对于许多细胞类型,定向运动取决于它们在前缘处保持丝状。丝状足缺乏任何Ca2结合结构蛋白,但对储存操作的Ca2进入(SOCE)作出反应。
SOCE是通过首先用环吡嗪酸(CPA)用无Ca2盐溶液代替培养基来诱导的。这降低了ER中的Ca2并导致基质相互作用分子(STIM)易位到细胞表面。在这个启动步骤之后,注册会计师被淘汰了,并且通过添加细胞外Ca2来恢复Ca2内流。通过钙橙荧光测量细胞内Ca2水平。通过药物治疗鉴定了调节机制。通过免疫荧光对介导SOCE的蛋白质进行定位,并在图像处理后进行分析。
ERCa2+耗尽会短暂增加丝足病患病率,随后是被内吞抑制剂阻断的自发下降。细胞内Ca2+持续增加约50分钟。在单独的区室中发现了STIM和瞬时受体电位规范(TRPC)蛋白,但是两者都存在与SOCE无关的水通道蛋白。携带STIM1-和TRPC1的囊泡在微管上运输。在耗尽期间,STIM1迁移到地表,与Orai重合,如预期。TRPC1与可快速释放的池标记Vamp2部分共定位,和磷脂酶(PLC)。TRPC1在内质网耗尽期间回落到内部隔室。细胞外Ca2+的补充改变了STIM1的分布,类似于未经处理的细胞。Vamp2和TRPC1经历了胞吐作用,并在细胞表面均匀分布。伴随着丝足病的患病率增加,被TRPC1/4/5抑制剂和内吞作用阻断。
因为培养基缺乏在消耗和补充Ca2+期间激活受体的配体,我们可以将filopodia扩展归因于SOCE。我们建议Orai电流刺激带有TRPC的囊泡的胞吐作用,并且Ca2+通过TRPC流入抑制PLC活性。这允许基材的再生,磷脂酰肌醇4,5双磷酸酯(PIP2),一个组装蛋白质的平台,启用和IRSp53。需要与PLC进行TRPC接触,但由于TRPC传播而中断。这解释了STIM1如何调节细胞对吸引力或排斥性线索的定向能力。视频摘要。
For many cell types, directional locomotion depends on their maintaining filopodia at the leading edge. Filopodia lack any Ca2+-binding structural protein but respond to store-operated Ca2+ entry (SOCE).
SOCE was induced by first replacing the medium with Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+ in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+ influx restored by addition of extracellular Ca2+. Intracellular Ca2+ levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing.
Depletion of the ER Ca2+ increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+ increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+ altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis.
Because the media were devoid of ligands that activate receptors during depletion and Ca2+ replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+ influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell\'s ability to orient itself in response to attractive or repulsive cues. Video Abstract.