The ability to chemically control protein-protein interactions would allow the interrogation of dynamic cellular processes and lead to a better understanding and exploitation of self-assembling protein architectures. Here we introduce a new engineering strategy--reverse metal-templated interface redesign (rMeTIR)--that transforms a natural protein-protein interface into one that only engages in selective response to a metal ion. We have applied rMeTIR to render the self-assembly of the cage-like protein ferritin controllable by divalent copper binding, which has allowed the study of the structure and stability of the isolated ferritin monomer, the demonstration of the primary role of conserved hydrogen-bonding interactions in providing geometric specificity for cage assembly and the uniform chemical modification of the cage interior under physiological conditions. Notably, copper acts as a structural template for ferritin assembly in a manner that is highly reminiscent of RNA sequences that template virus capsid formation.

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Currently, there are few methods to visualize cell cycle progression in leukemia stem cells (LSC). Human LSC are characterized as quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Additionally, current methods require sequential transduction and clonal selection. These methods and conditions are not feasible to study primary patient samples and LSC, which are generally limited in sample size. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI (fluorescent ubiquitination-based cell-cycle indicator) probes. Time-lapse confocal imaging of transduced 293 cells and SKNO-1 cells exhibited normal cell division and distinct nuclear staining of either green or red fluorescence. Further, analysis by flow cytometry of 293 cell confirms that S/G2/M and G1/G0 phases of cell cycle correlate to expression of fluorescent reporters expressed from bicistronic lentiviral vector and recapitulate FACS plots using traditional Ki67 and 7AAD staining. The development of this new reagent offers a new and more effective method to track cell cycle progression in limited mammalian cell populations. Future studies will utilize primary patient CD34+CD38- selected leukemic stem cell populations. Citation Format: Gabriel Pineda, Florence Lambert-Fliszar, Gennarina L. Riso, Kathleen M. Kane, Catriona Jamieson. Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-159. doi:10.1158/1538-7445.AM2014-LB-159