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. 2009 Jun 19;384(1):55-60.
doi: 10.1016/j.bbrc.2009.04.069. Epub 2009 Apr 23.

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and alpha-actinin2

Hyang-Sook Hoe  1 , Ji-Yun Lee, Daniel T S Pak
Affiliations

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and alpha-actinin2

Hyang-Sook Hoe et al. Biochem Biophys Res Commun. .

Abstract

Rap small GTPases regulate excitatory synaptic strength and morphological plasticity of dendritic spines. Changes in spine structure are mediated by the F-actin cytoskeleton, but the link between Rap activity and actin dynamics is unclear. Here, we report a novel interaction between SPAR, a postsynaptic inhibitor of Rap, and alpha-actinin, a family of actin-cross-linking proteins. SPAR and alpha-actinin engage in bidirectional structural plasticity of dendritic spines: SPAR promotes spine head enlargement, whereas increased alpha-actinin2 expression favors dendritic spine elongation and thinning. Surprisingly, SPAR and alpha-actinin2 can function in an additive rather than antagonistic fashion at the same dendritic spine, generating combination spine/filopodia hybrids. These data identify a molecular pathway bridging the actin cytoskeleton and Rap at synapses, and suggest that formation of spines and filopodia are not necessarily opposing forms of structural plasticity.

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Figures

Figure 1
Figure 1. Identification of α-actinin interaction with SPAR
(A) Y2H screens were performed using SPAR Act2 domain as bait in vector pBHA. Interaction strength was scored by β-gal activity (+++, 0-30 min; +, 30 min-2 hrs; -, >12 hrs). Interaction time for α-actinin-SPAR was consistently under 10 minutes. Residues encompassed by positive clones (HB15 and HB20) and α-actinin deletion constructs are indicated below α-actinin schematic diagram (interacting clones shown in black). SNK in pGAD10 vector was positive control for interaction with Act2-pBHA; NR1 C0-C1 exon-containing C-terminus in pBHA was positive control for the α-actinin interactions. PSD-95 PDZ1/2 in pGAD10 was negative control for all pBHA constructs. (B) α-Actinins bind specifically to the C-terminal portion of Act2 (Act2ΔN), whereas SNK binds primarily to the N-terminal part (Act2ΔC). (C) Schematic of SPAR domains and interactions. Act2, actin-associated domain 2. GKBD, guanylate kinase binding domain. ACTN, α-actinin. Double headed arrow indicates SPAR binding to α-actinin may displace NR1.
Figure 2
Figure 2. Biochemical and immunocytochemical association of SPAR and α-actinin
(A) Rat brain lysates were immunoprecipitated with SPAR V20 goat antibodies or with nonimmune goat IgG control, and precipitates were immunoblotted for SPAR, α-actinin and tubulin as indicated. (B) Rat brain lysates were immunoprecipitated with mouse monoclonal antibodies to α-actinin or with nonimmune mouse IgG control, and precipitates were immunoblotted for SPAR and α-actinin. IN represents 10% of input used in each immunoprecipitation reaction in (A, B). (C) COS-7 cells transfected with myc-tagged SPAR or α-actinin2 as indicated were immunoprecipitated with myc agarose and immunoblotted for α-actinin. 10% of input is shown as expression control. (D-E) COS-7 cells transfected with myc-tagged SPAR alone (D) or with mycAct2ΔN domain of SPAR together with α-actinin2 (E). Cells were immunostained with myc and α-actinin antibodies, as indicated. Arrows show examples of colocalization. Scale, 5 μm.
Figure 3
Figure 3. Colocalization of SPAR and α-actinin in neurons
(A-F) Cultured hippocampal neurons at 19 DIV were double immunostained for proteins as indicated, with merged images shown at right (C, F). Boxed regions are shown below each image at higher magnification. Arrow, example of α-actinin cluster lacking SPAR colocalization. Arrowhead, example SPAR cluster lacking α-actinin colocalization. Scale, 20 μm. (G, H) Linescans of cyan segments shown in (C, F) demonstrating partial colocalization of α-actinin with SPAR (G) and with Shank (H). (I) Pixel colocalization between Shank/α-actinin or SPAR/α-actinin, calculated as the percentage of the integrated intensity of the first marker overlapping with the integrated intensity of the second marker. (J) Percentage of puncta colocalized between Shank/α-actinin and SPAR/α-actinin. ‘Marker only’ represents puncta that contain only the marker indicated out of the pair examined.
Figure 4
Figure 4. SPAR and α-actinin2 regulation of dendritic spine morphogenesis
(A-E) Cultured hippocampal neurons (15 DIV) were transfected with DNA constructs as indicated and with pEGFP to visualize neuronal morphology. Insets show higher magnification views of representative dendritic protrusions. (E) Representative hybrid spine/filopodia from transfected neurons in (D), shown at same scale as higher magnification insets in (A-C). Scale bars: 5 μm for dendrite images and 1 μm for all higher magnification images. (F) Quantification of dendritic spine morphological classifications as indicated. Hybrid refers to combination spine/filopodia protrusions. (G) Dendritic protrusion length, width, and length-to-width ratio (L/W) for transfected neurons as indicated. *p<0.05, **p<0.01, ***p<0.001, compared to control.
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