Wilson Oliver J, Bradley Helen, Shaw Christopher S, Wagenmakers Anton J M
Exercise Metabolism Research Group, School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Histochem Cell Biol. 2014 Sep;142(3):245-56. doi: 10.1007/s00418-014-1212-3. Epub 2014 Mar 27.
Focal adhesion kinase (FAK) and paxillin are functionally linked hormonal- and mechano-sensitive proteins. We aimed to describe paxillin's subcellular distribution using widefield and confocal immunofluorescence microscopy and test the hypothesis that FAK and paxillin colocalise in human skeletal muscle and its associated microvasculature. Percutaneous muscle biopsies were collected from the m. vastus lateralis of seven healthy males, and 5-μm cryosections were stained with anti-paxillin co-incubated with anti-dystrophin to identify the sarcolemma, anti-myosin heavy chain type I for fibre-type differentiation, anti-dihydropyridine receptor to identify T-tubules, lectin UEA-I to identify the endothelium of microvessels and anti-α-smooth muscle actin to identify vascular smooth muscle cells (VSMC). Colocalisation of anti-paxillin with anti-dystrophin or anti-FAK was quantified using Pearson's correlation coefficient on confocal microscopy images. Paxillin was primarily present in (sub)sarcolemmal regions of skeletal muscle fibres where it colocalised with dystrophin (r = 0.414 ± 0.026). The (sub)sarcolemmal paxillin immunofluorescence intensity was ~2.4-fold higher than in sarcoplasmic regions (P < 0.001) with sarcoplasmic paxillin immunofluorescence intensity ~10 % higher in type I than in type II fibres (P < 0.01). In some longitudinally orientated fibres, paxillin formed striations that corresponded to the I-band region. Paxillin immunostaining was highest in endothelial and VSMC and distributed heterogeneously in both cell types. FAK and paxillin colocalised at (sub)sarcolemmal regions and within the microvasculature (r = 0.367 ± 0.036). The first images of paxillin in human skeletal muscle suggest paxillin is present in (sub)sarcolemmal and I-band regions of muscle fibres and within the microvascular endothelium and VSMC. Colocalisation of FAK and paxillin supports their suggested role in hormonal and mechano-sensitive signalling.
粘着斑激酶（FAK）和桩蛋白是功能上相关的激素敏感和机械敏感蛋白。我们旨在使用宽场和共聚焦免疫荧光显微镜描述桩蛋白的亚细胞分布，并验证FAK和桩蛋白在人类骨骼肌及其相关微血管中共定位的假设。从7名健康男性的股外侧肌采集经皮肌肉活检样本，将5μm厚的冰冻切片用抗桩蛋白染色，并与抗肌营养不良蛋白共同孵育以识别肌膜，用抗I型肌球蛋白重链进行纤维类型分化鉴定，用抗二氢吡啶受体识别T小管，用凝集素UEA-I识别微血管内皮，用抗α-平滑肌肌动蛋白识别血管平滑肌细胞（VSMC）。在共聚焦显微镜图像上，使用皮尔逊相关系数对抗桩蛋白与抗肌营养不良蛋白或抗FAK的共定位进行定量分析。桩蛋白主要存在于骨骼肌纤维的（亚）肌膜区域，在该区域它与肌营养不良蛋白共定位（r = 0.414 ± 0.026）。（亚）肌膜桩蛋白免疫荧光强度比肌浆区域高约2.4倍（P < 0.001），I型纤维中肌浆桩蛋白免疫荧光强度比II型纤维高约10%（P < 0.01）。在一些纵向排列的纤维中，桩蛋白形成与I带区域相对应的条纹。桩蛋白免疫染色在内皮细胞和VSMC中最高，且在两种细胞类型中分布不均一。FAK和桩蛋白在（亚）肌膜区域和微血管内共定位（r = 0.367 ± 0.036）。人类骨骼肌中桩蛋白的首张图像表明，桩蛋白存在于肌纤维的（亚）肌膜和I带区域以及微血管内皮和VSMC内。FAK和桩蛋白的共定位支持了它们在激素和机械敏感信号传导中所暗示的作用。
