Regulation of Spine Density and Morphology by IQGAP1 Protein Domains

<div><p>IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42.</p> </div

IQGAP1 stimulates spine formation and increases spine head size.

<p>(A) Confocal image showing an example of a 17 DIV cultured hippocampal neuron transfected with mock plasmid plus GFP (green) and double stained with MAP2 (red). (B) A high magnification view of a dendritic segment from another neuron of the same culture (C) A 3-D reconstruction of dendritic shafts from a sister culture; note the morphology and density of dendritic spines. (D–G) Confocal images showing a dendritic segment from a neuron co-transfected with myc-tagged-mock plasmid (blue) plus GFP-PSD95 (green) and stained for synaptophysin (red). (G) Merge image; note that GFP-PSD95 (+) spines colocalize with synaptophysin puncta (arrowheads). (H) A Confocal image showing an example of a 17 DIV cultured hippocampal neuron transfected with myc-tagged IQGAP1 WT+GFP (green) and double stained with MAP2 (red). (I) A high magnification view of a dendritic segment from another neuron of the same culture. (J) A 3-D reconstruction of dendritic shafts from a sister culture; note the increase in the number and size of spines. (K–N) Confocal images showing a segment of a dendritic shaft of a neuron co-transfected with myc-tagged-IQGAP1 WT (blue) plus GFP-PSD95 (green) and stained for synaptophysin (red). (N) Merge image. (O–Q) Graphs showing effects of ectopic expression of myc-tagged IQGAP1 WT on spine number, spine type and spine head size. Bars represent mean ± standard deviation. *p<0.0001. The effect of IQGAP1 mutants on spine number is also shown (O).</p

Δ-CT IQGAP1 stimulates spine formation.

<p>(A) Confocal image showing an example of a 17 DIV cultured hippocampal neuron transfected with myc-tagged Δ-CT-IQGAP1 plus GFP (green) and double stained with MAP2 (red). (B) A high magnification view of dendritic segments from another neuron of the same culture; (C) A 3-D reconstruction of dendritic shafts from a sister culture. (D–F) Confocal images showing a dendritic segment from a neuron co-transfected with myc-tagged-Δ-CT-IQGAP1 (blue) plus GFP-PSD95 (green) and stained for synaptophysin (red). (G) Merge image. (H–J) Graphs showing the effect of expressing myc-tagged-Δ-CT-IQGAP1 on spine number/type and spine head size and length. Bars represent mean ± standard deviation. *p<0.0001.</p

IQGAP1 CHD domain is required for spine head formation.

<p>(A) Confocal image showing an example of a 17 DIV cultured hippocampal neuron transfected with myc-tagged Δ-CHD IQGAP1 plus GFP (green) and double stained with MAP2 (red). (B) A high magnification view of dendritic segments from another neuron of the same culture; note the long filopodial-like protrusions merging from dendritic shafts (arrowheads). (C) A 3-D reconstruction of dendritic shafts from a sister culture; note the morphology and density of filopodial-like protrusions (arrowheads). (D–E) Confocal images showing a dendritic segment from a neuron co-transfected with myc-tagged-Δ-CHD-IQGAP1 (blue) plus GFP-PSD95 (green) and stained for synaptophysin (red). (G) Merge image; note that GFP-PSD95 (+) protrusions colocalize with synaptophysin puncta (arrowheads). (H) Graphs showing effects of the ectopic expression of myc-tagged Δ-CHD-IQGAP1 on the number of different types of dendritic spines; note that Δ-CHD-IQGAP1 significantly increases the number of thin spines and filopodial extensions, while decreases the number of mushroom-shaped spines. (I) Graphs showing the effect of scrambled-sh-Arp3, sh-Arp3, and sh-WASP on the number of dendritic protrusions. For this experiment, cultures were transfected with the corresponding GFP-or HcRed-sh plasmids at 17 DIV and fixed 24 h later. Note the significant decrease in the total number of dendritic protrusions in the sh-Arp3 and sh-WASP-treated groups. (J) Graphs showing the number of dendritic protrusions contacting synaptophysin puncta in neurons transfected with IQGAP1 WT, Δ-CHD-IQGAP1 and sh-Arp3 plus myc-tagged-IQGAP1 WT. Note the dramatic decrease in the number of dendritic protrusions contacting synaptophysin puncta in the cells treated with sh-Arp3 plus IQGAP1 WT; most of these protrusion resemble filopodial extensions. (K) Confocal image showing an example of a 17 DIV cultured hippocampal neuron transfected with HcRed-sh-Arp3 (red) plus myc-tagged IQGAP1 WT (green). (L) A high magnification view of a dendritic segment from another neuron of the same culture. (M) A 3-D reconstruction of dendritic shafts from a sister culture; note the presence of many filopodial-like protrusions. (N-P) Confocal images showing a segment of a dendritic shaft of a neuron co-transfected with myc-tagged-IQGAP1 WT (blue) plus GFP-sh-Arp3 (green) and stained for synaptophysin (red). (Q) Merge image. Note that many filopodial protrusions are not contacted by synaptophysin puncta. Bars represent mean ± standard deviation. * p<0.0001.</p

