stiffness-measurement-soft-silicone-substrates-for-mechanobiology

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Old Dominion University

Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na⁺, Ca, K⁺ channels). The membrane's biomechanical functions include sensing external forces and/or the rigidity of the external environment through force transmission, specific conformational changes and/or signaling through mechanoreceptors (e.g., platelet endothelial cell adhesion molecule (PECAM), vascular endothelial (VE)-cadherin, epithelial (E)-cadherin, integrin) embedded in the membrane. Certain mechanical stimulations through specific receptor complexes induce electrical and/or chemical impulses in cells and propagate across cells and tissues. These biomechanical sensory and biochemical responses have profound implications in normal physiology and disease. Here, we discuss the tools that facilitate the understanding of mechanosensitive adhesion receptors. This article is structured to provide a broad biochemical and mechanobiology background to introduce a freshman mechano-biologist to the field of mechanotransduction, with deeper study enabled by many of the references cited herein.

Biophysical Tools to Study Cellular Mechanotransduction.

Electrotaxis-the directional migration of cells in response to an electric field-is most evident in multicellular collectives and plays an important role in physiological contexts. While most cell types respond to applied electric fields of the order of a Volt per centimeter, our knowledge of the factors influencing this response is limited. This is especially true for collective cell electrotaxis, in which the subcellular migration response within a cell has to be coordinated with coupled neighboring cells. Here, we investigated the effect of the level of actin cytoskeleton polymerization and myosin activity on collective cell electrotaxis of Madin-Darby Canine Kidney (MDCK) cells in response to a weak electric field of physiologically relevant magnitude. We modulated the polymerization state of the actin cytoskeleton using the depolymerizing agent cytochalasin D or the polymerizing agent jasplakinolide. We also modulated the contractility of the cell using the myosin motor inhibitor blebbistatin or the phosphatase inhibitor calyculin A. While all the above pharmacological treatments altered cell speed to various extents, we found that only increasing the contractility and a high level of increase/stabilization of polymerized actin had a strong inhibitory effect specifically on the directedness of collective cell electrotaxis. On the other hand, even as the effect of the actin modulators on collective cell migration was varied, most conditions of actin and myosin pharmacological modulation-except for high level of actin polymerization/stabilization-resulted in cell speeds that were similar in the absence or presence of the electric field. Our results led us to speculate that the applied electric field may largely impact the cellular apparatus specifying the polarity of collective cell migration, rather than the functioning of the migratory apparatus. Bioelectromagnetics. 39:289-298, 2018. © 2018 Wiley Periodicals, Inc.

Venkat Maruthamuthu

Department of Mechanical & Aerospace Engineering

Old Dominion University

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Venkat Maruthamuthu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Mechanical & Aerospace Engineering

Old Dominion University

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Department of Mechanical & Aerospace Engineering