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Cells are highly sensitive to their geometrical and mechanical microenvironment that directly regulate cell shape, cytoskeleton and organelle, as well as the nucleus morphology and genetic expression. The emerging two-dimensional micropatterning techniques offer powerful tools to construct controllable and well-organized microenvironment for single-cell level investigations with qualitative analysis, cellular standardization, and in vivo environment mimicking. Here, we provide an overview of the basic principle and characteristics of the two most widely-used micropatterning techniques, including photolithographic micropatterning and soft lithography micropatterning. Moreover, we summarize the application of micropatterning technique in controlling cytoskeleton, cell migration, nucleus and gene expression, as well as intercellular communication. [ABSTRACT FROM AUTHOR]

Actin cytoskeleton plays crucial roles in various cellular functions. Extracellular matrix (ECM) can modulate cell morphology by remodeling the internal cytoskeleton. To define how geometry of ECM regulates the organization of actin cytoskeleton, we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes. It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern, but were absent from the opening edge of V-shaped pattern, indicating that the organization of actin cytoskeleton was dependent on the mechanical environment. Furthermore, a secondary actin ring was observed on 50 μ m circular pattern while did not appear on 30 μ m and 40 μ m pattern, showing a size-dependent organization of actin cytoskeleton. Finally, osteoblasts, MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern, suggesting a cell-type specificity in arrangement of actin cytoskeleton. Together, our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments. [ABSTRACT FROM AUTHOR]

Sarcolipin (SLN) is an important transmembrane (TM) protein encoded by long noncoding RNA. SLN is expressed in the sarcoplasmic reticulum and regulates cardiac and skeletal muscle contractions. SLN forms a pentameric hydrophobic ligand‐gated ion channel. The protonation of Glu7 (protonated SLN, pSLN) and mutation of Thr18 to Ala18 (T18A) have been reported to exert a significant influence on the permeability of the channel. In this study, the altered permeability of both the pSLN and T18A pentameric channels was simulated. Combined with molecular dynamics simulation, the free‐energy landscape for single ions, computational electrophysiology, diffusion coefficient, and pore geometrical characteristic analyses were performed to further understand the properties of amino acid modifications in the SLN pentameric channel. The results suggest that both the pSLN and T18A pentameric channels form stable hydrophobic ligand‐gated channels. The TM voltage has a positive effect on the permeability of water molecules and ions. By using pSLN and T18A, our study provides helpful information on the pore‐forming mechanism of SLN and furthers our understanding of the regulatory mechanisms underlying the permeation of ions and water molecules in the pentameric SLN channel. [ABSTRACT FROM AUTHOR]

Bone constantly remodels between resorption by osteoclasts and formation by osteoblasts; therefore the functions of osteoblasts are pivotal for maintaining homeostasis of bone mass. Transient receptor potential vanilloid 4 (TRPV4), a type of mechanosensitive channel, has been reported to be a key regulator in bone remodeling. However, the relationship between TRPV4 and osteoblast function remains largely elusive. Only little is known about the spatial distribution change of TRPV4 during osteoblastic differentiation and related signal events. Based on three-dimensional super-resolution microscopy, our results clearly showed a different distribution of TRPV4 in undifferentiated and differentiated osteoblasts, which reflected the plasma membrane translocation of TRPV4 along with prolonged differentiation. GSK1016790A (GSK101), the most potent agonist of TRPV4, triggered rapid calcium entry and calmodulin-dependent protein kinase II (CaMKII) phosphorylation via TRPV4 activation in a differentiation-dependent manner, indicating that the abundance of TRPV4 at the cell surface resulting from differentiation may be related to the modulation of Ca2+ response and CaMKII activity. These data provide compelling evidences for the plasma membrane translocation of TRPV4 during osteoblastic differentiation as well as demonstrate the regulation of downstream Ca2+/CaMKII signaling. [ABSTRACT FROM AUTHOR]

ABSTRACT: The intracellular calcium ([Ca2+]i) response induced by external forces can be diverse and complex. Using primary osteoblasts from Wistar rats, we found multiple patterns of [Ca2+]i responses induced by fluidic shear stress (Fss), including homogeneous non‐oscillation and heterogeneous oscillations. These multiple‐patterned [Ca2+]i responses could be influenced by Fss intensity, cell density, and cell differentiation. Our real‐time measurements with free calcium, ATP, ATP without calcium, suramin, apyrase, and thapsigargin confirmed homogeneous [Ca2+]i patterns and/or heterogeneous [Ca2+]i oscillations with respect to the combined degree of external Ca2+ influx, and intracellular Ca2+ release. Our theoretical model supported diverse Fss‐induced calcium activities as well. We concluded that a singular factor of Ca2+ influx or release dominated to produce smooth homogeneous patterns, but combined factors produced oscillatory heterogeneous patterns. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2039–2051, 2018. [ABSTRACT FROM AUTHOR]