Single-cell quantification of dynamic cytoskeletal proteins with microscale electrophoretic separations

Biological processes are often dynamic. On a macro-level, the transformation of a biological system from one state to another drive cell differentiation, migration, and basic functions. On a micro-level, the active arrangement of biological material is necessary for the generation of cellular machinery, critical signaling and binding events, and proteomic production. Across all levels, disruptions in these dynamic response processes underpin disease states and drug resistance. Advances in single-cell protein measurement tools are necessary to define functional heterogeneity in varying cell types, elucidate complex biological processes, and uncover new molecular targets for treatments and therapies/nThis doctoral research reports on the design and development of new bioanalytical techniques for the targeted interrogation of dynamic biological processes with single-cell resolution. On the micro-scale, we measure the dynamic arrangement of protein complexes in the cytoskeleton. On the macro-scale, we couple mechanical stimulus with multiplexed proteomic evaluation on a single-cell level. Both microfluidic platforms leverage protein electrophoresis to increase the molecular specificity of the measurement and report on structural and cytoskeletal proteins./nFirst, “Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout” (SIFTER) combines differential detergent fractionation, bi-directional electrophoresis, and immunoassay steps into an arrayed microdevice for the simultaneous detection of multimeric cytoskeletal protein complexes and their respective monomers in 100s of individual cells. To improve upon the molecular specificity of antibody-based detection, we physically fractionate protein complexes and monomers by size-exclusion electrophoresis in a polyacrylamide gel. During fractionation, the depolymerization of complexes is minimized by stabilizing lysis buffer, delivered by gel lid fluidics. We apply SIFTER to evaluate the distribution of monomers and complexes within a heterogeneous population of cells, and the change in distribution of cytoskeletal complexes upon cellular stimuli. Co-detection of actin filaments, microtubules, and intermediate filaments expands SIFTER to global cytoskeletal evaluation./nNext, to clarify the link between extracellular stiffness and resultant proteomic state of a cell, we report on ECM-PAGE, an assay that combines the molecular specificity of single-cell polyacrylamide gel electrophoresis (PAGE) with a tunable substrate for extracellular matrix (ECM) recapitulation. Two distinct functional layers permit optimization of the ECM layer without affecting the analytical layer, and vice versa. The ECM layer, made up of PDMS, is compatible with existing material formulations to generate material stiffnesses across several orders of magnitude and for stamp-off microcontact patterning of ECM proteins onto the surface. By using PAGE for size-based protein separation and immobilization, we report on the quantitative protein abundance of 8 proteins, ranging from actin (42 kDa) to Vinculin (117 kDa) within a 1 mm separation lane. Informed by separation resolution, we design antibody combinations (Actin/actinin and vimentin/paxillin) for the quantification of four proteins with only two fluorescent reporter molecules. Further optimization of the ECM-PAGE system is necessary to evaluate protein expression for a greater sample number, as a fluid layer between the two components causes cross-contamination between single-cell reservoirs with greater array occupancy. We anticipate that with further system characterization, the ECM-PAGE system could report on single-cell multidimensional data (stiffness, cell morphology, and proteomic expression) and elucidate the heterogeneous single-cell responses that drive cancerous tumor behavior./nGiven the molecular specificity necessary to report on protein complexes, we investigated means mitigation injection dispersion to improve separation performance. Furthermore, the miniaturization of these technologies to achieve high-throughput, single-cell quantification requires advancements in device materials and geometries. We consider the generation of large-surface polyacrylamide-polymer devices as a direction for new microscale device geometries./nBy leveraging microscale technologies, these new measurement methods can report on context-dependent processes with single-cell resolution.

Advisor: Herr, Amy E