Lock Jeffrey T, Parker Ian, Smith Ian F
Department of Neurobiology and Behavior, University of California, Irvine, CA, United States.
Department of Neurobiology and Behavior, University of California, Irvine, CA, United States; Department of Physiology and Biophysics, University of California, Irvine, CA, United States.
Cell Calcium. 2016 Oct;60(4):266-72. doi: 10.1016/j.ceca.2016.06.004. Epub 2016 Jun 20.
Tunneling membrane nanotubes (TNTs) are thin membrane projections linking cell bodies separated by many micrometers, which are proposed to mediate signaling and even transfer of cytosolic contents between distant cells. Several reports describe propagation of Ca(2+) signals between distant cells via TNTs, but the underlying mechanisms remain poorly understood. Utilizing a HeLa M-Sec cell line engineered to upregulate TNTs we replicated previous findings that mechanical stimulation elicits robust cytosolic Ca(2+) elevations that propagate to surrounding, physically separate cells. However, whereas this was previously interpreted to involve intercellular communication through TNTs, we found that Ca(2+) signal propagation was abolished - even in TNT-connected cells - after blocking ATP-mediated paracrine signaling with a cocktail of extracellular inhibitors. To then establish whether gap junctions may enable cell-cell signaling via TNTs under these conditions, we expressed sfGFP-tagged connexin-43 (Cx43) in HeLa M-Sec cells. We observed robust communication of mechanically-evoked Ca(2+) signals between distant but TNT-connected cells, but only when both cells expressed Cx43. Moreover, we also observed communication of Ca(2+) signals evoked in one cell by local photorelease of inositol 1,4,5-trisphosphate (IP3). Ca(2+) responses in connected cells began after long latencies at intracellular sites several microns from the TNT connection site, implicating intercellular transfer of IP3 and subsequent IP3-mediated Ca(2+) liberation, and not Ca(2+) itself, as the mediator between TNT-connected, Cx43-expressing cells. Our results emphasize the need to control for paracrine transmission in studies of cell-cell signaling via TNTs and indicate that, in this cell line, TNTs do not establish cytosolic continuity between connected cells but rather point to the crucial importance of connexins to enable communication of cytosolic Ca(2+) signals via TNTs.
隧道膜纳米管（TNTs）是连接相隔数微米的细胞体的薄膜突起，有人提出它们可介导信号传导，甚至介导远处细胞间胞质内容物的转移。有几份报告描述了Ca(2+)信号通过TNTs在远处细胞间的传播，但其潜在机制仍知之甚少。利用经过工程改造以上调TNTs的HeLa M-Sec细胞系，我们重复了之前的发现，即机械刺激会引发强大的胞质Ca(2+)升高，并传播到周围物理上分离 的细胞。然而，尽管此前认为这涉及通过TNTs进行的细胞间通讯，但我们发现，在用细胞外抑制剂混合物阻断ATP介导的旁分泌信号传导后，Ca(2+)信号传播被消除，即使在通过TNTs连接的细胞中也是如此。为了确定在这些条件下间隙连接是否可以通过TNTs实现细胞间信号传导，我们在HeLa M-Sec细胞中表达了带有sfGFP标签的连接蛋白43（Cx43）。我们观察到在远处但通过TNTs连接的细胞之间，机械诱发的Ca(2+)信号有强大的通讯，但仅当两个细胞都表达Cx43时才会出现。此外，我们还观察到由肌醇1,4,5-三磷酸（IP3）的局部光释放引发的一个细胞中的Ca(2+)信号的通讯。连接细胞中的Ca(2+)反应在距TNT连接位点数微米的细胞内位点经过长时间延迟后开始，这表明IP3的细胞间转移以及随后IP3介导的Ca(2+)释放，而不是Ca(2+)本身，是通过TNTs连接的、表达Cx43的细胞之间的介质。我们的结果强调在通过TNTs进行细胞间信号传导的研究中需要控制旁分泌传递，并表明，在这种细胞系中，TNTs不会在连接的细胞之间建立胞质连续性，而是指出连接蛋白对于通过TNTs实现胞质Ca(2+)信号通讯的至关重要性。
