and the analytical expression may be incorporated into larger scale models of cellular behavior to bridge the gap from molecular time scales to cellular and tissue time scales.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Morphogen gradients dictate the spatial patterning of multicellular organisms and are established via transport mechanisms. One of the best-characterized morphogens, Bicoid, acts as a polarity determinant in the Drosophila embryo through spatial-temporal control of gap gene expression. The prevailing model for establishment of the gradient has been localized anterior translation, subsequent diffusion, and spatially uniform degradation, consistent with the observed exponential anterior-posterior decay. However, a recent direct measurement of the Bicoid diffusion coefficient via fluorescence recovery after photobleaching (FRAP) resulted in a surprisingly low estimate, which challenged the prevailing model and led to more complicated active transport models. Here, we reassessed this conclusion using a detailed computational model of the FRAP experiment and analysis. In our model, we found disagreement between the input diffusion coefficient and the resulting estimated diffusion coefficient, as measured by previous methods. By using the model to reproduce the original data, we estimate that Bicoid's mitotic diffusion coefficient is 3-fold larger than the originally reported value. Thus, the long-standing diffusive transport model still holds.

Background: Breaking cell symmetry, known as polarization, requires dynamic reorganization of microtubules (MTs) and is essential to many cellular processes, including axon formation in neurons. A critical step in polarization is believed to be the "selective stabilization" of MTs, which hypothesizes a spatial and/or temporal shift toward net MT assembly in a preferred direction of growth., Results: We now find that a simpler "length-dependent" model, in which MT assembly parameters are spatially and temporally constant, predicts MT accumulation in the direction of growth because of longer mean first passage times in the longer direction. We experimentally tested both models by tracking MT assembly dynamics in polarizing embryonic chick forebrain neurons, and we confirmed that assembly is spatially and temporally constant during axon formation., Conclusion: Cell polarization occurs most simply through cell-length-dependent accumulation of MTs without MT stabilization or capture. In this way, F-actin-mediated cell shape and size changes can be read out by dynamic MTs undergoing simple dynamic instability to ultimately break cell symmetry., (2010 Elsevier Ltd. All rights reserved.)

Digital fluorescence microscopy is commonly used to track individual proteins and their dynamics in living cells. However, extracting molecule-specific information from fluorescence images is often limited by the noise and blur intrinsic to the cell and the imaging system. Here we discuss a method called "model-convolution," which uses experimentally measured noise and blur to simulate the process of imaging fluorescent proteins whose spatial distribution cannot be resolved. We then compare model-convolution to the more standard approach of experimental deconvolution. In some circumstances, standard experimental deconvolution approaches fail to yield the correct underlying fluorophore distribution. In these situations, model-convolution removes the uncertainty associated with deconvolution and therefore allows direct statistical comparison of experimental and theoretical data. Thus, if there are structural constraints on molecular organization, the model-convolution method better utilizes information gathered via fluorescence microscopy, and naturally integrates experiment and theory.

Computational modeling can be extremely useful in interpreting experimental results. Here we describe how a relatively sophisticated stochastic model for microtubule dynamic instability in the mitotic spindle can be developed starting with straightforward rules and simple programming code. Once this model is developed, the method for comparing simulation results to experimental data must be carefully considered. The ultimate utility of any computational model relies on its predictive power and the ability to assist in designing new experiments. We describe how "deconstructing" the model through the use of quantitative animations contributes to a better qualitative understanding of model behavior. By extracting key qualitative elements of the model in this fashion, model predictions and new experiments can be more easily extracted from model results.

Cells sense the environment's mechanical stiffness to control their own shape, migration, and fate. To better understand stiffness sensing, we constructed a stochastic model of the "motor-clutch" force transmission system, where molecular clutches link F-actin to the substrate and mechanically resist myosin-driven F-actin retrograde flow. The model predicts two distinct regimes: (i) "frictional slippage," with fast retrograde flow and low traction forces on stiff substrates and (ii) oscillatory "load-and-fail" dynamics, with slower retrograde flow and higher traction forces on soft substrates. We experimentally confirmed these model predictions in embryonic chick forebrain neurons by measuring the nanoscale dynamics of single-growth-cone filopodia. Furthermore, we experimentally observed a model-predicted switch in F-actin dynamics around an elastic modulus of 1 kilopascal. Thus, a motor-clutch system inherently senses and responds to the mechanical stiffness of the local environment.

Recent studies suggest that the human Kinesin-8 molecular motor Kif18A has a role in chromosome congression. Specifically, these studies find that Kif18A promotes chromosome congression by attenuating chromosome oscillation magnitudes. Together with recent modeling work, in vitro studies, and the analysis of in vivo yeast data, these reports reveal how Kinesin-8 molecular motors might control chromosome oscillation amplitudes by spatially regulating the dynamic instability of microtubule plus-ends within the mitotic spindle.

Intracellular protein concentration gradients are generally thought to be unsustainable at steady-state due to diffusion. Here we show how protein concentration gradients can theoretically be sustained indefinitely through a relatively simple mechanism that couples diffusion to a spatially segregated kinase-phosphatase system. Although it is appreciated that such systems can theoretically give rise to phosphostate gradients, it has been assumed that they do not give rise to gradients in the total protein concentration. Here we show that this assumption does not hold if the two forms of protein have different diffusion coefficients. If, for example, the phosphorylated state binds selectively to a second larger protein or protein complex then a steady state gradient in total protein concentration will be created. We illustrate the principle with an analytical solution to the diffusion-reaction problem and by stochastic individual-based simulations using the Smoldyn program. We argue that protein gradients created in this way need to be considered in experiments using fluorescent probes and could in principle encode spatial information in the cytoplasm.

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and approximately 8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.

We developed a stochastic model of microtubule (MT) assembly dynamics that estimates tubulin-tubulin bond energies, mechanical energy stored in the lattice dimers, and the size of the tubulin-GTP cap at MT tips. First, a simple assembly/disassembly state model was used to screen possible combinations of lateral bond energy (DeltaG(Lat)) and longitudinal bond energy (DeltaG(Long)) plus the free energy of immobilizing a dimer in the MT lattice (DeltaG(S)) for rates of MT growth and shortening measured experimentally. This analysis predicts DeltaG(Lat) in the range of -3.2 to -5.7 k(B)T and DeltaG(Long) plus DeltaG(S) in the range of -6.8 to -9.4 k(B)T. Based on these estimates, the energy of conformational stress for a single tubulin-GDP dimer in the lattice is 2.1-2.5 k(B)T. Second, we studied how tubulin-GTP cap size fluctuates with different hydrolysis rules and show that a mechanism of directly coupling subunit addition to hydrolysis fails to support MT growth, whereas a finite hydrolysis rate allows growth. By adding rules to mimic the mechanical constraints present at the MT tip, the model generates tubulin-GTP caps similar in size to experimental estimates. Finally, by combining assembly/disassembly and cap dynamics, we generate MT dynamic instability with rates and transition frequencies similar to those measured experimentally. Our model serves as a platform to examine GTP-cap dynamics and allows predictions of how MT-associated proteins and other effectors alter the energetics of MT assembly.