RESUMEN
The shift of funding organizations to prioritize interdisciplinary work points to the need for workflow models that better accommodate interdisciplinary studies. Most scientists are trained in a specific field and are often unaware of the kind of insights that other disciplines could contribute to solving various problems. In this paper, we present a perspective on how we developed an experimental pipeline between a microscopy and image analysis/bioengineering lab. Specifically, we connected microscopy observations about a putative mechanosensing protein, obscurin, to image analysis techniques that quantify cell changes. While the individual methods used are well established (fluorescence microscopy; ImageJ WEKA and mTrack2 programs; MATLAB), there are no existing best practices for how to integrate these techniques into a cohesive, interdisciplinary narrative. Here, we describe a broadly applicable workflow of how microscopists can more easily quantify cell properties (e.g., perimeter, velocity) from microscopy videos of eukaryotic (MDCK) adherent cells. Additionally, we give examples of how these foundational measurements can create more complex, customizable cell mechanics tools and models.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Animales , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía FluorescenteRESUMEN
The N-terminal half of the giant cytoskeletal protein obscurin is comprised of more than 50 Ig-like domains, arranged in tandem. Domains 18-51 are connected to each other through short 5-residue linkers, and this arrangement has been previously shown to form a semi-flexible rod in solution. Domains 1-18 generally have slightly longer ~7 residue interdomain linkers, and the multidomain structure and motion conferred by this kind of linker is understudied. Here, we use NMR, SAXS, and MD to show that these longer linkers are associated with significantly more domain/domain flexibility, with the resulting multidomain structure being moderately compact. Further examination of the relationship between interdomain flexibility and linker length shows there is a 5 residue "sweet spot" linker length that results in dual-domain systems being extended, and conversely that both longer or shorter linkers result in a less extended structure. This detailed knowledge of the obscurin N terminus structure and flexibility allowed for mathematical modeling of domains 1-18, which suggests that this region likely forms tangles if left alone in solution. Given how infrequently protein tangles occur in nature, and given the pathological outcomes that occur when tangles do arise, our data suggest that obscurin is likely either significantly scaffolded or else externally extended in the cell.
Asunto(s)
Difracción de Rayos X , Dispersión del Ángulo Pequeño , Espectroscopía de Resonancia MagnéticaRESUMEN
We used particle-based computer simulations to study the emergent properties of the actomyosin cytoskeleton. Our model accounted for biophysical interactions between filamentous actin and non-muscle myosin II and was motivated by recent experiments demonstrating that spatial regulation of myosin activity is required for fibroblasts responding to spatial gradients of platelet derived growth factor (PDGF) to undergo chemotaxis. Our simulations revealed the spontaneous formation of actin asters, consistent with the punctate actin structures observed in chemotacting fibroblasts. We performed a systematic analysis of model parameters to identify biochemical steps in myosin activity that significantly affect aster formation and performed simulations in which model parameter values vary spatially to investigate how the model responds to chemical gradients. Interestingly, spatial variations in motor stiffness generated time-dependent behavior of the actomyosin network, in which actin asters continued to spontaneously form and dissociate in different regions of the gradient. Our results should serve as a guide for future experimental investigations.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actomiosina/fisiología , Quimiotaxis , Simulación por Computador , Fibroblastos/citología , Animales , Movimiento Celular , Humanos , Modelos Biológicos , Miosina Tipo II/metabolismo , Reconocimiento de Normas Patrones Automatizadas , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
Filamentous actin (F-actin) and non-muscle myosin II motors drive cell motility and cell shape changes that guide large scale tissue movements during embryonic morphogenesis. To gain a better understanding of the role of actomyosin in vivo, we have developed a two-dimensional (2D) computational model to study emergent phenomena of dynamic unbranched actomyosin arrays in the cell cortex. These phenomena include actomyosin punctuated contractions, or "actin asters" that form within quiescent F-actin networks. Punctuated contractions involve both formation of high intensity aster-like structures and disassembly of those same structures. Our 2D model allows us to explore the kinematics of filament polarity sorting, segregation of motors, and morphology of F-actin arrays that emerge as the model structure and biophysical properties are varied. Our model demonstrates the complex, emergent feedback between filament reorganization and motor transport that generate as well as disassemble actin asters. Since intracellular actomyosin dynamics are thought to be controlled by localization of scaffold proteins that bind F-actin or their myosin motors we also apply our 2D model to recapitulate in vitro studies that have revealed complex patterns of actomyosin that assemble from patterning filaments and motor complexes with microcontact printing. Although we use a minimal representation of filament, motor, and cross-linker biophysics, our model establishes a framework for investigating the role of other actin binding proteins, how they might alter actomyosin dynamics, and makes predictions that can be tested experimentally within live cells as well as within in vitro models.
Asunto(s)
Actinas/química , Actomiosina/química , Citoesqueleto de Actina/química , Adenosina Trifosfato/química , Animales , Fenómenos Biomecánicos , Movimiento Celular , Simulación por Computador , Reactivos de Enlaces Cruzados/química , Citoplasma/química , Drosophila , Hidrólisis , Proteínas de Microfilamentos/química , Proteínas Motoras Moleculares/química , Contracción Muscular , Miosinas/química , Polímeros , Viscosidad , Xenopus laevisRESUMEN
Sculpting organism shape requires that cells produce forces with proper directionality. Thus, it is critical to understand how cells orient the cytoskeleton to produce forces that deform tissues. During Drosophila gastrulation, actomyosin contraction in ventral cells generates a long, narrow epithelial furrow, termed the ventral furrow, in which actomyosin fibres and tension are directed along the length of the furrow. Using a combination of genetic and mechanical perturbations that alter tissue shape, we demonstrate that geometrical and mechanical constraints act as cues to orient the cytoskeleton and tension during ventral furrow formation. We developed an in silico model of two-dimensional actomyosin meshwork contraction, demonstrating that actomyosin meshworks exhibit an inherent force orienting mechanism in response to mechanical constraints. Together, our in vivo and in silico data provide a framework for understanding how cells orient force generation, establishing a role for geometrical and mechanical patterning of force production in tissues.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actomiosina/fisiología , Forma de la Célula/fisiología , Modelos Biológicos , Animales , Animales Modificados Genéticamente , Simulación por Computador , Drosophila , Embrión no Mamífero , Femenino , Gastrulación/fisiología , Microscopía Intravital , Proteínas Luminiscentes/química , Microtúbulos/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors.