HSPC300 and its role in neuronal connectivity - PubMed (original) (raw)
HSPC300 and its role in neuronal connectivity
Abrar Qurashi et al. Neural Dev. 2007.
Abstract
Background: The WAVE/SCAR complex, consisting of CYFIP (PIR121 or Sra1), Kette (Nap1), Abi, SCAR (WAVE) and HSPC300, is known to regulate the actin nucleating Arp2/3 complex in a Rac1-dependent manner. While in vitro and in vivo studies have demonstrated that CYFIP, Kette, Abi and SCAR work as subunits of the complex, the role of the small protein HSPC300 remains unclear.
Results: In the present study, we identify the HSPC300 gene and characterize its interaction with the WAVE/SCAR complex in the Drosophila animal model. On the basis of several lines of evidence, we demonstrate that HSPC300 is an indispensable component of the complex controlling axonal and neuromuscular junction (NMJ) growth. First, the Drosophila HSPC300 expression profile resembles that of other members of the WAVE/SCAR complex. Second, HSPC300 mutation, as well as mutations in the other complex subunits, results in identical axonal and NMJ growth defects. Third, like with other complex subunits, defects in NMJ architecture are rescued by presynaptic expression of the respective wild-type gene. Fourth, HSPC300 genetically interacts with another subunit of the WAVE/SCAR complex. Fifth, HSPC300 physically associates with CYFIP and SCAR.
Conclusion: Present data provide the first evidence for HSPC300 playing a role in nervous system development and demonstrate in vivo that this small protein works in the context of the WAVE/SCAR complex.
Figures
Figure 1
HSPC300 expression profile. (a) Quantitative analysis of HSPC300 mRNA levels by light cycler at indicated developmental stages. Quantification is relative to the housekeeping ribosomal protein 49 (rp49) mRNA. Bars indicate SEM. APF: After puparium formation. (b) Western blot analysis on protein extracts prepared from the Drosophila S2 cell line (S2 extract), using anti-HSPC300 antibody (HSPC300). The right lane represents equivalent amounts of protein extract from the S2 cell line, which transiently overexpresses HSPC300 upon transfection (S2 extract + HSPC300). β-tubulin (β-tub) represents a loading control. (c) HSPC300 immunolabeling of a whole mount embryo at stage 16; ventral view, anterior to the left. HSPC300 shows specific accumulation in CNS longitudinal connectives and commissures. The arrow and arrowheads show midline and motor neuron labeling, respectively. Scale bar: 50 μm.
Figure 2
HSPC300 locus and mutants. (a) Exon/intron organization and resulting transcripts are shown for the genes PebIII, CG3163 and HSPC300. ATG and Stop (in grey) indicate open reading frames. EP indicates the P element insertion in the EP(2R)0506 line and UAS indicates the upstream activating sequence contained in the P element. (b) Molecular characterization of three excision lines. Line HSPC300_Δ_96.1(as well as non-depicted HSPC300_Δ_52.1 , HSPC300_Δ_31.1 alleles) represent an excision event affecting the entire HSPC300 gene and part of the PebIII gene. Line HSPC300_Δ_54.3 displays intact adjacent genes (PebIII and CG3163) and P element. The red line represents integrated _HSPC300_-unrelated sequence, and the dotted line represents uncharacterized sequence. (c) RT-PCR on the wild-type (wt) and the HSPC300_Δ_54.3 excision line. The left three lanes show products obtained from wild-type third instar larvae; the right three lanes show product obtained from HSPC300_Δ_54.3 larvae. Note that HSPC300 transcripts are detected in the wild type (third lane from the left), but not in HSPC300_Δ_54.3 larvae (sixth lane from the left). In contrast, both PebIII (fourth lane from the left) and CG3163 (fifth lane from the left) transcripts are detected in the wild type and in mutant larvae.
