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. 2006 Mar;17(3):1273-85.
doi: 10.1091/mbc.e05-07-0700. Epub 2006 Jan 4.

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Takashi Nishimura  1 , Tomoya Yamaguchi, Akinori Tokunaga, Akitoshi Hara, Tomonari Hamaguchi, Katsuhiro Kato, Akihiro Iwamatsu, Hideyuki Okano, Kozo Kaibuchi
Affiliations

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Takashi Nishimura et al. Mol Biol Cell. 2006 Mar.

Abstract

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.

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Figures

Figure 1.
Figure 1.
Localization of Numb in dendritic spines. (A–C) Localization of Numb in adult rat brain. Sections were incubated with anti-Numb antibody (green), anti-MAP-2 antibody (blue), DiI (red), and Hoechst. (A) cerebral cortex; (B) CA1 region of the hippocampus; and (C) cerebellar cortex. Insets, the enlarged images of the boxed area. Arrows, colocalization of Numb with MAP-2-positive dendrites. SO; stratum oriens, SP; stratum pyramidale, SR, stratum radiatum; GL, granular cell layer; P, Purkinje cells; ML, molecular layer. Bar, 50 μm. (D) High magnification images of the layer V pyramidal neurons of cerebral cortex (top panel) and the pyramidal neurons of the CA1 region (bottom panel). Red, green, and blue indicate DiI, Numb, and Hoechst, respectively. Bar, 10 μm. (E) Double-staining of Numb with PSD95 (top panel) and synaptophysin (bottom panel). High magnification images of the pyramidal neurons of the CA1 region are shown. Bar, 5 μm. (F) Expression of Numb in brain lysate. Equal amounts of the whole brain lysate at the indicated periods were subjected to the immunoblot analysis. (G) Localization of Numb in cultured rat hippocampal neurons. DIV21 mature neurons were fixed and then stained with anti-Numb antibody (green), anti-PSD95 antibody (blue), and phallodin (red) to stain F-actin. Enlarged images of the boxed area are shown (bottom). Arrowhead, the axon, as determined by its morphology; arrows, the dendritic spines where Numb colocalized with PSD95 and F-actin. Bar, 10 μm.
Figure 2.
Figure 2.
Suppression of Numb affects spine development. (A) Suppression of Numb and Numb-like by siRNAs in COS7 cells. COS7 cells were cotransfected with the indicated plasmid and siRNA. After 24 h of transfection, the expression level of each protein was analyzed by immunoblotting with anti-myc and anti-GEP antibody. (B) Suppression of Numb in hippocampal neurons. DIV12 primary neurons were transfected with the indicated siRNA along with the plasmid encoding GST as a fill. After 4 d of transfection, neurons were fixed and stained with anti-Numb antibody (red) and anti-GST antibody (green). Arrows indicate the transfected cells. Bar, 10 μm. (C) Quantitative analysis for the expression level of Numb. Fluorescence intensity of the cell body was measured to confirm the efficiency of Numb siRNAs. (D) Morphology of control and Numb-suppressed dendrites, as revealed by staining of cotransfected GST. Bar, 5 μm. (E) Number of dendritic protrusions in Numb-suppressed cells. Under the same conditions as those in D, the number of dendritic protrusions was counted. The data are means ± SE of pooled samples from at least three independent experiments. (F and G) The line graphs show the cumulative frequency distribution of protrusion lengths (F) and area of spine-head (G) for neurons transfected with siScramble (blue) or siNumb and siNumb-like (red). Insets show the mean spine length and mean area of spine-head for the two groups.
Figure 3.
Figure 3.
