Research Projects

Classically, metastatic mechanisms focus predominantly on biochemical signaling, neglecting the role of plasma membrane architecture. Contrary to the traditional depiction of mammalian cells as smooth and spherical, the plasma membrane exhibits a dynamic spiky topography, characterized by projections such as microvilli. Recent studies in immune cells, including T cells, B cells, and mast cells, reveal that microvilli, the finger-like membrane protrusions, serve as the key signaling hub. Crucial immune signaling proteins are pre-organized on these signaling hubs through a spatio-structural interplay between membrane topography, lipid rafts, and actin scaffolding proteins. A strong correlation between spikier membrane topography and metastatic potential has been reported for decades. However, the underlying molecular mechanisms remain obscure. Notably, metastasis-promoting proteins, such as Epidermal Growth Factor Receptor (EGFR), are known to localize on lipid rafts whose abundance increases during metastasis. We hypothesize that lipid rafts selective localization of EGFR causes its enrichment within the ultrathin (~100 nm diameter) microvillar domains, where nanoscale confinement promotes dimerization and activation of EGFR. This will facilitate the metastatic microenvironment, inducing cell growth, survival, and migration. Our key objectives are 1. quantitatively characterize membrane topographical changes and spatial redistribution of EGFR during metastasis. 2. Evaluate the potential of membrane architecture as a driver of metastasis. 3. Identify metastasis-associated structural signatures and decipher membrane architecture-related targets for therapeutic intervention. To investigate this, we propose to develop the ‘Microvillar Cartography’ (MVC) setup, a super-resolution imaging platform, by combining total internal reflection fluorescence microscopy (TIRFM) and stochastic localization microscopy (SLN). MVC uniquely maps the localization of membrane protein relative to the three-dimensional membrane architecture of cells. Integration of Sparsity-Based Super-Resolution Correlation Microscopy (SPARCOM) analysis with MVC will further enable live cell imaging compatibility. Only immune-gold electron microscopy matches MVC in terms of resolution and protein localization mapping capability, but it involves harsh sample preparation, preventing live imaging, and risking structural artifacts. We will use biochemical perturbations in combination with MVC to investigate the role of membrane architecture in metastatic mechanisms. These include modulation of lipid raft abundance using C2 ceramide, alteration of microvillar composition and abundance using methyl-β-cyclodextrin (MβCD), and increasing metastatic potential of MCF-7 cells using chemotherapy agents (Adriamycin and FUdR). Understanding the role of membrane architecture in metastasis holds substantial clinical implications. The parametrization of membrane topographical changes across different stages of metastasis, along with EGFR localization relative to the 3D membrane landscape, may identify structural signatures that distinguish metastatic from non-metastatic cells. This will overcome the limitation of the current biomarker-based circulating tumor cell (CTC) detection arises due to the heterogeneity in the biomarker expression and develop innovative membrane architecture-based CTC diagnostics. Moreover, targeting membrane architecture to suppress EGFR-driven metastasis could become a new therapeutic avenue if membrane topography-specific EGFR localization is verified as a metastasis driver. For instance, agents like MβCD, proven to alter microvillar density and composition, may act as potential anti-metastatic drugs. Backed by prior expertise in developing the MVC setup, along with strengths in cell signaling and cancer biology, this project is both technically feasible and well-positioned for success.