Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA.
J Cell Sci. 2023 Nov 15;136(22). doi: 10.1242/jcs.261511. Epub 2023 Nov 27.
The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.
收缩泡复合体（CVC）是一种动态的、形态复杂的膜细胞器，由一个大囊泡（膀胱）与一个管状网状结构（海绵体）相连。CVC 在不同的真核生物谱系中提供关键的渗透调节作用，但由于缺乏用于体内分析的有限工具，探测其结构和功能的机制受到了阻碍。在实验上可处理的纤毛虫 Tetrahymena thermophila 中，我们描述了四个蛋白质，它们作为内源性标记构建体，特异性定位于不同的 CVC 区域。DOPEY 同源物 Dop1p 和 CORVET 亚基 Vps8Dp 都定位于膀胱和海绵体，但分布不同，对渗透胁迫敏感，而脂质翻转酶 Scr7p 与 Vps8Dp 共定位。H+-ATPase 亚基 Vma4 是海绵体特异性的。这些探针的实时成像揭示了多个尺度的动力学，包括 CVC 定位和可溶性蛋白库的快速交换与海绵体中的侧向扩散、海绵体的延伸和分支以及有丝分裂期间 CVC 的形成。尽管与 DOP1 和 VPS8D 的关联表明 CVC 参与了内体运输，但膀胱和海绵体可能与批量内吞输入隔离。
