Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to ...

Sam Houston State University

The mechanical microenvironment of an endothelial cell includes a stable protein scaffold on the basal side, flowing blood on the apical side and contractile cells on the lateral sides. Interaction with the protein scaffold and flowing blood modulates the ability of endothelial cells to migrate, align and maintain barrier function. Interaction with neighbors provides the endothelial monolayer unique "collective" properties. However, the nature of local mechanical signaling - i.e., the local functional consequence of a cell interacting with its contractile neighbors - remains unclear. Using an advancing sheet of pulmonary microvascular endothelial cells, here we examine the mechanical properties of an individual cell and its neighboring region. By combining Monolayer Stress Microscopy (MSM) with a novel analysis, we assessed several mechanical properties of an individual cell and its neighboring region. Across the monolayer, mechanical properties of the neighboring region defined multicellular "subdivisions" wherein constituent cells were exposed to a similar mechanical microenvironment. Adjacent subdivisions were separated by a narrow interface where adjoining cells were exposed to remarkably different mechanical microenvironments. Comparison of temporal fluctuations in mechanical properties of individual cells and those of their neighboring regions suggested three distinct intercellular mechanical signaling processes. These processes indicated that change in size, shape and speed of individual cells is associated with change in contractile forces in their neighboring regions. In summary, we present a novel approach to assess the mechanical interactions of individual cells with their contractile neighbors and identify potential functional consequences of such interactions.

Mechanical signaling in a pulmonary microvascular endothelial cell monolayer.

In the field of endothelial biology, the term "shear forces" is tied to the forces exerted by the flowing blood on the quiescent cells. But endothelial cells themselves also exert physical forces on their immediate and distant neighbors. Specific factors of such intrinsic mechanical signals most relevant to immediate neighbors include normal (F) and shear (F) components of intercellular tractions, and those factors most relevant to distant neighbors include contractile or dilatational (M) and shear (M) components of the moments of cytoskeletal forces. However, for cells within a monolayer, F, F, M, and M remain inaccessible to experimental evaluation. Here, we present an approach that enables quantitative assessment of these properties. Remarkably, across a collectively migrating sheet of pulmonary microvascular endothelial cells, F was of the same order of magnitude as F. Moreover, compared to the normal components (F, M) of the mechanical signals, the shear components (F, M) were more distinctive in the cells closer to the migration front. Individual cells had an innately collective tendency to migrate along the axis of maximum contractile moment - a collective migratory process we referred to as cellular plithotaxis. Notably, larger F and M were associated with stronger plithotaxis, but dilatational moment appeared to disengage plithotactic guidance. Overall, cellular plithotaxis was more strongly associated with the "shear forces" (F, M) than with the "normal forces" (F, M). Finally, the mechanical state of the cells with fast migration speed and those with highly circular shape were reminiscent of fluid-like and solid-like matter, respectively. The results repeatedly pointed to neighbors imposing shear forces on a cell as a highly significant event, and hence, the term "shear forces" must include not just the forces from flowing fluid but also the forces from the substrate and neighbors. Collectively, these advances set the stage for deeper understanding of mechanical signaling in cellular monolayers.

Unleashing shear: Role of intercellular traction and cellular moments in collective cell migration.

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a  transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models. The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.

Development of an endothelial cell-restricted transgenic reporter rat: a resource for physiological studies of vascular biology.

The Gram-negative, opportunistic pathogen  utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A (PLA) that hydrolyses host cell membranes, and  strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized  strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with  strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from -infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during  infection that may inhibit endothelial barrier and inflammatory functions.

Diego F. Alvarez

Department of Physiology & Pharmacology,
College of Osteopathic Medicine,
Department of Physiology & Pharmacology, College of Osteopathic Medicine

Sam Houston State University

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Diego F. Alvarez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology & Pharmacology,College of Osteopathic Medicine,Department of Physiology & Pharmacology, College of Osteopathic Medicine

Sam Houston State University

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Department of Physiology & Pharmacology,
College of Osteopathic Medicine,
Department of Physiology & Pharmacology, College of Osteopathic Medicine