Domain organization of IQGAP1 and representation of the deletion mutants.

<p>CHD: Calponin-homology domain. IRS: WW: protein domain containing two highly conserved triptophans that bind proline- rich peptide motifs; responsible for interaction with ERKs. IQ: calmodulin-binding motif; the term refers to the first two amino acids of the motif: isoleucine and glutamine. GRD: Ras GTP related activating protein domain; responsible for interactions with Cdc42 and Rac. CT: C-terminus; responsible for interactions with cadherin, CLIP-170, APC, etc.</p

Identification, Localization, and Quantification of Neuronal Cell Membrane Receptors with Plasmonic Probes: Role of Protein Kinase D1 in Their Distribution

Detecting, imaging, and being able to localize the distribution of several cell membrane receptors on a single neuron are very important topics in neuroscience research. In the present work, the distribution of metabotropic glutamate receptor 1a (mGluR1a) density on neuron cells on subcellular length scales is determined by evaluating the role played by protein kinase D1 (PKD1) in the trafficking of membrane proteins, comparing the distribution of mGluR1a in experiments performed in endogenous PKD1 expression with those in the presence of kinase-inactive protein kinase D1 (PKD1-kd). The localization, distribution, and density of cell surface mGluR1a were evaluated using 90 nm diameter Au nanoparticle (NP) probes specifically functionalized with a high-affinity and multivalent labeling function, which allows not only imaging NPs where this receptor is present but also quantifying by optical means the NP density. This is so because the NP generates a density (ρ)-dependent SERS response that facilitated a spatial mapping of the mGluR1a density distribution on subcellular length scales (dendrites and axons) in an optical microscope. The measured ρ values were found to be significantly higher on dendrites than on axons for endogenous PKD1, while an increase of ρ on axons was observed when PKD1 is altered. The spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the results obtained by fluorescence bright-field analysis and dark-field spectroscopy and provided additional structural details. In addition, it is shown using electrodynamic simulations that SERS spectroscopy could be a very sensitive tool for the spatial mapping of cell membrane receptors on subcellular length scales, as SERS signals are almost linearly dependent on NP density and therefore give indirect information on the distribution of cell membrane proteins. This result is important since the calibration of the ρ-dependent near-field enhancement of the Au immunolabels through correlation of SERS and SEM paves the way toward quantitative immunolabeling studies of cell membrane proteins involved in neuron polarity. From the molecular biology point of view, this study shows that in cultured hippocampal pyramidal cells mGluR1a is predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive protein kinase D1 (PKD1-kd) dramatically and selectively alters the intracellular trafficking and membrane delivery of mGluR1a-containing vesicles