Amato P A, Unanue E R, Taylor D L
J Cell Biol. 1983 Mar;96(3):750-61. doi: 10.1083/jcb.96.3.750.
The distribution of actin in proteose peptone-elicited murine peritoneal macrophages is examined with fluorescent analog cytochemistry (FAC), immunofluorescence, and electron microscopy (EM). Living adherent macrophages, microinjected with 5- iodoacetamidofluorescence-labeled actin, show a rather uniform distribution of actin with punctuate and linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of the microinjected actin into the cell's actin cytoskeleton is also demonstrated when microinjected cells are subsequently examined for fluorescein fluorescence after fixation and extraction. However, a substantial perinuclear pool of actin, observed with FAC, is lost when microinjected cells are prepared for immunofluorescence using standard fixation methods. These results suggest that part of the cellular actin, possibly nonfilamentous or oligomeric, can be extracted during the normal preparative steps for immunofluorescence. When the dynamic distributin of actin structures is examined in living cells, extension of the cell's periphery is associated with the formation of punctuate structures. The distribution of the most stable, nonextractable actin structures in fixed cells at different stages of spreading is quantified using rhodamine-labeled phalloidin and antiactin indirect immunofluorescence. At early stages, the rounded cells show cortical bands of fluorescence surrounding the nuclear region with punctuate structures directly above the plane of the attached plasma membrane. At later time periods, fully spread cells contain both punctuate and linear fluorescent structures. Adherent macrophage membranes, a preparation in which the attached membrane and membrane-cortex are isolated by shearing away the unattached plasma membrane and underlying cytoplasm, show punctuate and linear fluorescence when stained with rhodamine-labeled phalloidin. When the same cell remnant is negatively stained and examined with EM, the fluorescent punctuate structures coincide with electron-dense foci and associated radiating thin filaments. We suggest that the optimal approach for elucidating the distribution of cytoskeletal and contractile proteins involved in motile processes is a combined approach using all three techniques. Although each technique is subject to potential artifacts and limitations, the use of FAC can permit the visualization of both the soluble and stabilized components of the cytoskeleton in living, functional cells. A qualitative method for determining differences in local concentrations of proteins is also presented.
运用荧光类似物细胞化学（FAC）、免疫荧光和电子显微镜（EM）技术，研究了蛋白胨诱导的小鼠腹腔巨噬细胞中肌动蛋白的分布情况。对活体贴壁巨噬细胞微量注射5-碘乙酰氨基荧光标记的肌动蛋白后，可见肌动蛋白在细胞周边薄区域呈相当均匀的分布，伴有点状和线性荧光。当对微量注射后的细胞进行固定和抽提处理，随后检测其荧光素荧光时，还证实了微量注射的部分肌动蛋白明显掺入到细胞的肌动蛋白细胞骨架中。然而，使用标准固定方法对微量注射细胞进行免疫荧光制备时，通过FAC观察到的大量核周肌动蛋白池消失了。这些结果表明，在免疫荧光的正常制备步骤中，细胞内部分肌动蛋白，可能是非丝状或寡聚体形式的，会被抽提出来。当在活细胞中检测肌动蛋白结构的动态分布时，细胞周边的延伸与点状结构的形成相关。使用罗丹明标记的鬼笔环肽和抗肌动蛋白间接免疫荧光，对处于不同铺展阶段的固定细胞中最稳定、不可抽提的肌动蛋白结构的分布进行了定量分析。在早期，圆形细胞在核区域周围显示出皮质荧光带，在附着质膜平面上方有一些点状结构。在后期，完全铺展的细胞同时含有点状和线性荧光结构。贴壁巨噬细胞膜是一种通过剪去未附着的质膜和下方细胞质来分离附着膜和膜皮质的制备物，用罗丹明标记的鬼笔环肽染色时显示出点状和线性荧光。当对同一细胞残余物进行负染色并用电子显微镜检查时，荧光点状结构与电子致密灶及相关的放射状细丝相吻合。我们认为，阐明参与运动过程的细胞骨架和收缩蛋白分布的最佳方法是结合使用这三种技术。尽管每种技术都可能存在潜在的假象和局限性，但FAC的使用能够使我们在有功能的活细胞中观察到细胞骨架的可溶性和稳定成分。本文还介绍了一种确定蛋白质局部浓度差异的定性方法。
