Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA.
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA.
J Biol Chem. 2024 May;300(5):107279. doi: 10.1016/j.jbc.2024.107279. Epub 2024 Apr 7.
Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.
肌动蛋白成束蛋白将纤维交联成极化结构，这些结构塑造并支撑着膜突起，包括丝状伪足、微绒毛和静纤毛。在肠上皮细胞的微绒毛中，有丝分裂纺锤体定位蛋白（MISP）是一种肌动蛋白成束蛋白，它特异性地定位于基底根毛，即核心束纤维的尖端会聚的地方。以前的研究已经证实，MISP 被阻止与其他肌动蛋白结合蛋白结合，从而无法与核心束的更远端结合。然而，MISP 是否更倾向于直接与根毛肌动蛋白结合仍然是一个悬而未决的问题。通过对天然肠组织切片进行免疫染色，我们发现微绒毛根毛上装饰有丝切蛋白 cofilin，这表明这些结构中存在高水平的 ADP-肌动蛋白。通过全内反射荧光显微镜检测，我们还发现纯化的 MISP 表现出对 ADP-肌动蛋白的结合偏好，而不是 ADP-Pi-肌动蛋白。这与以下结果一致：在有丝分裂生长的肌动蛋白纤维的检测中发现，MISP 结合在纤维的尖端或附近。此外，尽管附着在底物上的 MISP 可以平行和反平行的方式组装纤维束，但在溶液中，MISP 可以组装由多个具有均匀极性的纤维组成的平行束。这些发现强调了核苷酸状态感应作为一种机制，可以沿纤维分拣肌动蛋白成束蛋白，并将其聚集在纤维末端附近。这种局部结合可能驱动平行束的形成和/或局部调节微绒毛和相关突起中的束状机械性能。
