Cells respond to mechanical stress by rapid disassembly of caveolae.

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.

Use of a multi-method approach to rapidly assess the impact of public health policies at the state and local level: a case study of flavored e-cigarette policies.

BACKGROUND: E-cigarettes are the most-commonly used tobacco product by youth since 2014. To prevent youth access and use of e-cigarettes, many U.S. states and localities have enacted policies over a relatively short period of time. The adoption of these policies has necessitated timely data collection to evaluate impacts. METHODS: To assess the impact of flavored e-cigarette policies in select states and local jurisdictions across the United States, a multi-method, complementary approach was implemented from July 2019 to present, which includes analyses of cross-sectional online surveys of young people ages 13-24 years with retail sales data. RESULTS: From February 2020 through February 2023, cross-sectional surveys have been conducted in three cities, one county, and eight states where policy changes have been enacted or are likely to be enacted. Data collection occurred every six months to provide near real-time data and examine trends over time. Additionally, weekly retail sales data were aggregated to showcase monthly sales trends at the national level and for the selected states. DISCUSSION: This rapid and efficient method of coupling online survey data with retail sales data provides a timely and effective approach for monitoring a quickly changing tobacco product landscape, particularly for states and localities where rapidly-available data is often not available. This approach can also be used to monitor other health behaviors and relevant policy impacts.

Student tobacco use, secondhand smoke exposure, and policy beliefs before and after implementation of a tobacco-free campus policy: Analysis of five U.S. college and university campuses.

The adoption of comprehensive tobacco policies by colleges and universities may help reduce student tobacco use. To this end, The American Cancer Society's Tobacco-Free Generation Campus Initiative (TFGCI) awarded grants to 106 higher learning institutions to adopt 100% tobacco-free campus policies. This study measured changes in student tobacco use, reported exposure to secondhand smoke, and support for types of tobacco policies among five TFGCI grantee institutions who implemented 100% tobacco-free policies. Students at five U.S. TFGCI grantee institutions completed two independent cross-sectional online surveys regarding tobacco use, exposure to secondhand smoke, and policy attitudes, once before (n = 2499) and once after (n = 1667) their campuses adopted a tobacco-free policy. Students were less likely to report current cigarette smoking (aOR: 0.73, 95% C.I.: 0.63, 0.85) and exposure to secondhand smoke on campus (aOR: 0.42, 95% C.I.: 0.23, 0.76) following the policy change. In contrast, students were more likely to report past 30-day use of electronic nicotine delivery systems (ENDS) (aOR: 2.16, C.I.: 1.77, 2.63) following the policy change, despite the policy's inclusion of all tobacco and nicotine products. Tobacco-free campus policies can be associated with decreases in tobacco product use and environmental smoke exposure. The extent of their effectiveness may vary by product and the inclusion of tailored messaging, cessation support, and enforcement approaches. To discourage use of these products among students, colleges and universities should adopt 100% tobacco-free policies, monitor product use trends, offer cessation support and messaging customized for specific groups and products, and utilize a comprehensive enforcement strategy.

Smoke-free and tobacco-free colleges and universities in the United States.

OBJECTIVE: To describe the number and proportion of accredited, degree-granting institutions with 100% smoke-free and 100% tobacco-free protections across the USA and by state. METHODS: Data on postsecondary education institutions from the US Department of Education National Center for Education Statistics Integrated Postsecondary Education Data System 2015, and smoke-free and tobacco-free campus protections from the American Nonsmokers' Rights Foundation's Smokefree and Tobacco-Free Colleges and Universities List 2017, were integrated to calculate the number and proportion of: (1) smoke-free and tobacco-free accredited, degree-granting institutions and (2) students and staff protected by campus policies and state laws. Campus protections are given a 100% smoke-free designation if smoking is not allowed on campus anywhere, at any time; 100% tobacco-free designations extend smoke-free protections to include non-combustible products such as smokeless tobacco. RESULTS: 823 accredited, degree-granting institutions (16.7%) representing 1816 individual campuses, sites and schools have either 100% smoke-free or 100% tobacco-free protections. An estimated 14.9 million college students (26.9%) and 8.9 million faculty and staff (25.4%) are protected by campus policies and state laws. Only three states and two territories have 100% smoke-free or 100% tobacco-free protections in over half of their institutions; four states and six territories have no known 100% smoke-free or 100% tobacco-free campus protections. CONCLUSIONS: In 2017, just 16.7% of accredited, degree-granting institutions in the USA had 100% smoke-free or 100% tobacco-free protections. Despite progress, more efforts can ensure that students and staff benefit from comprehensive 100% smoke-free and 100% tobacco-free protections at US colleges and universities.

