Ghosh Shirsendu, Wagenknecht-Wiesner Alice, Desai Shriya, Vyphuis Jada, Ramos Mariena Silvestry, Grazul John L, Baird Barbara A
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.
Department of Chemistry, Indian Institute of Technology, Jammu, Jagti 181221, Jammu and Kashmir, India.
Proc Natl Acad Sci U S A. 2025 Jul 22;122(29):e2424427122. doi: 10.1073/pnas.2424427122. Epub 2025 Jul 17.
Similar to T cells and B cells, mast cell surfaces are dominated by microvilli, and like these other immune cells we showed with microvillar cartography (MVC) that key signaling proteins for RBL mast cells localize to these topographical features. Although stabilization of ordered lipid nanodomains around antigen-crosslinked IgE-FcεRI is known to facilitate necessary coupling with Lyn tyrosine kinase to initiate transmembrane signaling in these mast cells, the relationship of ordered-lipid nanodomains to membrane topography had not been determined. With nanoscale resolution provided by MVC, standard error of the mean (SEM), and colocalization probability (CP) analysis, we found that FcεRI and Lyn kinase are positioned primarily on the microvilli of resting mast cells in separate nano-assemblies. Upon antigen-activation, FcεRI and Lyn merge into overlapping populations together with the LAT scaffold protein, accompanied by merger of microvilli into ridge-like ruffles. With selective lipid probes, we further found that ordered-lipid nanodomains preferentially occupy microvillar membranes, contrasting with localization of disordered lipids to flatter regions. With this proximity of signaling proteins and ordered lipid nanodomains in microvilli, the mast cells are poised to respond sensitively and efficiently to antigen but only in the presence of this stimulus. Use of a short chain ceramide to disrupt ordered-lipid regions of the plasma membrane and evaluation with MVC, CP, and flow cytometry provided strong evidence that the microvillar selective localization of signaling proteins and membrane environments is facilitated by the interplay between ordered-lipid nanodomains and actin attachment proteins, ERM (ezrin, radixin, moesin), and cofilin.
与T细胞和B细胞类似，肥大细胞表面以微绒毛为主，并且与我们用微绒毛绘图（MVC）展示的其他免疫细胞一样，RBL肥大细胞的关键信号蛋白定位于这些拓扑特征上。虽然已知抗原交联的IgE - FcεRI周围有序脂质纳米域的稳定有助于与Lyn酪氨酸激酶进行必要的偶联，从而在这些肥大细胞中启动跨膜信号传导，但有序脂质纳米域与膜拓扑结构的关系尚未确定。通过MVC提供的纳米级分辨率、平均标准误差（SEM）和共定位概率（CP）分析，我们发现FcεRI和Lyn激酶主要定位在静息肥大细胞的微绒毛上，处于不同的纳米组装体中。在抗原激活后，FcεRI和Lyn与LAT支架蛋白一起合并为重叠群体，同时微绒毛合并为脊状褶皱。使用选择性脂质探针，我们进一步发现有序脂质纳米域优先占据微绒毛膜，这与无序脂质定位于较平坦区域形成对比。由于微绒毛中信号蛋白和有序脂质纳米域如此接近，肥大细胞随时准备对抗原做出敏感而有效的反应，但前提是存在这种刺激。使用短链神经酰胺破坏质膜的有序脂质区域，并通过MVC、CP和流式细胞术进行评估，提供了有力证据，表明信号蛋白和膜环境的微绒毛选择性定位是由有序脂质纳米域与肌动蛋白附着蛋白、ERM（埃兹蛋白、根蛋白、膜突蛋白）和丝切蛋白之间的相互作用所促进的。
