Kumar G Aditya, Bagheri Yousef, Puthenveedu Manojkumar A
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109.
Program in Biophysics, University of Michigan, Ann Arbor, MI 48109.
Mol Biol Cell. 2025 Jul 1;36(7):br17. doi: 10.1091/mbc.E25-02-0081. Epub 2025 Apr 30.
Clathrin-mediated endocytosis involves the remodeling of membranes via the initiation, maturation, and scission of clathrin-coated pits (CCPs). How CCP initiation and dynamics are regulated has been addressed primarily from the perspective of the core proteins that mediate endocytosis. Whether and how the physical properties of the membrane regulate CCP initiation and dynamics are heavily underexplored. Here, we used a solvatochromic probe to readout membrane order in the plasma membrane in live cells undergoing endocytosis, using total internal reflection fluorescence microscopy. Cholesterol depletion decreased membrane order, and reduced CCP initiation and increased lifetimes of CCPs. In unperturbed cells, however, membrane order was correlated to CCP lifetimes, but not their initiation. When membrane order was decreased or increased independently without extracting lipids, CCP lifetimes were affected, but CCP initiation was not. Together, by reading out membrane order in living cells undergoing endocytosis and manipulating membrane order in both directions, we show that membrane order primarily regulates CCP dynamics, and that cholesterol extraction has additional effects on CCP initiation independent of its effect on order.
网格蛋白介导的内吞作用涉及通过网格蛋白包被小窝（CCP）的起始、成熟和切割来重塑膜结构。CCP的起始和动力学如何调控，主要是从介导内吞作用的核心蛋白的角度进行研究的。膜的物理性质是否以及如何调控CCP的起始和动力学，目前仍未得到充分探索。在这里，我们使用一种溶剂化显色探针，通过全内反射荧光显微镜，来读取正在进行内吞作用的活细胞质膜中的膜有序性。胆固醇耗竭会降低膜有序性，减少CCP的起始，并延长CCP的寿命。然而，在未受干扰的细胞中，膜有序性与CCP的寿命相关，但与它们的起始无关。当膜有序性在不提取脂质的情况下独立降低或增加时，CCP的寿命会受到影响，但CCP的起始不受影响。总之，通过读取正在进行内吞作用的活细胞中的膜有序性，并在两个方向上操纵膜有序性，我们表明膜有序性主要调控CCP的动力学，并且胆固醇提取对CCP起始有独立于其对膜有序性影响的额外作用。
