A study of lipid-mediated clustering of oncogenic Ras proteins on cell membrane using nanotopography engineering

Dysregulation of Ras, a dominant oncogenic protein, in cell proliferation, differentiation, and survival contributes to ~20% of human cancers. Despite great efforts paid in identifying anti-Ras therapeutics for the past decades, it remains a challenging task to provide effective anti-Ras drugs for clinical use, with only 1 FDA-approved inhibitor last year over thousands of candidates. To tackle the problem, one promising anti-Ras strategy is to target the lateral segregation of Ras on the plasma membrane (PM) that partitions Ras into guanine nucleotide- dependent dimers and higher-order clusters, the localization of which ensures the fidelity of isoform-specific signaling. Recently, membrane curvature has been identified to be a key membrane property to regulate Ras recruitment and nanoclustering by coordinating with lipids and Ras binding partners. However, due to the limited techniques to manipulate the curvature of the PM effectively, the live-cell characterization of the curvature effect on Ras clustering remains elusive. Here, a nanotopography-based approach to generate nanoscale membrane curvature for the direct visualization of Ras clustering changes in live cells was established. Using vertically aligned nanobar arrays, Ras clusters were guided into quantifiable patterns along the contour of the nanobars. Enriching Ras clusters at the curved nanobar ends validates that membrane curvature acts as a common modulator of Ras sorting on the PM regardless of Ras isoforms. Within the Ras structure itself, the lipidated membrane anchors of Ras at the C-terminus were shown to be the dominant region causing the curvature-guided Ras clustering. Furthermore, the lipidation-based modification of the palmitoyl tail at Cysteine 181 instead of at Cysteine 184 was found to be essential in H-Ras curvature sensing. Beyond Ras, the nanotopography further demonstrated its capability in reorganizing the membrane microenvironment to mediate curvature-guided Ras clustering. Various lipid species were found to be accumulated to the curved membranes, including phosphatidylinositol 4,5-bisphosphate (PIP2), cholesterol, phosphatidylserine (PS), and phosphatidic acid (PA). Specifically, PIP2 depletion abolished the curvature sensing behaviors of H-Ras and tK. Together, these results suggest lipids to be a key coregulator of Ras curvature sensing. Overall, with the help of surface nanotopography, membrane curvature was validated as a significant modulator of Ras spatial localizations through lipidation-based membrane anchoring and reorganizing the local lipid microenvironment in live cells.It can be envisioned that the nanotopography-induced membrane curvature provides a promising venue for the fundamental investigations and the drug development related to Ras clustering.Doctor of Philosoph