Cholesterol Depletion by MßCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity.

Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) remodels the plasma membrane's mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it's unclear if MßCD alters membrane tension or only enhances membrane-cytoskeleton interactions-and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MßCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations' frequency spectrum. This helps us conclude that MßCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MßCD also has a strong impact on the cell membrane's tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters-an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.

Mapping Cell Membrane Fluctuations Reveals Their Active Regulation and Transient Heterogeneities.

Shape fluctuations of the plasma membrane occur in all cells, are incessant, and are proposed to affect membrane functioning. Although studies show how membrane fluctuations are affected by cellular activity in adherent cells, their spatial regulation and the corresponding change in membrane mechanics remain unclear. In this article, we study how ATP-driven activities and actomyosin cytoskeleton impact basal membrane fluctuations in adherent cells. Using interference imaging, we map height fluctuations within single cells and compare the temporal spectra with existing theoretical models to gain insights about the underlying membrane mechanics. We find that ATP-dependent activities enhance the nanoscale z fluctuations but stretch out the membrane laterally. Although actin polymerization or myosin-II activity individually enhances fluctuations, the cortex in unperturbed cells stretches out the membrane and dampens fluctuations. Fitting with models suggest this dampening to be due to confinement by the cortex. However, reduced fluctuations on mitosis or on ATP-depletion/stabilization of cortex correlate with increased tension. Both maps of fluctuations and local temporal autocorrelation functions reveal ATP-dependent transient short-range (<2 µm) heterogeneities. Together, our results show how various ATP-driven processes differently affect membrane mechanics and hence fluctuations, while creating distinct local environments whose functional role needs future investigation.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

The Plasma Membrane and Mechanoregulation in Cells.

Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal role in this process of adaptation. In this Review, we begin by examining well-studied processes where mechanoregulation proves significant. Specifically, we highlight examples from the immune system and stem cells, besides discussing processes involving fibroblasts and other cell types. Subsequently, we discuss the common molecular players that facilitate the sensing of the mechanical signal and transform it into a chemical response covering integrins YAP/TAZ and Piezo. We then review how this understanding of molecular elements is leveraged in drug discovery and tissue engineering alongside a discussion of the methodologies used to measure mechanical properties. Focusing on the processes of endocytosis, we discuss how cells may respond to altered membrane mechanics using endo- and exocytosis. Through the process of depleting/adding the membrane area, these could also impact membrane mechanics. We compare pathways from studies illustrating the involvement of endocytosis in mechanoregulation, including clathrin-mediated endocytosis (CME) and the CLIC/GEEC (CG) pathway as central examples. Lastly, we review studies on cell-cell fusion during myogenesis, the mechanical integrity of muscle fibers, and the reported and anticipated roles of various molecular players and processes like endocytosis, thereby emphasizing the significance of mechanoregulation at the PM.

Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells.

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

Lessons learned about post-secondary institution tobacco policy change by Tobacco-Free Generation Campus Initiative Grantees, 2018-2020.

OBJECTIVE: The Tobacco-Free Generation Campus Initiative (TFGCI) granted funding to U.S. post-secondary institutions to assist with creating 100% smoke- and tobacco-free campus policies to prevent young adult tobacco use. This study assessed grantee experiences working on campus tobacco policy change. Participants: Sixty U.S. post-secondary institutions completing TFGCI funded work between 2018 and 2020. Methods: An online survey assessment was completed by each institution at the end of their 18-month grant period to share facilitators and barriers to policy change, lessons learned, and advice for future efforts. Results: Many institutions faced challenges of time and capital constraints and pushback from campus constituents. Collaboration, diverse constituent engagement, and educational efforts throughout the advocacy process were important facilitators of policy change. Conclusions: Adopting and implementing comprehensive tobacco policy on college campuses is challenging. Regardless of institution type, commitment to the long-term goal and diverse stakeholder support guided movement toward 100% smoke- and tobacco-free campus policies.Supplemental data for this article can be accessed online at https://doi.org/10.1080/07448481.2021.1953032 .

Connexin 43 mediated collective cell migration is independent of Golgi orientation.

