ABSTRACT

Extracellular vesicles (EVs) are small membrane vesicles that serve as important intercellular signalling intermediaries in both malignant and non-malignant cells. For EVs formed by the plasma membrane, their biogenesis is characterized by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. EV-production is significantly higher in malignant cells relative to non-malignant cells and previous work suggests this is dependent on increased calcium mobilization and activity of calpain. However, calcium-signalling pathways involved in malignant and non-malignant EV biogenesis remain unexplored. Here we demonstrate; malignant cells have high basal production of plasma membrane EVs compared to non-malignant cells and this is driven by a calcium–calpain dependent pathway. Resting vesiculation in malignant cells occurs via mobilization of calcium from endoplasmic reticulum (ER) stores rather than from the activity of plasma membrane calcium channels. In the event of ER store depletion however, the store-operated calcium entry (SOCE) pathway is activated to restore ER calcium stores. Depleting both ER calcium stores and blocking SOCE, inhibits EV biogenesis. In contrast, calcium signalling pathways are not activated in resting non-malignant cells. Consequently, these cells are relatively low vesiculators in the resting state. Following cellular activation however, an increase in cytosolic calcium and activation of calpain increase in EV biogenesis. These findings contribute to furthering our understanding of extracellular vesicle biogenesis. As EVs are key mediators in the intercellular transfer of deleterious cancer traits such as cancer multidrug resistance (MDR), understanding the molecular mechanisms governing their biogenesis in cancer is the crucial first step in finding novel therapeutic targets that circumvent EV-mediated MDR.

摘要

细胞外囊泡（EVs）是小的膜囊泡，在恶性和非恶性细胞中均充当重要的细胞间信号传导中介。对于由质膜形成的电动汽车，其生物发生的特征是细胞内钙的增加，随后是膜和细胞骨架的连续变化。相对于非恶性细胞，恶性细胞的EV产生显着更高，以前的研究表明，这取决于增加的钙动员和钙蛋白酶的活性。然而，涉及恶性和非恶性EV生物发生的钙信号通路尚待探索。在这里我们演示；与非恶性细胞相比，恶性细胞具有较高的基础膜质膜电动车基础产量，这是由钙-钙蛋白酶依赖性途径驱动的。恶性细胞中的静息囊泡形成是通过动员来自内质网（ER）的钙而不是通过质膜钙通道的活动来实现的。但是，在ER储存库耗尽的情况下，会激活储存库操作的钙进入（SOCE）途径以恢复ER钙库。耗尽ER钙库和阻断SOCE均可抑制EV生物发生。相反，钙信号通路在静止的非恶性细胞中不被激活。因此，这些细胞在静止状态下是相对较低的囊泡器。然而，在细胞活化之后，胞质钙的增加和钙蛋白酶的活化在EV生物发生中增加。这些发现有助于进一步了解细胞外囊泡的生物发生。