Wong Timothy H, Khater Ismail M, Joshi Bharat, Shahsavari Mona, Hamarneh Ghassan, Nabi Ivan R
Life Sciences Institute, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
School of Computing Science, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
Sci Rep. 2021 Apr 8;11(1):7810. doi: 10.1038/s41598-021-86770-6.
Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.
小窝蛋白-1（CAV1）是小窝的包被蛋白，也与非小窝支架结构域相关联。单分子定位显微镜（SMLM）网络分析可区分小窝和三个支架结构域，即半球形S2支架以及较小的S1B和S1A支架。小窝蛋白支架结构域（CSD）是一个高度保守的疏水区域，介导CAV1与多种效应分子的相互作用。F92A/V94A突变会破坏CSD功能，然而CSD突变对小窝或支架的结构影响仍不清楚。在此，SMLM网络分析定量显示，在CRISPR/Cas CAV1基因敲除的MDA-MB-231乳腺癌细胞中表达CAV1 CSD F92A/V94A突变体，会减小小窝和支架结构域的尺寸和体积，并增加其伸长率，对S2和S1B支架的影响更为明显。对CAV1点云外表面的凸包分析证实了CSD突变体CAV1斑点的尺寸减小，并表明CSD突变在收缩值增加时减少了S2和S1B CAV1斑点之间的体积变化，这可能反映了CAV1 N端向膜的回缩，可能阻止了CSD的可及性。检测点突变引起的CAV1结构域变化，突出了SMLM网络分析在其天然环境中对寡聚体进行中尺度结构分析的实用性。
