Robert L. Steward

Unjamming and cell shape in the asthmatic airway epithelium

From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems-both inert and living-have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches

The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell–material interactions.

Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology

Mechanotransduction of sensory neurons is of great interest to the scientific community, especially in areas such as pain, neurobiology, cardiovascular homeostasis and mechanobiology. We describe a method to investigate stretch-activated mechanotransduction in sensory nerves through subcellular stimulation. The method imposes localized mechanical stimulation through indentation of an elastomeric substrate and combines this mechanical stimulation with whole-cell patch clamp recording of the electrical response to single-nerve stretching. One significant advantage here is that the neurites are stretched with limited physical contact beyond their attachment to the polymer. When we imposed specific mechanical stimulation through the substrate, the stretched neurite fired and an action potential response was recorded. In addition, complementary protocols to control the molecules at the cell–substrate interface are presented. These techniques provide an opportunity to probe neurosensory mechanotransduction with a defined substrate, whose physical and molecular context can be modified to mimic physiologically relevant conditions. The entire process from fabrication to cellular recording takes 5 to 6 d.

Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts

It was our objective to study the role of mechanical stimulation on fibronectin (FN) reorganization and recruitment by exposing fibroblasts to shear fluid flow and equibiaxial stretch. Mechanical stimulation was also combined with a Rho inhibitor to probe their coupled effects on FN. Mechanically stimulated cells revealed a localization of FN around the cell periphery as well as an increase in FN fibril formation. Mechanical stimulation coupled with chemical stimulation also revealed an increase in FN fibrils around the cell periphery. Complimentary to this, fibroblasts exposed to fluid shear stress structurally rearranged pre-coated surface FN, but unstimulated and stretched cells did not. These results show that mechanical stimulation directly affected FN reorganization and recruitment, despite perturbation by chemical stimulation. Our findings will help elucidate the mechanisms of FN biosynthesis and organization by furthering the link of the role of mechanics with FN.

Fluid shear, intercellular stress, and endothelial cell alignment.

Endothelial cell alignment along the direction of laminar fluid flow is widely understood to be a defining morphological feature of vascular homeostasis. While the role of associated signaling and structural events have been well studied, associated intercellular stresses under laminar fluid shear have remained ill-defined and the role of these stresses in the alignment process has remained obscure. To fill this gap, we report here the tractions as well as the complete in-plane intercellular stress fields measured within the human umbilical vein endothelial cell (HUVEC) monolayer subjected to a steady laminar fluid shear of 1 Pa. Tractions, intercellular stresses, as well as their time course, heterogeneity, and anisotropy, were measured using monolayer traction microscopy and monolayer stress microscopy. Prior to application of laminar fluid flow, intercellular stresses were largely tensile but fluctuated dramatically in space and in time (317 ± 122 Pa). Within 12 h of the onset of laminar fluid flow, the intercellular stresses decreased substantially but continued to fluctuate dramatically (142 ± 84 Pa). Moreover, tractions and intercellular stresses aligned strongly and promptly (within 1 h) along the direction of fluid flow, whereas the endothelial cell body aligned less strongly and substantially more slowly (12 h). Taken together, these results reveal that steady laminar fluid flow induces prompt reduction in magnitude and alignment of tractions and intercellular stress tensor components followed by the retarded elongation and alignment of the endothelial cell body. Appreciably smaller intercellular stresses supported by cell-cell junctions logically favor smaller incidence of gap formation and thus improved barrier integrity.

