Kuo Joe Chin-Hun, Colville Marshall J, Sorkin Michelle R, Kuo Jacky Lok Ka, Huang Ling Ting, Thornlow Dana N, Beacham Gwendolyn M, Hollopeter Gunther, DeLisa Matthew P, Alabi Christopher A, Paszek Matthew J
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States.
Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, United States.
ACS Cent Sci. 2024 Nov 23;11(2):193-207. doi: 10.1021/acscentsci.4c01061. eCollection 2025 Feb 26.
Complex carbohydrates called glycans play crucial roles in regulating cell and tissue physiology, but how they map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type -glycans throughout the entirety of the model organism. We constructed a library of multifunctional linkers to probe and anchor metabolically labeled glycans in expansion microscopy (ExM). A flexible strategy was demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing as a test bed, metabolically labeled -glycans were resolved on the gut microvilli and other nanoscale anatomical features. Transmission electron microscopy images of nanoanatomy validated the fidelity and isotropy of gel expansion. Whole organism maps of -glycosylation in the first larval stage revealed -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond , we validated ExM protocols for nanoscale imaging of metabolically labeled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labeled biomolecules at enhanced resolutions with ExM.
被称为聚糖的复合碳水化合物在调节细胞和组织生理过程中发挥着关键作用，但它们如何映射到纳米级解剖特征仍有待解决。在这里，我们展示了整个模式生物中粘蛋白型聚糖的首张纳米级图谱。我们构建了一个多功能连接体文库，用于在膨胀显微镜（ExM）中探测和锚定代谢标记的聚糖。展示了一种灵活的策略，用于化学合成具有广泛生物正交官能团、荧光团、锚定化学和连接体臂的连接体。以[具体生物]作为测试平台，在肠道微绒毛和其他纳米级解剖特征上解析了代谢标记的聚糖。[具体生物]纳米解剖结构的透射电子显微镜图像验证了凝胶膨胀的保真度和各向同性。第一幼虫阶段的全生物体糖基化图谱揭示了在意外解剖位置的聚糖“热点”，包括体壁沟。除此之外，我们验证了用于在培养的哺乳动物细胞上对代谢标记聚糖进行纳米级成像的ExM方案。总之，我们的结果表明多功能试剂在通过ExM以更高分辨率对聚糖和其他代谢标记生物分子进行成像方面具有广泛的适用性。
