The regulation of exocytosis is physiologically vital in cells and requires a variety of distinct proteins and lipids that facilitate efficient, fast, and timely release of secretory vesicle cargo. Growing evidence suggests that regulatory lipids act as important lipid signals and regulate various biological processes including exocytosis. Though functional roles of many of these regulatory lipids has been linked to exocytosis, the dynamic behavior of these lipids during membrane fusion at sites of exocytosis in cell culture remains unknown. Total internal reflection fluorescence microscopy (TIRF) was used to observe the spatial organization and temporal dynamics (i.e. spatial positioning and timing patterns) of several lipids, and accessory proteins, like lipid kinases and protein kinases, in the form of protein kinase C (PRKC) associated with sites of exocytosis of matrix metalloproteinase-9 (MMP-9) in living MCF-7 cancer cells. Following stimulation with phorbol myristate acetate (PMA) to promote exocytosis, a transient accumulation of several distinct regulatory lipids, lipid kinases, and protein kinases at exocytic sites was observed. This transient accumulation centered at the time of membrane fusion is followed by a rapid diffusion away from the fusion sites. Additionally, the synthesis of these regulatory lipids, degradation of these lipids, and the downstream effectors activated by these lipids, are also achieved by the recruitment and accumulation of key enzymes at exocytic sites (during the moment of cargo release). This includes key enzymes like lipid kinases, protein kinases, and phospholipases that facilitate membrane fusion and exocytosis of MMP-9. This work suggests that these regulatory lipids and associated effector proteins are locally synthesized and/or recruited to sites of exocytosis, during membrane fusion and cargo release. More importantly, their enrichment at fusion sites serves as an important spatial and temporal organizing “element” defining individual exocytic sites.

中文翻译：

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在MCF-7细胞中MMP-9胞吐作用的部位，在膜融合过程中成像调节脂质，脂质激酶和蛋白激酶的快速而短暂的积累。

胞吐作用的调节在细胞中是生理上至关重要的，并且需要多种不同的蛋白质和脂质，以促进有效，快速和及时地释放分泌性囊泡货物。越来越多的证据表明，调节脂质起着重要的脂质信号的作用，并调节各种生物过程，包括胞吐作用。尽管许多这些调节脂质的功能作用与胞吐作用有关，但是在细胞培养中胞吐作用部位的膜融合过程中这些脂质的动态行为仍然未知。使用全内反射荧光显微镜（TIRF）来观察几种脂质以及脂质激酶和蛋白激酶等辅助蛋白的空间组织和时间动态（即空间定位和时序模式），蛋白激酶C（PRKC）的形式与活MCF-7癌细胞中基质金属蛋白酶9（MMP-9）的胞吐作用相关。在用佛波肉豆蔻酸酯乙酸酯（PMA）刺激以促进胞吐作用之后，观察到几种不同的调节脂质，脂质激酶和蛋白激酶在胞外位点的短暂积累。集中在膜融合时的这种瞬时积累之后是迅速扩散离开融合部位。另外，这些调节脂质的合成，这些脂质的降解以及被这些脂质激活的下游效应子也可以通过关键酶在胞外位点的聚集和积累来实现（在货物释放的那一刻）。其中包括关键酶，例如脂质激酶，蛋白激酶，以及促进膜融合和MMP-9胞吐作用的磷脂酶。这项工作表明，在膜融合和货物释放过程中，这些调节脂质和相关的效应蛋白是局部合成的和/或募集到胞吐部位。更重要的是，它们在融合位点的富集是定义单个外生位点的重要时空组织“元素”。