Interaction of Numb with intersectin in vivo and in vitro. (A) Isolation of Numb-binding proteins by affinity column chromatography. GST or GST-Numb fragment immobilized beads were incubated with rat brain lysate containing ionic detergents. The bound proteins were eluted by the addition of glutathione and analyzed by SDS-PAGE followed by silver staining. Red dots, the specific binding proteins to GST-Numb-PRR; yellow dot, the eluted GST-Numb-PRR protein. Identities of each bound protein, as revealed by molecular mass analysis, are shown on the right. (B) Coimmunoprecipitation of Numb and intersectin from rat brain lysate. Extract of developing rat brain was incubated with rabbit IgG, anti-Numb, mouse IgG, or anti-intersectin antibody. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (C) Domain structure and deletion constructs of Numb. Numbers refer to amino acid positions. (D) Identification of the binding region of Numb with intersectin. GST-Numb fragment immobilized beads were incubated with rat brain lysate. The bound proteins were analyzed by immunoblotting with anti-intersectin antibody. (E) Domain structure and deletion constructs of intersectin. Numbers refer to amino acid positions. (F) Mapping of the region in intersectin required for binding to Numb. GST-intersectin fragment immobilized beads were incubated with rat brain lysate or recombinant His-Numb-PRR. The bound proteins were analyzed by immunoblotting with anti-Numb antibody or anti-His antibody. (G) Deletion constructs of intersectin-SH3 domains. Numbers refer to amino acid positions. (H) Mapping of the region in intersectin-SH3 domains required for binding to Numb. GST-intersectin fragment immobilized beads were incubated with rat brain lysate or recombinant His-Numb-PRR. The bound proteins were analyzed by immunoblotting with anti-Numb antibody, anti-dynamin antibody, or anti-His antibody.
Figure 4.
Figure 4.
Effects of Numb and intersectin mutants on dendritic spine morphology in hippocampal neurons. (A) Colocalization of endogenous intersectin and Numb. DIV21 neurons were fixed and stained with anti-Numb antibody (green), anti-intersectin antibody (red), and phalloidin (blue). Arrows indicate dendritic spines where Numb and intersectin were colocalized. (B) Localization of Numb and intersectin at the postsynaptic site. DIV14 neurons were transfected with GFP-intersectin-L (green) and fixed at DIV21. Neurons were stained with anti-Numb antibody (red) and anti-synaptophysin antibody (blue). Arrows indicate the colocalization of Numb and GFP-intersectin-L at the dendritic spines. Enlarged images of single spine are shown below. Bars, 5 μm. (C) Cell morphology of control GST- and Numb-full-expressing neurons. DIV14 neurons were transfected with the indicated plasmid along with GST as a fill and fixed at DIV21. Enlarged images of the boxed area are shown in (D). Bar, 10 μm. (D) Morphology of dendritic spines. Under the same conditions those in (C), various constructs were examined. Arrow indicates a filopodial protrusion of control GST expressing neurons. (E) Quantitative analysis of the morphology of dendritic spines. Spine length, percentage of protrusions with a spine-head, and spine density were analyzed. The data are means ± SE of pooled samples from at least three independent experiments. Asterisks indicate a difference from the value of control GST-expressing cells at p < 0.01.
Figure 5.
Figure 5.
Numb enhances intersectin GEF activity toward Cdc42 in vivo. (A) Filopodia and lamellipodia formation by intersectin mutants in N1E-115 cells. Cells were transfected with the indicated constructs, fixed 24 h after transfection, and stained with anti-GST antibody to visualize the transfected cells by immunostaining of cotransfected GST. Bar, 20 μm. (B) Effects of Numb and intersectin mutants on cell morphology. Under the same conditions as those in A, GST-positive cells were scored for the presence of filopodia or lamellipodia. The data are means ± SD of at least three independent experiments. Asterisks indicate a statistical difference at p < 0.01. (C) Activation of Cdc42 by Numb and intersectin. COS7 cells transfected with the indicated constructs and GFP-Cdc42-WT were incubated with GST-PAK-CRIB to precipitate the GTP form of Cdc42. The amounts of GTP-bound and total Cdc42 were determined by immunoblotting with anti-GFP antibody (top). The ratio of GTP-Cdc42 to total Cdc42 