Cell migration is vital for multiple physiological functions and is involved in the metastatic dissemination of tumour cells in various cancers. For effective directional migration, cells often reorient their Golgi apparatus and, therefore, the secretory traffic towards the leading edge. However, not much is understood about the regulation of Golgi's reorientation. Herein, we address the role of gap junction protein Connexin 43 (Cx43), which connects cells, allowing the direct exchange of molecules. We utilized HeLa WT cells lacking Cx43 and HeLa 43 cells, stably expressing Cx43, and found that functional Cx43 channels affected Golgi morphology and reduced the reorientation of Golgi during cell migration. Although the migration velocity of the front was reduced in HeLa 43, the front displayed enhanced coherence in movement, implying an augmented collective nature of migration. On BFA treatment, Golgi was dispersed and the high heterogeneity in inter-regional front velocity of HeLa WT cells was reduced to resemble the HeLa 43. HeLa 43 had higher vimentin expression and stronger basal F-actin. Furthermore, non-invasive measurement of basal membrane height fluctuations revealed a lower membrane tension. We, therefore, propose that reorientation of Golgi is not the major determinant of migration in the presence of Cx43, which induces collective-like coherent migration in cells.

Early tension regulation coupled to surface myomerger is necessary for the primary fusion of C2C12 myoblasts.

Here, we study the time-dependent regulation of fluctuation-tension during myogenesis and the role of the fusogen, myomerger. We measure nanometric height fluctuations of the basal membrane of C2C12 cells after triggering differentiation. Fusion of cells increases fluctuation-tension but prefers a transient lowering of tension (at ~2-24 h). Cells fail to fuse if early tension is continuously enhanced by methyl-ß-cyclodextrin (MßCD). Perturbing tension regulation also reduces fusion. During this pre-fusion window, cells that finally differentiate usually display lower tension than other non-fusing cells, validating early tension states to be linked to fate decision. Early tension reduction is accompanied by low but gradually increasing level of the surface myomerger. Locally too, regions with higher myomerger intensity display lower tension. However, this negative correlation is lost in the early phase by MßCD-based cholesterol depletion or later as differentiation progresses. We find that with tension and surface-myomerger's enrichment under these conditions, myomerger clusters become pronouncedly diffused. We, therefore, propose that low tension aided by clustered surface-myomerger at the early phase is crucial for fusion and can be disrupted by cholesterol-reducing molecules, implying the potential to affect muscle health.

Effect of heterogeneous substrate adhesivity of follower cells on speed and tension profile of leader cells in primary keratocyte collective cell migration.

In single keratocyte motility, membrane tension is reported to be high at cell-fronts and believed to establish front coherence. To understand role of membrane mechanics in collective cell migration, we study membrane height fluctuations in cell sheets from fish scales using interference reflection microscopy (IRM). We report the monolayer to have cells lacking substrate adhesion and show that such 'non-sticky' cells can form bridges between leader cells and far-away follower cells. Do such interactions alter motility and membrane mechanics in such leaders? We find non-significant, but reduced speed for leaders with 'non-sticky' followers in comparison to other leaders. Cells show high phenotypic variability in their membrane fluctuation tension profiles. On average, this tension is found to be lower at cell fronts than the mid-section. However, leaders with non-sticky followers are more prone to display higher tension at their front and have a negative correlation between cell speed and front-mid tension difference. Thus, we conclude that intracellular tension gradients are heterogeneous in cell sheets and substrate adhesivity of followers can control the coupling of the gradient to cell speed.

Two Consecutive Prolines in the Fusion Peptide of Murine ß-Coronavirus Spike Protein Predominantly Determine Fusogenicity and May Be Essential but Not Sufficient to Cause Demyelination.

Combined in silico, in vitro, and in vivo comparative studies between isogenic-recombinant Mouse-Hepatitis-Virus-RSA59 and its proline deletion mutant, revealed a remarkable contribution of centrally located two consecutive prolines (PP) from Spike protein fusion peptide (FP) in enhancing virus fusogenic and hepato-neuropathogenic potential. To deepen our understanding of the underlying factors, we extend our studies to a non-fusogenic parental virus strain RSMHV2 (P) with a single proline in the FP and its proline inserted mutant, RSMHV2 (PP). Comparative in vitro and in vivo studies between virus strains RSA59(PP), RSMHV2 (P), and RSMHV2 (PP) in the FP demonstrate that the insertion of one proline significantly resulted in enhancing the virus fusogenicity, spread, and consecutive neuropathogenesis. Computational studies suggest that the central PP in Spike FP induces a locally ordered, compact, and rigid structure of the Spike protein in RSMHV2 (PP) compared to RSMHV2 (P), but globally the Spike S2-domain is akin to the parental strain RSA59(PP), the latter being the most flexible showing two potential wells in the energy landscape as observed from the molecular dynamics studies. The critical location of two central prolines of the FP is essential for fusogenicity and pathogenesis making it a potential site for designing antiviral.

