High-Definition Biotechnology

SLAS2018 Innovation Award Finalist: Electrophoretic Cytometry Isolates Cytoskeleton Molecular Complexes of Single Cancer Cells

Tuesday, February 6
3:00 PM - 3:30 PM
Location: 6E

Changes in molecular interactions underpin disease and drug treatment alike. To regulate actin polymerization and depolymerization, over 100 binding proteins complex with monomeric actin (G-actin, 42 kDa) and filamentous actin (F-actin, up to 100s of monomers) [1,2]. In cancer progression, actin polymerization is disrupted, impacting numerous essential cellular processes (from cell motility to proliferation [3]). Consequently, oncology drugs targeting stabilization of F-actin filaments have been studied [4]. However, actin binding proteins and oncology drugs compete with small-molecule stains for actin binding sites. Thus gold-standard F and G-actin stains have limited utility in cancer studies of actin polymerization and binding protein complexation [5]. Furthermore, assays (e.g., bulk ultracentrifugation) that physically separate F and G-actin and dissociate inhibiting drugs require millions of cells [6]. Single-cell resolution of actin polymerization state and binding protein complexes would inform drug development, but is currently unfeasible.
We have developed a micro-scale electrophoretic cytometry assay that preserves chemical interactions to separate and detect molecular complexes in up to 1000s of single-cells. As a first demonstration, we fractionate F and G-actin from single cancer cells in a microwell array patterned in polyacrylamide gel. We use gel lid fluidics [7] to introduce a series of lysis buffers, first containing non-ionic detergents to preserve interactions, followed by a depolymerization buffer. G-actin is electrophoresed in interaction-stabilizing lysis buffer and immobilized in the gel, while F-actin is size-excluded from the gel. Upon delivery of depolymerization buffer, F-actin is electrophoresed in the opposite direction of the G-actin and immobilized. Antibody detection of the actin species yields quantitation of previously unmeasured heterogeneity in F and G actin at the single-cell level.
We will discuss application of electrophoretic cytometry molecular complex separation to evaluate single-cell response to stimuli that alter actin polymerization state and molecular interactions. Future work will assess efficacy of drugs targeting actin maintenance, as well as their impact on the binding protein machinery responsible for actin polymerization/depolymerization.
References:
(1) Masai, J.; Ishiwata, S.; Fujime, S. Biophys. Chem. 1986, 25 (3), 253–269.
(2) dos Remedios, C. G.; Chhabra, D.; Kekic, M.; Dedova, I. V; Tsubakihara, M.; Berry, D. A.; Nosworthy, N. J. Physiol. Rev. 2003, 83 (2), 433–473.
(3) Rao, K. M. K.; Cohen, H. J. Mutat. Res. 1991, 256 (2–6), 139–148.
(4) Senderowicz, A. M.; Kaur, G.; Sainz, E.; Laing, C.; Inman, W. D.; Rodríguez, J.; Crews, P.; Malspeis, L.; Grever, M. R.; Sausville, E. A. J. Natl. Cancer Inst. 1995, 87 (1), 46–51.
(5) Bubb, M. R.; Senderowicz, A. M.; Sausville, E. A.; Duncan, K. L.; Korn, E. D. J. Biol. Chem. 1994, 269 (21), 14869–14871.
(6) Heacock, C. S.; Bamburg, J. R. Anal. Biochem. 1983, 135 (1), 22–36.
(7) Yamauchi, K.; Herr, A. Microsystems Nanoeng. 2017, 3, 16079.

Julea Vlassakis

Graduate Student
UC Berkeley/UCSF Joint Graduate Program in Bioengineering

Julea Vlassakis is a PhD candidate in the Herr Lab for Quantitative Bioinstrumentation in the UC Berkeley/UCSF Joint Graduate Program in Bioengineering. Julea received her BA in Physics and Chemistry from Smith College, and was awarded an NSF Graduate Research Fellowship. She is also the recipient of the 2017 SLAS Graduate Education Fellowship Grant. Julea develops micro-scale technologies for selective probing of protein isoforms and complexes at the single-cell level.

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SLAS2018 Innovation Award Finalist: Electrophoretic Cytometry Isolates Cytoskeleton Molecular Complexes of Single Cancer Cells



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