is shown on the bottom. (D) Intramolecular interaction between intersectin-SH3 and intersectin-CT. GST-intersectin immobilized beads were incubated with COS7 cell lysate expressing GFP-intersectin-CT. Bound proteins were analyzed by immunoblotting with anti-GFP antibody. (E) Competition assay of Numb with intramolecular interaction of intersectin. GST, GST-intersectin-SH3, and GST-intersectin-SH3D immobilized beads were incubated with a mixture of COS7 cell lysate expressing GFP-intersectin-CT and excess recombinant His-Numb-PTB or His-Numb-PRR. Bound proteins were analyzed by immunoblotting with anti-GFP antibody. Number indicates the amount of His-Numb proteins against GST-intersectin proteins.
Figure 6.
Figure 6.
Numb associates with EphB2 and NMDA receptors at dendritic spines. (A) Isolation of PSD fraction from rat adult cerebral cortices. Subcellular fractionation was performed to isolate the SPM fraction. Aliquots of subcellular fractions (see Materials and Methods) were analyzed by immunoblotting with the indicated antibodies. RhoGDI was used as a control for supernatant. G1 to G4 fractions of the density gradient contain floating myelin (myelin), light membrane mixture (light mem.), synaptosomal plasma membrane (SPM), and mitochondrial pellet (mito.) from top to bottom. SPM were further extracted in 0.5% Triton X-100 to yield the PSD Triton-1 pellet (P) and supernatant (S). The PSD Triton-1-P was extracted in 0.5% Triton X-100 again or 3% N-lauroyl sarcosine to yield PSD Triton-2 and sarcosyl pellets and supernatants, respectively. (B) Interaction of Numb and intersectin with EphB2 and NMDA receptors. GST-Numb or GST-intersectin fragment immobilized beads were incubated with rat brain lysate. Bound proteins were analyzed by immunoblotting with the indicated antibodies. (C) Coimmunoprecipitation of Numb with EphB2 and NMDA receptors. Solubilized SPM lysate was incubated with rabbit IgG or anti-Numb antibody. Immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (D) Colocalization of Numb with clustered EphB2 receptor. DIV14 neurons were treated with clustered ephrin-B1-Fc for 4 h and then fixed. Neurons were stained with anti-Numb antibody (red), anti-EphB2 antibody (green), and phalloidin (blue). Arrows indicate the colocalization of Numb with EphB2 at dendritic spines. Bar, 5 μm.
Figure 7.
Figure 7.
Suppression of Numb impairs ephrin-B1-induced spine development and maturation. (A) The effects of Numb suppression on the localization of intersectin, EphB2, and NR1 on dendritic spines. DIV10 neurons were transfected with indicated siRNA along with GST. Four days after transfection, neurons were fixed and stained with anti-GST (green) and the indicated antibodies (red). Arrows indicate the position of dendritic spines. Bar, 5 μm. (B) Quantification of average fluorescence intensity in spines. Average fluorescence intensity of each protein per spine area was analyzed in neurons transfected with siScramble and neurons transfected with siNumb and siNumb-like. The data are means ± SE of pooled samples. Asterisks indicate a difference from the value of siScramble-transfected neurons at p < 0.001. (C) Changes in dendritic protrusions after stimulation of clustered ephrin-B1-Fc. DIV10 neurons were transfected with indicated siRNA along with GST. Four days after transfection, neurons were treated with clustered ephrin-B1-Fc or control Fc for 4 h and then fixed. Neurons were stained with anti-GST antibody (green) and anti-PSD95 antibody (red). Enlarged images of the boxed area are shown below. Bar, 10 μm. (D), (E) Quantitative analysis of the morphology of dendritic protrusions. Number of dendritic protrusions (D) and PSD95-positive protrusions with a spine-head (E) were analyzed. The data are means ± SE of pooled samples from at least three independent experiments. Asterisks indicate a difference from the value of siScramble-transfected neurons with ephrin-B1-Fc at p < 0.01.
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