Cell spread area and traction forces determine myosin-II-based cortex thickness regulation.

Actomyosin network under the plasma membrane of cells forms a cortical layer that regulates cellular deformations during different processes. What regulates the cortex? Characterized by its thickness, it is believed to be regulated by actin dynamics, filament-length regulators and myosin motor proteins. However, its regulation by cellular morphology (e.g. cell spread area) or mechanical microenvironment (e.g. substrate stiffness) has remained largely unexplored. In this study, super- and high-resolution imaging of actin in CHO cells demonstrates that at high spread areas (>450 µm2), the cortex is thinner, better separated as layers, and sensitive to deactivation of myosin II motors or reduction of substrate stiffness (and traction forces). In less spread cells (<400 µm2) such perturbations do not elicit a response. Myosin IIA's mechanosensing is limited here due to its lowered actin-bound fraction and higher turnover rate. Cofilin, in line with its competitive inhibitory role, is found to be overexpressed in these cells. To establish the causal relation, we initiate a spread area drop by de-adhesion and find enhanced actin dynamics and fragmentation along with oscillations and increase in thickness. This is more correlated to the reduction of traction forces than the endocytosis-based reduction in cell volume. Cortex thickness control by spread area is also found be true during differentiation of THP-1 monocytes to macrophages. Thus, we propose that spread area regulates cortex and its thickness by traction-based mechanosensing of myosin II.

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells.

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.

Cellular and Nuclear Forces: An Overview.

Biological cells sample their surrounding microenvironments using nanoscale force sensors on the cell surfaces. These surface-based force and stress sensors generate physical and chemical responses inside the cell. The inherently well-connected cytoskeleton and its physical contacts with the force elements on the nuclear membrane lead these physicochemical responses to cascade all the way inside the cell nucleus, physically altering the nuclear state. These physical alterations of the cell nucleus, through yet-unknown complex steps elicit physical and functional response from the chromatin in the form of altered gene expression profiles. This mechanism of force/stress sensing by the cell and then its nuclear response has been shown to play a vital role in maintaining robust cellular homeostasis, controlling gene expression profiles during developmental phases as well as cell differentiation. Over the last few years, there has been appreciable progress toward identification of the molecular players responsible for force sensing. However, the actual sensing mechanism of cell surface bound force sensors and more importantly cascading of the signals, both physical (via cytosolic force sensing elements such as microtubule and actin framework) and chemical (cascade of biochemical signaling from cell surface to nuclear surface and further to the chromatin), inside the cell is poorly understood. In this chapter, we present a review of the currently known molecular players in cellular as well as nuclear force sensing repertoire and their possible mechanistic aspects. We also introduce various biophysical concepts that are used to describe the force/stress sensing and response of a cell. We hope this will help asking clearer questions and designing pointed experiments for better understanding of the force-dependent design principles of the cell surface, nuclear surface, and gene expression.

"Love Your Love Life": Disadvantaged African American Adolescents Cocreate Psychoeducational Romantic and Sexual Health Resources.

Sexually transmitted disease transmission, dating violence, trafficking, and unplanned pregnancy disproportionately afflict disadvantaged African American adolescents, and social services to effectively remedy this crisis need improvement. Moreover, disadvantaged African American adolescents have been significantly underrepresented in mental health, social services, and best practices research, so existing evidence-based practice models are insufficiently inclusive of these adolescents' perspectives. As a remedy, this study describes a formative evaluation of a youth-led, participatory action-based summer and after-school program, Love Your Love Life. Over eight successive semesters, 155 African American adolescents authored instructional materials including a guide to dating, a documentary, a workbook, a PowerPoint presentation, social media messages, and skits, and then led seminars for their peers. The highly engaged adolescents contributed valuable information about their strengths, challenges, and preferences, and suggested improvements to make sexual and romantic health curricula more relevant for their peers.

Dynamic organization of chromatin assembly and transcription factories in living cells.

The interphase nucleus is an active organelle involved in processing genetic information. In higher order eukaryotes, information control is compartmentalized - for example at the scale of inter-chromosome territories and nuclear bodies. Regulatory proteins, nuclear bodies and chromatin assembly are found to be highly dynamic within the nucleus of primary cells and through cellular differentiation programs. In this chapter we describe live-cell fluorescence based techniques and single particle tracking analysis, to probe the spatio-temporal dimension in nuclear function.