Apical Sarcomere-like Actomyosin Contracts Nonmuscle Drosophila Epithelial Cells

Actomyosin networks generate contractile force that changes cell and tissue shape. In muscle cells, actin filaments and myosin II appear in a polarized structure called a sarcomere, in which myosin II is localized in the center. Nonmuscle cortical actomyosin networks are thought to contract when nonmuscle myosin II (myosin) is activated throughout a mixed-polarity actin network. Here, we identified a mutant version of the myosin-activating kinase, ROCK, that localizes diffusely, rather than centrally, in epithelial cell apices. Surprisingly, this mutant inhibits constriction, suggesting that centrally localized apical ROCK/myosin activity promotes contraction. We determined actin cytoskeletal polarity by developing a barbed end incorporation assay for Drosophila embryos, which revealed barbed end enrichment at junctions. Our results demonstrate that epithelial cells contract with a spatially organized apical actomyosin cortex, involving a polarized actin cytoskeleton and centrally positioned myosin, with cell-scale order that resembles a muscle sarcomere.National Institutes of Health (U.S.) (GM06806)American Heart Association (Grant-in-aid 14GRNT1888005)American Cancer Society (grant 125792-RSG-14-039-01-CS)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant T32GM00728

Actomyosin Pulsing in Tissue Integrity Maintenance during Morphogenesis

The actomyosin cytoskeleton is responsible for many changes in cell and tissue shape. For a long time, the actomyosin cytoskeleton has been known to exhibit dynamic contractile behavior. Recently, discrete actomyosin assembly/disassembly cycles have also been observed in cells. These so-called actomyosin pulses have been observed in a variety of contexts, including cell polarization and division, and in epithelia, where they occur during tissue contraction, folding, and extension. In epithelia, evidence suggests that actomyosin pulsing, and more generally, actomyosin turnover, is required to maintain tissue integrity during contractile processes. This review explores possible functions for pulsing in the many instances during which pulsing has been observed, and also highlights proposed molecular mechanisms that drive pulsing

BioMeT and algorithm challenges: A proposed digital standardized evaluation framework

Technology is advancing at an extraordinary rate. Continuous flows of novel data are being generated with the potential to revolutionize how we better identify, treat, manage, and prevent disease across therapeutic areas. However, lack of security of confidence in digital health technologies is hampering adoption, particularly for biometric monitoring technologies (BioMeTs) where frontline healthcare professionals are struggling to determine which BioMeTs are fit-for-purpose and in which context. Here, we discuss the challenges to adoption and offer pragmatic guidance regarding BioMeTs, cumulating in a proposed framework to advance their development and deployment in healthcare, health research, and health promotion. Furthermore, the framework proposes a process to establish an audit trail of BioMeTs (hardware and algorithms), to instill trust amongst multidisciplinary users

Verification, Analytical Validation, and Clinical Validation (V3): The Foundation of Determining Fit-for-Purpose for Biometric Monitoring Technologies (BioMeTs)

Digital medicine is an interdisciplinary field, drawing together stakeholders with expertize in engineering, manufacturing, clinical science, data science, biostatistics, regulatory science, ethics, patient advocacy, and healthcare policy, to name a few. Although this diversity is undoubtedly valuable, it can lead to confusion regarding terminology and best practices. There are many instances, as we detail in this paper, where a single term is used by different groups to mean different things, as well as cases where multiple terms are used to describe essentially the same concept. Our intent is to clarify core terminology and best practices for the evaluation of Biometric Monitoring Technologies (BioMeTs), without unnecessarily introducing new terms. We focus on the evaluation of BioMeTs as fit-for-purpose for use in clinical trials. However, our intent is for this framework to be instructional to all users of digital measurement tools, regardless of setting or intended use. We propose and describe a three-component framework intended to provide a foundational evaluation framework for BioMeTs. This framework includes (1) verification, (2) analytical validation, and (3) clinical validation. We aim for this common vocabulary to enable more effective communication and collaboration, generate a common and meaningful evidence base for BioMeTs, and improve the accessibility of the digital medicine field

Stable Force Balance between Epithelial Cells Arises from F-Actin Turnover

The propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells’ contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. Here, we identified the role for F-actin turnover in regulating the contractile cytoskeletal network’s attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. Our work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues.National Institute of General Medical Sciences (U.S.) (F32GM113425)National Institute of General Medical Sciences (U.S.) (R01GM084947)National Institute of General Medical Sciences (U.S.) (R01GM105984

Optogenetic tuning reveals rho amplification-dependent dynamics of a cell contraction signal network

Local cell contraction pulses play important roles in tissue and cell morphogenesis. Here, we improve a chemo-optogenetic approach and apply it to investigate the signal network that generates these pulses. We use these measurements to derive and parameterize a system of ordinary differential equations describing temporal signal network dynamics. Bifurcation analysis and numerical simulations predict a strong dependence of oscillatory system dynamics on the concentration of GEF-H1, an Lbc-type RhoGEF, which mediates the positive feedback amplification of Rho activity. This prediction is confirmed experimentally via optogenetic tuning of the effective GEF-H1 concentration in individual living cells. Numerical simulations show that pulse amplitude is most sensitive to external inputs into the myosin component at low GEF-H1 concentrations and that the spatial pulse width is dependent on GEF-H1 diffusion. Our study offers a theoretical framework to explain the emergence of local cell contraction pulses and their modulation by biochemical and mechanical signals

Molecular organization of the actin cortex in apical constriction and epithelial folding

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (pages 120-139).Actin and myosin generate contractile forces to change tissue and cell shape. These shape changes are essential for many biological functions, ranging from muscle contraction to tissue morphogenesis in development. While the spatial organization and composition of the actin and myosin contractile force generating machine is well known in muscle, it is less understood in nonmuscle epithelia, which change shape during development and form functional barriers on an organism's inner surfaces. Prevailing models for nonmuscle contractility suggest that the intrinsic ability of mixed polarity actin networks and uniformly distributed myosin to contract into asters drives nonmuscle contractility. Here, I provide insight into the mechanism of nonmuscle contraction by demonstrating that the apical actin cortex and associated proteins are spatially organized in epithelia. In addition, I demonstrate that this spatial organization forms a sarcomere-like actomyosin apparatus, which is essential for epithelial contractility. This updated model is likely to inform our understanding of a wide range of contractile force-generating systems, and may lead to advances in understanding of pathologies that involve defects in contractility, like cardiovascular disease and pulmonary fibrosis.by Jonathan S. Coravos.Ph. D