IQGAP1 scaffolding links phosphoinositide kinases to cytoskeletal reorganization.

IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling. We find that IQGAP1 and PIPKIγ interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKIγ complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKIγ and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P3 that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKIγ association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKIγ), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.

Live Cell Fluorescence Imaging Shows Neurotransmitter Activation Promotes Aggregation of the Intracellular Domain of Amyloid Precursor Protein.

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCß signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCß1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCß reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

The Gαq/phospholipase Cß signaling system represses tau aggregation.

Alzheimer's disease is typified by calcium dysfunction and neurofibrillary tangles of tau aggregates along with mitotic proteins. Using PC12 cells as a model system, we determined whether the Gαq/PLCß/ calcium signaling pathway impacts the manifestation of Alzheimer's disease. Down-regulating PLCß significantly increases tau protein expression and causes a large increase in tau aggregation. Stimulating Gαq to activate PLCß results in a modest reduction in tau aggregation while inhibiting PLCß activity results in a modest enhancement of tau aggregation. These results suggest that PLCß may effect tau aggregation by an additional mechanism that is independent of its ability to transduce calcium signals. To this end, we found that a cytosolic population of PLCß binds to a mitotic protein found in neurofibrillary tangles, CDK18, which promotes tau phosphorylation and aggregation. Taken together, our studies show that the loss of PLCß1 can promote Alzheimer's disease by a combination of its catalytic activity and its interaction with mitotic proteins thus offering an orthogonal method to control tau aggregation.

Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

The Breast Cancer Susceptibility Gene Product (γ-Synuclein) Alters Cell Behavior through its [corrected] Interaction with Phospholipase Cß.

The breast cancer susceptibility gene protein, also known as γ-synuclein, is highly expressed in human breast cancer in a stage-specific manner, with highest expression in late stage cancer. In model systems, γ-synuclein binds phospholipase Cß2 which is regulated by Gαq to generate intracellular Ca(2+) signals. PLCß2, which is also absent in normal tissue but highly expressed in breast cancer, is additionally regulated by Rac to promote migration pathways. We have found that γ-synuclein binds to the same region of PLCß2 as Gαq. Using cells that mimic stage 4 breast cancer (MDA MB 231), we show that down-regulation of γ-synuclein reduces the protein level of PLCß but increases the transcript level over 40 fold. γ-Synuclein down-regulation also promotes the interaction between Gαq and PLCß resulting in a stronger Ca(2+) response to Gαq agonists. The ability of γ-synuclein to interfere with Gαq-PLCß interactions allows more PLCß to colocalize with Rac impacting Rac-mediated pathways that may give rise to cancerous phenotypes.

HIV-1 Nucleocapsid Mimics the Membrane Adaptor Syntenin PDZ to Gain Access to ESCRTs and Promote Virus Budding.

HIV-1 recruits cellular endosomal sorting complexes required for transport (ESCRTs) to bud virions from the membrane. Disruption of the viral nucleocapsid (NC) domain integrity affects HIV-1 budding. However, the molecular mechanisms of NC's involvement in HIV budding remain unclear. We find that NC mimics the PDZ domains of syntenin, a membrane-binding adaptor involved in cell-to-cell contact/communication, to capture the Bro1 domain of ALIX, which is an ESCRTs recruiting cellular adaptor. NC binds membranes via basic residues in either the distal or proximal zinc fingers, and NC-membrane binding is essential for Bro1 capture and HIV-1 budding. Removal of RNA enhances NC membrane binding, suggesting a dynamic competition between membrane lipids and RNA for the same binding sites in NC. Remarkably, syntenin PDZ can substitute for NC function in HIV-1 budding. Thus, NC mimics syntenin PDZs to function as a membrane-binding adaptor critical for HIV-1 budding at specific microdomains of the membrane.