Response of an Actin Filament Network Model under Cyclic Stretching through a Coarse Grained Monte Carlo Approach

Cells are complex, dynamic systems that actively adapt to various stimuli including mechanical alterations. Central to understanding cellular response to mechanical stimulation is the organization of the cytoskeleton and its actin filament network. In this manuscript, we present a minimalistic network Monte Carlo based approach to model actin filament organization under cyclic stretching. Utilizing a coarse-grained model, a filament network is prescribed within a two-dimensional circular space through nodal connections. When cyclically stretched, the model demonstrates that a perpendicular alignment of the filaments to the direction of stretch emerges in response to nodal repositioning to minimize net nodal forces from filament stress states. In addition, the filaments in the network rearrange and redistribute themselves to reduce the overall stress by decreasing their individual stresses. In parallel, we cyclically stretch NIH 3T3 fibroblasts and find a similar cytoskeletal response. With this work, we test the hypothesis that a first-principles mechanical model of filament assembly in a confined space is by itself capable of yielding the remodeling behavior observed experimentally. Identifying minimal mechanisms sufficient to reproduce mechanical influences on cellular structure has important implications in a diversity of fields, including biology, physics, medicine, computer science, and engineering.

Illuminating human health through cell mechanics.

Cells reside in mechanically rich and dynamic microenvironments, and the complex interplay between mechanics and biology is widely acknowledged. Recent research has yielded insights linking the mechanobiology of cells, human physiology, and pathophysiology. In particular, we have learned of the cells astounding ability to sense and respond to its mechanical microenvironment. This seemingly innate behaviour of the cell has driven efforts to characterise precisely the cellular behaviour from a mechanical viewpoint. Here we present an overview of technologies used to probe cell mechanical and material properties, how they have led to the discovery of seemingly strange cellular mechanical behaviours, and their influential role in health and disease, including asthma, cancer, and glaucoma. The properties reviewed here have implications in physiology and pathology and raise questions that will fuel research opportunities for years to come.

2D and 3D Mechanobiology in Human and Nonhuman Systems.

Mechanobiology involves the investigation of mechanical forces and their effect on the development, physiology, and pathology of biological systems. The human body has garnered much attention from many groups in the field, as mechanical forces have been shown to influence almost all aspects of human life ranging from breathing to cancer metastasis. Beyond being influential in human systems, mechanical forces have also been shown to impact nonhuman systems such as algae and zebrafish. Studies of nonhuman and human systems at the cellular level have primarily been done in two-dimensional (2D) environments, but most of these systems reside in three-dimensional (3D) environments. Furthermore, outcomes obtained from 3D studies are often quite different than those from 2D studies. We present here an overview of a select group of human and nonhuman systems in 2D and 3D environments. We also highlight mechanobiological approaches and their respective implications for human and nonhuman physiology.

Cellular force signal integration through vector logic gates

The multi-signal mechanical environment mammalian cells experience is often unaccounted for in current mechanical stimulation studies. To address this we developed a novel technique to induce dual integrated force inputs, uniaxial stretch and fluid shear stress and present here for the first time a vector logic-gate framework to characterize cellular response as a function of cytoskeletal reorganization. Using this framework we found that under fluid shear stress and uniaxial stretch NIH 3T3 fibroblasts responded by the Stretch OR Shear vector logic-gate and HUVECs responded by the NOT Stretch OR Shear vector logic-gate. We further developed a parsimonious model of cellular response to multiple mechanical stimuli, which provides a unifying model that captured the experimental response of both cell types.

Bio-Inspired Microdevices that Mimic the Human Vasculature

Blood vessels may be found throughout the entire body and their importance to human life is undeniable. This is evident in the fact that a malfunctioning blood vessel can result in mild symptoms such as shortness of breath or chest pain to more severe symptoms such as a heart attack or stroke, to even death in the severest of cases. Furthermore, there are a host of pathologies that have been linked to the human vasculature. As a result many researchers have attempted to unlock the mysteries of the vasculature by performing studies that duplicate the physiological structural, chemical, and mechanical properties known to exist. While the ideal study would consist of utilizing living, blood vessels derived from human tissue, such studies are not always possible since intact human blood vessels are not readily accessible and there are immense technical difficulties associated with such studies. These limitations have opened the door for the development of microdevices modeled after the human vasculature as it is believed by many researchers in the field that such devices can one day replace tissue models. In this review we present an overview of microdevices developed to mimic various types of vasculature found throughout the human body. Although the human body contains a diverse array of vascular systems for this review we limit our discussion to the cardiovascular system and cerebrovascular system and discuss such systems that have been fabricated in both 2D and 3D configurations.