Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States.
Department of Biochemistry and Molecular Biology, The Institute for Biophysical Dynamics, University of Chicago, Chicago, United States.
Elife. 2024 Oct 31;12:RP90603. doi: 10.7554/eLife.90603.
Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.
肌球蛋白 10（Myo10）是一种已知在丝状伪足形成中起作用的马达蛋白。尽管已经描述了 Myo10 驱动的丝状伪足动力学，但关于丝状伪足生命周期中 Myo10 分子的绝对数量尚无信息。为了更好地了解丝状伪足中的分子化学计量和包装限制，我们测量了这些结构中的 Myo10 丰度。我们将 SDS-PAGE 密度测定法与荧光显微镜相结合，定量分析 U2OS 细胞中 HaloTag 标记的 Myo10。大约 6%的总细胞内 Myo10 定位于丝状伪足，在那里它在细胞的相对两端富集。数百个 Myo10 位于一个典型的丝状伪足中，它们在丝状伪足中的分布呈对数正态分布。一些丝状伪足的尖端甚至包含比肌动蛋白丝束上可及的结合位点更多的 Myo10。活细胞电影揭示了一个由一百多个 Myo10 分子组成的密集簇，该簇起始丝状伪足的延伸。在丝状伪足生长过程中会不断积累数百个 Myo10 分子，但当开始回缩时积累就会停止。丝状伪足延伸、第二阶段延伸和回缩的速度与 Myo10 数量呈反比。我们对丝状伪足中 Myo10 分子的估计为在物理上包装 Myo10、其货物以及其他与丝状伪足相关的蛋白质在狭窄的膜隔室中提供了深入的了解。我们的方案为未来分析扰动时 Myo10 丰度和分布的工作提供了框架。
