Illuminating Epidermal Growth Factor Receptor Densities on Filopodia through Plasmon Coupling

Abstract

Filopodia have been hypothesized to act as remote sensors of the cell environment but many details of the sensor function remain unclear. We investigated the distribution of the epidermal growth factor (EGF) receptor (EGFR) density on filopodia and on the dorsal cell membrane of A431 human epidermoid carcinoma cells using a nanoplasmonic enabled imaging tool. We targeted cell surface EGFR with 40 nm diameter Au nanoparticles (NPs) using a high affinity multivalent labeling strategy and determined relative NP binding affinities spatially resolved through plasmon coupling. Distance dependent near-field interactions between the labels generated a NP density (ρ) dependent spectral response that facilitated a spatial mapping of the EGFR density distribution on subcellular length scales in an optical microscope in solution. The measured ρ values were significantly higher on filopodia than on the cellular surface, which is indicative of an enrichment of EGFR on filopodia. A detailed characterization of the spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the findings of the all-optical plasmon coupling studies and provided additional structural details. The NPs exhibited a preferential association with the sides of the filopodia. We calibrated the ρ-dependent spectral response of the Au immunolabels through correlation of optical spectroscopy and SEM. The experimental dependence of the measured plasmon resonance wavelength (λ res ) of the interacting immunolabels on ρ was well described by the fit λ res = 595.0nm − 46.36nm*exp(−ρ/51.48) for ρ ≤ 476 NPs/μm 2 . The performed correlated spectroscopic/SEM studies pave the way towards quantitative immunolabeling studies of EGFR and other important cell surface receptors in an optical microscope. KeywordsEpidermal Growth Factor Receptor; Filopodia; Immunolabels; Molecular Imaging; Nanoplasmonics; Receptor Clustering; Nano-Bio Interface Cell motility is a key requirement for natural growth processes, such as wound healing and embryonic development, but dysregulated cell motility enables aggressive tumor invasion in cancer. 1 Any cell translocation during normal or pathological growth requires a propelling force. The latter is generated by a branched actin network in a cytoplasmic extension -the lamellipodium -at the leading edge of moving cells. 2 Lamellipodia can contain spike-like protrusions with parallel actin bundles, which are referred to as filopodia. 3,4 A series of diverse biological functions have been attributed to filopodia, but in many cases the exact mechanisms underlying the biological function remain insufficiently characterized. 5 One proposed filopodia function is that of sensory antennae for chemotaxis in the extended environment of the cell body. In normal growth processes in vivo, individual cells usually bmr@bu.edu. Supporting Information available: Figures S1-S4. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript ACS Nano. Author ma...

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