Micron-scale dynamics of the scaffold protein IQGAP1 and its cytoskeletal regulatory signaling partners in living cells

dc.contributor.advisorDiehl, Michael Ren_US
dc.creatorMcLaughlin, Tyleren_US
dc.date.accessioned2019-05-17T15:30:58Zen_US
dc.date.available2019-05-17T15:30:58Zen_US
dc.date.created2018-05en_US
dc.date.issued2018-04-20en_US
dc.date.submittedMay 2018en_US
dc.date.updated2019-05-17T15:30:58Zen_US
dc.description.abstractScaffold proteins are a hallmark of signaling pathways in eukaryotic cells. They are analogous to molecular circuit boards that wire the native signaling circuits which are composed of information-transferring enzymes like kinases and GTPases. IQGAP1 is a ubiquitously expressed scaffold that regulates cell state and morphology by tuning protein signaling pathways at the crux of phenotypic transitions in cancer biology and immunology. With > 6 domains, several proposed conformational states, and almost 2000 amino acids, IQGAP1 is known to bind and regulate actin, GTPases, MAP kinases, PI3K, E-cadherin, and numerous other proteins in diverse pathways. It has been shown theoretically and experimentally that scaffold proteins can activate or inhibit signaling pathways depending on their relative concentrations. Despite numerous experimental studies, very little is known about how living cells regulate scaffold protein concentrations, and how these ‘scaffolded’ protein complexes evolve dynamically. The Diehl lab has discovered novel endosomal compartments in epithelial cells, and these small compartments are enriched in IQGAP1, actin, phospholipids, and various membrane-binding proteins and are highly amenable to time-lapse microscopy for dynamical measurements. Using these compartments, we extracted micron-scale, multi-protein time series data with 60-second temporal resolution and used this data to resolve the extremely detailed coordination between IQGAP1, actin, membrane, and GTPases Cdc42 and Rac1, including how specific domains and residues of the scaffold contribute to its local concentration and compartment lifecycle-specific dynamics. By characterizing dynamics of mutant scaffolds, we discovered that IQGAP1 domains confer novel opposing behaviors: the GAP-related domain promotes IQGAP1 compartmental dissociation whereas the calponin homology domain limits its dissociation. We developed statistical models of compartmental protein dynamics to show how the dynamics of actin and the membrane can be predicted by observing a combination of wild type (WT) and mutant scaffold dynamics. While IQGAP1 is highly correlated with WT Rac1, we observed oscillatory dynamics between WT IQGAP1 and constitutively active Rac1, which suggests that there is a negative feedback loop involving IQGAP1 and the GTP-bound state of Rac1. This work presents the first detailed examination of the micron-scale dynamics of a scaffold protein with its signaling partners in living cells.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationMcLaughlin, Tyler. "Micron-scale dynamics of the scaffold protein IQGAP1 and its cytoskeletal regulatory signaling partners in living cells." (2018) Diss., Rice University. <a href="https://hdl.handle.net/1911/105783">https://hdl.handle.net/1911/105783</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/105783en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectscaffold proteinen_US
dc.subjectcell signalingen_US
dc.subjectactinen_US
dc.subjectcytoskeletonen_US
dc.subjectendosomeen_US
dc.subjectimagingen_US
dc.subjectIQGAP1en_US
dc.subjectcytoskeletal regulationen_US
dc.subjectsystems biologyen_US
dc.subjectscaffold dynamicsen_US
dc.titleMicron-scale dynamics of the scaffold protein IQGAP1 and its cytoskeletal regulatory signaling partners in living cellsen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentSystems, Synthetic and Physical Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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