As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, Vpeel, was measured as a continuously increasing function of the imposed tension, Tpeel, which ranges from ∼0 to 50 nN/μm. For each cell, peeling proved highly heterogeneous, with Vpeel fluctuating between 0 μm/s (∼80% of time) and ∼10 μm/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.
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机译:作为典型的锚定依赖性细胞,心肌细胞必须在可收缩性与足够的粘附力之间取得平衡。机械分离成肌管一端后,在微图案玻璃上从成肌细胞分离的条状生长的骨骼肌管表现出自发剥离。这样的结果表明细胞中预应力的发展。为了评估这种预应力并研究单个肌细胞的动态粘附强度,然后使用抽吸到大口径微量移液管中的流体的剪切应力来强行剥离肌管。测量细胞从表面剥离的速度Vpeel作为施加的张力Tpeel的连续增加函数,其范围为〜0到50 nN /μm。对于每个单元,剥离表现出高度异质性,Vpeel在0μm/ s(约80%的时间)和〜10μm/ s之间波动。对表达GFP-paxillin的平滑肌细胞的平行研究也显示了不连续的剥离,其中粘连在强附着部位上方破裂(当使用小口径移液管剥离压力时)。此处描述的剥离方法有助于洞察收缩-粘附平衡,并且可以通过物理检测和使用GFP标记物来研究应激粘附的实时动态。该方法在比较正常和营养不良的肌肉细胞中应被证明是有用的。
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