Keiji Naruse

Mechanical behavior in living cells consistent with the tensegrity model

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

Subcellular positioning of small molecules.

Localized perturbation of processes that take place inside the living cell depends on molecular and spatial discrimination on a micrometre scale. Here we report the use of multiple laminar streams in a microfluidic channel to deliver membrane-permeable molecules to selected subcellular microdomains. This technique opens up avenues for non-invasively visualizing, probing and manipulating the cellular metabolic and structural machinery.

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca2+ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca2+ concentration and ATP release. Stretch stimulation evoked intracellular Ca2+ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca2+ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca2+ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca2+. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

Selective Chemical Treatment of Cellular Microdomains Using Multiple Laminar Streams

There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent. It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds. This paper demonstrates experimentally, and analyzes theoretically, the use of multiple laminar fluid streams in microfluidic channels to deliver reagents to, and remove them from, cells with subcellular spatial selectivity. The technique made it possible to label different subpopulations of mitochondria fluorescently, to disrupt selected regions of the cytoskeleton chemically, to dislodge limited areas of cell-substrate adhesions enzymatically, and to observe microcompartmental endocytosis within individual cells. This technique does not require microinjection or immobilization of reagents onto nondiffusive objects; it opens a new window into cell biology.

Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch

The present work was designed to elucidate the involvement of Ca(2+)-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicone membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and > 90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+]i) transiently increased in response to uniaxial stretch, and the basal [Ca2+]i gradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.The present work was designed to elucidate the involvement of Ca2+-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicone membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and >90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+]i) transiently increased in response to uniaxial stretch, and the basal [Ca2+]igradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

Calcium regulates the PI3K-Akt pathway in stretched osteoblasts

Mechanical loading plays a vital role in maintaining bone architecture. The process by which osteoblasts convert mechanical signals into biochemical responses leading to bone remodeling is not fully understood. The earliest cellular response detected in mechanically stimulated osteoblasts is an increase in intracellular calcium concentration ([Ca2+]i). In this study, we used the clonal mouse osteoblast cell line MC3T3‐E1 to show that uniaxial cyclic stretch induces: (1) an immediate increase in [Ca2+]i, and (2) the phosphorylation of critical osteoblast proteins that are implicated in cell proliferation, gene regulation, and cell survival. Our data suggest that cyclic stretch activates the phosphoinositide 3‐kinase (PI3K) pathway including: PI3K, Akt, FKHR, and AFX. Moreover, cyclic stretch also causes the phosphorylation of stress‐activated protein kinase/c‐Jun N‐terminal kinase. Attenuation in the level of phosphorylation of these proteins was observed by stretching cells in Ca2+‐free medium, using intra‐ (BAPTA‐AM) and extracellular (BAPTA) calcium chelators, or gadolinium, suggesting that influx of extracellular calcium plays a significant role in the early response of osteoblasts to mechanical stimuli.

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor β

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

Pp125FAK is required for stretch dependent morphological response of endothelial cells

In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uni-axial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis. We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation. The major phosphorylated proteins had molecular masses of approximately 120–135 kDa, and 70 kDa. Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125FAK) and pp130CAS were included in the 70 kDa and 120–135 kDa bands, respectively. The morphological change was inhibited by herbimycin A and genistein, inhibitors of tyrosine kinases, suggesting that tyrosine phosphorylation was required for the morphological change. In addition, the kinase activation of pp125FAK was observed in response to cyclic stretch. Moreover, suppression of pp125FAK expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125FAK play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.

Activation of pp60 src is critical for stretch-induced orienting response in fibroblasts

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

UNI-AXIAL CYCLIC STRETCH INDUCES C-SRC ACTIVATION AND TRANSLOCATION IN HUMAN ENDOTHELIAL CELLS VIA SA CHANNEL ACTIVATION

The kinase activity of c‐src increased and peaked at 15 min after an application of uni‐axial cyclic stretch in HUVECs followed by a translocation of c‐src to Triton‐insoluble fraction. Suppression of c‐src by an antisense S‐oligodeoxynucleotide inhibited the stretch‐induced tyrosine phosphorylation and morphological changes. The stretch‐induced increase in c‐src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd3+, a blocker for stretch activated channels, and by the extracellular Ca2+ depletion suggesting the involvement of SA channels. These results strongly suggest c‐src plays an important role in the downstream of SA channel activation followed by the morphological changes.