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

Abstract

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.

Figure 1

IQGAP1 disassociation precedes Arp2/3- and SNX9-dependent polymerization of actin around endosomes. (A) Live cell images and kymographs of MCF10A cells transfected with the indicated fluorescent constructs are shown. Data are representative of n > 30 cells and three separate transfections; arrows indicate IQGAP1-containing compartments. Scale bars, 10 μm. Kymographs (right panels) show rapid bursts in Arp3 and SNX9 intensity immediately after IQGAP1 dissociates from the compartments. (B) Time-dependent fluorescence intensity trajectories and their time derivatives display similar dissociation and bursting behaviors found in kymographs. Shaded area indicates standard error of the mean (for Arp3: n = 45 trajectories from seven cells; SNX9: n = 8 trajectories from four cells). (C) Dynamic cross correlation coefficients (r) calculated using Eq. 2 show IQGAP1 is correlated negatively with Arp3 and SNX9 (^∗∗∗p < 0.0001). Error bars indicate standard deviation. To see this figure in color, go online.

Figure 2

Endosomes exhibit prominent PIP₂ localization and membrane tubule formation. (A) Shown is the co-localization of IQGAP1 with PIP₂ lipids detected using the PH^PLCδ sensor with regards to IQGAP1 fluorescence. Scale bars, 10 μm. (B) Average fluorescence intensity and derivative of fluorescence intensity plots show the preemptive loss of IQGAP1 and retention of PIP₂ until the endosomes are disassembled by an actin burst (n > 30 compartments in eight cells). Shaded area indicates standard error of the mean. (C) Membrane tubule formation on PIP₂-positive endosomes is revealed by three-dimensional SIM. To see this figure in color, go online.

Figure 3

PIP₃ exhibits weak and nonuniform localization on endosomes. (A) Example of summed intensity images of IQGAP1 and PH^Btk co-localization is shown here. Scale bars, 10 μm. (B) Average intensity trajectories show that PH^Btk disassociates before IQGAP1. Shaded area indicates standard error of the mean (n = 30 compartment, eight cells). To see this figure in color, go online.

Figure 4

(A) (left) Kymographs and their corresponding normalized intensity-time trajectories (middle) for the ΔCHD mutant, WT IQGAP1, and F-Tractin, intensity trajectory for a representative compartment with an actin burst, and (right) time-derivative plots averaged over multiple trajectories showing mean ± SE for each marker (n = 14 compartments). (B) Shown are the intensity-time data for ΔCHD, widtype IQGAP1, and Exo70 aligned using the minimal of the derivative of the Exo70 signal. n = 241 inflection points. (C) Dynamic cross correlation coefficients (r) are calculated for the combination of WT and mutant IQGAP1 scaffolds and either actin or Exo70 (Actin: n = 234 compartments, 19 cells; Exo70: 183 compartments, 17 cells; ^∗∗∗ denotes p < 0.0001). (D–F) Intensity-time and cross correlation plots for the T1050AX2 mutant are shown here (Actin: n = 71 bursts, 354 compartments, 11 cells; Exo70: n = 163 inflection points, 363 compartments, and 12 cells; ^∗∗ denotes p = 0.005). To see this figure in color, go online.

Figure 5

PI3K and Rho GTPase inhibition affects actin bust amplitudes and IQGAP1 disassociation behaviors. PI3K was targeted using wortmannin (A) and Ly294002 (B). The Rho GTPases Rac1 and Cdc42 were targeted using NSC23766 (C) and ML141 (D), respectively. Plots represent the averages of trajectories in which actin-busting events were observed (n = 21 bursts in six cells; n = 102 bursts in 14 cells; n = 151 bursts in 19 cells; and n = 21 bursts in four cells for (A–D), respectively). Shaded area indicate standard error of the mean. Drug concentrations are indicated in Table 2. To see this figure in color, go online.

Figure 6

Three-state localization model for IQGAP1. The influence of the CHD and GRD-RGCT domains on scaffold localization dynamics implies that IQGAP1 can associate via three different tethered states in which the scaffold is either fully tethered to both the endosome membrane and actin (i) or partially tethered to either the membrane through a combination of C-terminal domains (ii) or to actin via the CHD (ii). The coupling of PI kinase enzymes in the membrane tethered states (i or ii) has the potential to facilitate sequential lipid signaling events and channeling of PIP₃ to downstream signaling proteins like Akt. Drug inhibition experiments point to the GTPase activating and deactivating GEFs and GAPs as drivers of scaffold binding and unbinding at the membrane. They also suggest that these associations regulate the active states of Rho GTPases within the membrane bound complex. The three-state localization mechanism supporting these interactions may allow IQGAP1 to coordinate signaling events at different timescales by enabling fast responses to rapid Rho GTPase signaling cascades while being responsive and even managing slower changes in other signaling events on the endosomal membrane. To see this figure in color, go online.