Membrane and Actin Tethering Transitions Help IQGAP1 Coordinate GTPase and Lipid Messenger Signaling.

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.

A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT.

Next-generation DNA sequencing is currently limited by an inability to accurately count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting to reconstruct the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPTs of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. The analytical sensitivity and specificity of sgNIPT was >98% and >99%, respectively. Validation of sgNIPTs was further performed with maternal blood samples collected during pregnancy, and sgNIPTs were 100% concordant with newborn follow-up.

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks.

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.

Delineating cooperative responses of processive motors in living cells.

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. Though in vitro assays using 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 suggests 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 with those of other cytoskeletal motors.