Browsing by Author "Trenton, Nicholaus James"

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    Multiplexed imaging and cell engineering strategies to understand complex molecular dynamics
    (2018-04-11) Trenton, Nicholaus James; Diehl, Michael R
    IQGAP1 is a multi-domain, 190 kDa scaffold protein that is critical for regulating a number of cellular processes, including migration, cytoskeletal reorganization, and membrane sorting. It is also often overexpressed or dysregulated in certain types of cancers and neurological diseases. Despite the interest in understanding IQGAP1’s function for clinical purposes, there remains confusion as to its relationship to phosphatidylinositol phosphate kinases (PIPK), critical regulators of membrane lipid metabolism. Because of the confusion in using endpoint analyses such as co-immunoprecipitation to relate IQGAP1 to these kinases and lipid components, it is unclear exactly what is IQGAP1’s role in this process. In this study, we expand on our group’s use of dynamic, epi-fluorescent live-cell imaging analyses to identify IQGAP1 as a sensor and regulator of phosphatidylinositol phosphate (PIP) lipids and PIPK reorganization. We show that IQGAP1 negatively regulates Arp2/3-mediated actin polymerization through PI(4,5)P2 recruitment, and that PI3-kinase acts as a switch for IQGAP1 to properly regulate actin dynamics. Furthermore, we found that inhibiting IQGAP1 behavior through treatment with neomycin, a known PI(4,5)P2 inhibitor, corresponded to an acceleration of dissociation of PI(4,5)P2 and wildtype IQGAP1 from endosomal sorting stations, while treatment with wortmannin, a PI3-kinase inhibitor, caused a loss of IQGAP1 localization completely. We then found that at short drug treatment time scales, the correlation between actin and ∆CHD, an IQGAP1 mutant lacking its actin-binding CH domain, becomes less correlated with actin when treated with wortmannin, and becomes more negatively correlated with actin when treated with neomycin. Lastly, we found that the PI(4,5)P2-binding deficient mutant AA3 mimics actin behavior more than wildtype IQGAP1, and that the PI3K-binding deficient mutant ∆CC-WW no longer responses to normal IQGAP1 cues. Our results overall suggest that binding and processing of the membrane is likely a switch that allows for IQGAP1 actin regulation through its CH domain. This experimental framework could be used to probe similar scaffolds, and disruption of PI3K through these techniques could be further investigated for therapeutic effects.