Browsing by Author "Diehl, Michael R"

Now showing 1 - 6 of 6
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    Investigations in Dynamic Protein Interactions and Steady-State Protein Abundance in Mammalian Cells
    (2021-03-26) Bellamkonda, Satya Kamala; Diehl, Michael R
    The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. Multi-domain scaffold proteins orchestrate the activities of multiple signaling intermediates and regulatory proteins on organelles. Investigating scaffold functions is challenging because these interactions occur at different timescales that are not well understood through standard static colocalization analyses. This work employs live-cell imaging to probe how the multi-domain scaffold IQGAP1 coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling, and point to a scaffold tethering mechanism. This work also focuses on engineering mammalian cells to modulate steady-state protein expression. Protein abundance is a highly regulated cellular process, and variations in protein levels leads to changes in cellular phenotypes and functions. These changes may cause human genetic diseases and changes in immune response, and it is important to build tunable mammalian cell libraries that can be used for systematic analyses to study the link between protein expression levels and cellular function. In this project, mammalian cell libraries with subpopulations of variable steady-state expression of a fluorescent protein or membrane ligand were made by multiplexing synthetic biology strategies in a modular, plug-and-play, high-throughput system. These libraries generated cell lines with tunable, stable, robust expression with quick turnaround times, which can be employed for future functional analyses of protein expression levels on cellular activity.
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    Investigations of macromolecular complex functions in living cells
    (2017-04-21) Tsao, David; Diehl, Michael R
    Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. While in vitro assays employing synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances and position noise on these parameters suggest that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared to those of other cytoskeletal motors. IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks. Using these endosomes as a model system of IQGAP1 regulation and function, in our next investigation we express a series of IQGAP1 domain mutations in MCF10A cells that allow us to investigate the effects of removing or enhancing specific IQGAP1 interactions while otherwise preserving the overall regulatory network surrounding IQGAP1 activities. The strength, directionality, and timing of interactions between IQGAP1 domains and endosomal cytoskeleton and membrane constituents are evaluated via time-lapse imaging of endosomal dynamics. We identify IQGAP1 states in which domain-level interactions with F-actin and Exo70 are coordinated through the GRD. Our findings are summarized in a proposed model of IQGAP1 domain activation and coordination.
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    IQGAP1 mediates the structure and dynamics of a novel multi-vesicular compartment
    (2016-08-08) Samson, Edward B; Diehl, Michael R
    IQGAP1 is a master cytoskeletal regulatory protein that connects extracellular signaling to changes in cell polarity, motility, and adhesion with adjacent cells. IQGAP1 achieves these fundamental outcomes by acting as a scaffolding protein that coordinates a wide variety of signaling cascades in a highly spatially-dependent manner. This dissertation details the use of multiple imaging modalities to characterize localized, highly-dynamic IQGAP1-related processes in epithelial MCF-10A cells. This led to the discovery of a novel multi-vesicular compartment that is surrounded by an outer layer of IQGAP1-associated actin filaments. Further studies showed that this compartment shares many common identifiers with traditional multi-vesicular bodies and participates in the internalization of cell-cell adhesion proteins via endocytic and recycling pathways. Live-cell imaging studies were conducted to correlate local cytoskeletal remodeling of this outer layer to various dynamic behaviors of the multi-vesicular core. These studies showed that IQGAP1 localization negatively correlates with actin polymerization during compartment formation and stabilization. During this time, rapid actin assembly appears to be constrained by a negative feedback mechanism. In contrast, IQGAP1 dissociation from the compartment’s surface is followed by a rapid, non-linear increase in actin polymerization that coincides with compartment disassembly and the release of multiple, high-motile intraluminal vesicles. Taken together, these results suggest a potential regulatory role of IQGAP1 in the trafficking of cell-cell adhesion proteins by promoting the stabilization of a novel multi-vesicular sorting compartment.
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    Micron-scale dynamics of the scaffold protein IQGAP1 and its cytoskeletal regulatory signaling partners in living cells
    (2018-04-20) McLaughlin, Tyler; Diehl, Michael R
    Scaffold 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.
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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.
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    Multiplexed Spatial Analyses in situ and in living cells
    (2015-12-04) Zimak, Jan; Diehl, Michael R; Qutub, Amina A; Wagner, Daniel S
    High-content spatial analyses are critical to understanding the structural organization and dynamics of many complex biological processes. Increasing the number of cellular components that can be visualized will help delineate the functions of many interacting and competing cellular pathways. However, the physical limitations of spectral bandwidth and the experimental difficulty of genomic manipulation have hampered traditional approaches to multiplex molecular analyses in both fixed samples and live cells. The programmable and predictable nature of the DNA molecule makes it a tantalizing candidate for an engineering tool to help alleviate some of these limitations. This thesis seeks to harness both the chemical and biological utility of DNA as a building block to multiplex the color and control the number and location of fluorescent reporters in biological samples. First in the context of in situ immunofluorescence imaging of fixed cells or tissues, And second in the context of live-cell imaging of genomically engineered cells. In the first case, by utilizing the stand displacement chemical reaction between dynamic DNA complexes and DNA-conjugated antibodies we selectively couple fluorophores to, and then remove them, from their protein targets. We leverage this mechanism to facilitate multiple sequential round of fluorescence microscopy where the same color dye molecules are used reiteratively to visualize different antibody-tagged markers. By optimizing the DNA-antibody conjugation chemistry and incubation protocol we now routinely perform 9 marker analyses of paraffin-fixed tissue sections with these DNA probes. Then automating the sequence design process enabled more complex probe designs to be use for balancing marker levels appropriately for hyperspectral imaging experiments. Here, discrete and reconfigurable control over amplification gains, greatly improved the spectral un-mixing of different antibody signals. Secondly, we focus on dissecting network-level functions of cytoskeletal regulatory proteins during epithelial cell polarization and morphogenesis. DNA-based STORM microscopy revealed that a scaffold protein, IQGAP1, associates with specialized actin filaments within cell-cell junctions and with basket-like structures in the basal actin cortex of normal epithelial cells. This work uses IQGAP1 as a platform, as it lies at the nexus of cell signaling and cytoskeletal regulatory networks. We construct multi-gene systems that simultaneously sense and control intracellular expression levels of IQGAP1 and track the actin cytoskeleton. By combining novel molecular biology techniques to manipulate the DNA in live cells. We use a barcoded self-assembly technique to construct large vectors that contain several transcriptional elements. These multi-gene systems are then stably incorporated into cells engineered with genomic ‘landing pads’ using locus-specific integration. Finally, we demonstrate functional circuits by linearly controlling intracellular IQGAP1 levels. These results will support future single-cell and multiplexed population-level analyses of IQGAP1 functions in epithelial cells, allow us to study IQGAP1 recruitment to epithelial cell-cell junctions and to examine how it influences cellular transitions.