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ABSTRACT: On rigid surfaces, the cytoskeleton of migrating cells is polarized, but tissue matrix is normally soft. We show that nonmuscle MIIB (myosin-IIB) is unpolarized in cells on soft matrix in 2D and also within soft 3D collagen, with rearward polarization of MIIB emerging only as cells migrate from soft to stiff matrix. Durotaxis is the tendency of cells to crawl from soft to stiff matrix, and durotaxis of primary mesenchymal stem cells (MSCs) proved more sensitive to MIIB than to the more abundant and persistently unpolarized nonmuscle MIIA (myosin-IIA). However, MIIA has a key upstream role: in cells on soft matrix, MIIA appeared diffuse and mobile, whereas on stiff matrix, MIIA was strongly assembled in oriented stress fibers that MIIB then polarized. The difference was caused in part by elevated phospho-S1943-MIIA in MSCs on soft matrix, with site-specific mutants revealing the importance of phosphomoderated assembly of MIIA. Polarization is thus shown to be a highly regulated compass for mechanosensitive migration.
The Journal of Cell Biology 11/2012; · 10.26 Impact Factor
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ABSTRACT: Almost every laboratory that grows mammalian cells today grows their cells on tissue culture plastic, which was introduced to cell culture decades ago based on properties such as inertness, transparency, and so forth. However, plastic is rigid and unlike the many soft tissues in the body. Polymer gel systems that mimic the softness of various tissues have been developed over the past decade to test and understand the effects of rigidity on cells such as muscle cells. One recent study even shows that muscle stem cells expand much better in vitro on muscle-mimetic gels and that such cells prove optimal for engraftment in muscle.
Stem Cell Research & Therapy 01/2010; 1(5):38. · 3.21 Impact Factor
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ABSTRACT: Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A 'cysteine shotgun' method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.
Journal of Cell Science 12/2008; 121(Pt 22):3794-802. · 6.11 Impact Factor
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ABSTRACT: Shrinking biosensors down to microscale dimensions enables increases in sensitivity and the ability to analyze minute samples such as the contents of individual cells. The goal of the present study is to create mobile microscale biosensors by attaching molecular beacons to microtubules and using kinesin molecular motors to transport these functionalized microtubules across two-dimensional surfaces. Previous work has shown that microfluidic channels can be functionalized with kinesin motors such that microtubules can be transported and directed through these channels without the need for external power or pressure-driven pumping. In this work, we show that molecular beacons can be attached to microtubules such that both the fluorescence reporting capability of the beacon and the motility of the microtubules are retained. These molecular beacon-functionalized microtubules were able to bind ssDNA target sequences, transport them across surfaces, and report their presence by an increase in fluorescence that was detected by fluorescence microscopy. This work is an important step toward creating hybrid microdevices for sensitive virus detection or analyzing mRNA profiles of individual cells.
Biotechnology and Bioengineering 04/2008; 99(4):764-73. · 3.95 Impact Factor