Figure 3
HSPC300 is maternally provided and is required in the morphology of embryonic CNS. (a-j) Embryos of indicated genotypes labeled with the axon-specific FasII and BP102 antibodies. All images show a portion of the ventral nerve cord (ventral views, anterior to the top) of stage 16/17 embryos. In wild-type embryos, anti-FasII reveals six longitudinal bundles (a), and BP102 two commissural bundles (b) in each segment. (c,d) HSPC300 embryos show CNS axon morphology that appears wild type. (e-h) Embryos that lack the HSPC300 zygotic component and are maternal hypomorphs show abnormal midline crossing (arrowhead) (e). In the most severe cases (g), axons ectopically cross the midline several times. (f,h) Commissures and longitudinal connectives are not properly formed (arrows). (i,j) Embryos that completely lack zygotic as well as maternal HSPC300 components show strongly disturbed CNS development, broken longitudinal connectives and commissures (asterisks). Scale bar: 20 μm. (k-m) Comparative analysis of HSPC300 expression in embryos of indicated genotypes; ventral views of stage 16 embryos, anterior to the left, anti-HSPC300 labeling. This experiment indicates that HSPC300 is maternally contributed and confirms the specificity of antibody. Scale bar: 50 μm.
Figure 4
HSPC300 controls synaptogenesis at NMJs. (a-f) Anti-Disc Large immunolabeling of muscle 4 synaptic terminals of third instar larvae of the following genotypes: (a-c) wild type; (d-f)HSPC300. Compared to the wild type (WT), HSPC300 NMJs are shorter and display supernumerary buds. Insets in (a,d) show high magnifications of marked regions; (c,f) represent high magnifications of marked regions in (b,e), respectively. Arrows indicate buds. Scale bar: 20 μm (a,d); 14 μm (b,e); 8 μm (c,f). (g,h) Statistical evaluation of the NMJ phenotype of the following genotypes: wild type (WT); HSPC300 (HSPC300_Δ_54.3); HSPC300 heterozygous (HSPC300_Δ_54.3 /+); Revertant, precise excision event (HSPC300 86.1); and Rescue (elav-Gal4; UAS-HSPC300, HSPC300_Δ_54.3). (g) Length of synaptic terminals (μm) normalized over respective muscle surface area (μm2). (h) Number of buds per synapse. The sample size (number of muscle 4 junctions scored) was 25 per genotype. Error bars indicate SEM. Statistical significance (p) was calculated using ANOVA and post hoc analysis (see Materials and methods). Asterisks indicate _p_-value; *p < 0.05; ***p < 0.0001. (i-n) Wild type (i-k) and HSPC300 (l-n) NMJs labeled with anti-Disc Large (DLG; green) and active zone-specific marker NC82 antibody (red). Arrows indicate the region of low NC82 accumulation located between boutons, and arrowheads the supernumerary buds in the mutant synapse. Scale bar: 20 μm (i,j,l,m); 4 μm (k,n).
Figure 5
Protein levels of WAVE/SCAR complex subunits in different mutant contexts. (a) Immunoprecipitation experiments in Drosophila S2 cytoplasmic cell extracts using the anti-HSPC300 antibody. From left to right: anti-HSPC300 immunoprecipitation, IgG immunoprecipitation, input (cytoplasmic extract). Proteins are indicated to the right, corresponding molecular weights to the left. (b) Quantitative analysis of CYFIP, Kette, SCAR, Abi and HSPC300 protein levels by western blot on third instar larval extracts of the following genotypes: wild type (WT); HSPC300 zygotic null; HSPC300 zygotic null and maternal hypomorph; CYFIP zygotic null. Proteins are indicated to the right, corresponding molecular weights to the left. β-tubulin represents a loading control.
Figure 6
HSPC300 Bristle phenotype. (a) Scanning electron microscope images of the dorso-posterior portion of the Drosophila head and (b) light microscope images of thoracic bristles. On the left are wild-type (wt), and on the right HSPC300 pharate adults. Occasionally, bristles in HSPC300 pharate adults show typical bent morphology (arrows). Note that HSPC300 pharate adults display normal specification of sensory bristles on the head, notum and scutellum. Scale bars: 100 μm.
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