Hallows K R, Law F Y, Packman C H, Knauf P A
Department of Biophysics, University of Rochester Medical Center, New York 14642, USA.
J Cell Physiol. 1996 Apr;167(1):60-71. doi: 10.1002/(SICI)1097-4652(199604)167:1<60::AID-JCP7>3.0.CO;2-A.
Cell volume regulation occurs via the regulated fluxes of ions and solutes across the cell membrane in response to cell volume perturbations under anisotonic conditions. Our earlier studies in human promyelocytic leukemic HL-60 cells showed that volume-dependent changes in total cellular F-actin content occur concomitantly as an inverse function of acute cell volume changes in anisotonic media (Hallows et al., 1991, Am. J. Physiol., 261:C1154-C1161). Although treatment with cytochalasin under anisotonic conditions significantly reduced total cellular F-actin levels, cytochalasin did not significantly affect the ability of cells to undergo normal volume regulation responses, which suggested that these volume-dependent changes in F-actin content may not play a critical role in HL-60 cell volume regulation. To examine more closely the possible role of the actin cytoskeleton in HL-60 cell volume regulation, we quantitated changes in Triton-insoluble cytoskeletal actin in the presence and absence of cytochalasin and also observed changes in F-actin distribution and surface morphology during volume regulation. The quantity of cytoskeletal-associated F-actin, like total F-actin, shifts inversely with initial cell volume changes in anisotonic media; however, subsequent changes in cytoskeletal actin levels during volume regulation are not significant. The soluble F-actin pool in HL-60 cells may thus be more susceptible to the physicochemical effects of shifts in cell volume than the insoluble (cytoskeletal) F-actin pool. Twenty-five micromolar dihydrocytochalasin B (DHB) treatment dramatically lowers cellular cytoskeletal actin levels by approximately 75% under resting (isotonic) conditions, but there are no significant further changes in cytoskeletal actin as cells undergo anisotonic volume regulation in the presence of DHB. These results suggest that volume-dependent changes in the absolute amounts of cytoskeletal-associated F-actin are not critical for HL-60 cell volume regulation. However, because some portions of the actin cytoskeleton are resistant to cytochalasin disruption during volume regulation, a role for the cytoskeleton in the sensing and signaling of HL-60 cell volume regulatory responses cannot be rigorously excluded. Particular F-actin distribution patterns, as observed using confocal fluorescent microscopy, were correlated with particular phases of volume regulation. Also, comparison of cellular F-actin distribution with surface morphology (observed by scanning electronic microscopy) of cells during volume regulation reveals a positive correlation between surface blebs and increased cortical F-actin staining intensity.
细胞体积调节是通过离子和溶质在细胞膜上的调节通量来实现的，以响应等渗条件下的细胞体积扰动。我们早期对人早幼粒细胞白血病HL-60细胞的研究表明，细胞总F-肌动蛋白含量的体积依赖性变化与等渗介质中急性细胞体积变化呈反比函数同时发生（Hallows等人，1991年，《美国生理学杂志》，261:C1154-C1161）。尽管在等渗条件下用细胞松弛素处理显著降低了细胞总F-肌动蛋白水平，但细胞松弛素并未显著影响细胞进行正常体积调节反应的能力，这表明F-肌动蛋白含量的这些体积依赖性变化可能在HL-60细胞体积调节中不发挥关键作用。为了更仔细地研究肌动蛋白细胞骨架在HL-60细胞体积调节中的可能作用，我们在有和没有细胞松弛素的情况下定量了Triton不溶性细胞骨架肌动蛋白的变化，并观察了体积调节过程中F-肌动蛋白分布和表面形态的变化。与总F-肌动蛋白一样，细胞骨架相关F-肌动蛋白的量与等渗介质中初始细胞体积变化呈反比；然而，在体积调节过程中细胞骨架肌动蛋白水平的后续变化并不显著。因此，HL-60细胞中的可溶性F-肌动蛋白池可能比不溶性（细胞骨架）F-肌动蛋白池更容易受到细胞体积变化的物理化学影响。在静息（等渗）条件下，25微摩尔双氢细胞松弛素B（DHB）处理可使细胞细胞骨架肌动蛋白水平显著降低约75%，但在DHB存在的情况下，当细胞进行等渗体积调节时，细胞骨架肌动蛋白没有进一步的显著变化。这些结果表明，细胞骨架相关F-肌动蛋白绝对量的体积依赖性变化对HL-60细胞体积调节并不关键。然而，由于在体积调节过程中肌动蛋白细胞骨架的某些部分对细胞松弛素破坏具有抗性，因此不能严格排除细胞骨架在HL-60细胞体积调节反应的感知和信号传导中的作用。使用共聚焦荧光显微镜观察到的特定F-肌动蛋白分布模式与体积调节的特定阶段相关。此外，在体积调节过程中，将细胞F-肌动蛋白分布与细胞表面形态（通过扫描电子显微镜观察）进行比较，发现表面泡与皮质F-肌动蛋白染色强度增加之间存在正相关。
