Fig. 1
Two main spastin isoforms are synthesized during zebrafish development through alternative use of two initiation codons. (A) Alignment of spastin cDNA sequences surrounding the first and second ATG (bold) from different vertebrate species. Kozak sequences surrounding the first and second ATG are boxed in red and blue, respectively. Asterisks indicate conserved nucleotides. (B) Schematic representation of the different spastin constructs used in the present study. Crosses indicate mutated ATG. (C-E) Zebrafish spastin transcript drives the synthesis of two main spastin isoforms through alternative translation start sites. (C) Western blot analysis of spastin expression in COS-7 cells transfected or not (not transfected, NT) with the different spastin constructs (see B) using HA and spastin86-340 antibodies. (D) Both zebrafish spastin isoforms display a microtubule-severing activity in vitro. Immunolabelling of spastin and microtubules in COS-7 cells overexpressing DrM1 or DrM61-HA using HA (red) and tyrosinated tubulin (green) antibodies. Dotted lines surround transfected cells. Both overexpressed DrM1 and DrM61 dismantle the microtubule network. Scale bar: 20 µm. (E) Western blot analysis of exogenous spastin expression from protein extracts of tailbud embryos injected or not with in vitro transcribed mRNAs from the different spastin constructs using HA antibody. (F) The expression ratio between spastin isoforms varies during zebrafish development. Western blot analysis of spastin main isoforms during zebrafish development, using spastin86-340 antibody. Protein extracts from tailbud embryos injected with spastin-HA mRNA and revealed using an HA antibody, were used as DrM1 and DrM61 benchmark. (G) Spastin exon 4 is alternatively spliced during zebrafish development. RT-PCR analysis of spastin transcripts at both the tailbud stage and 24 hpf using primers flanking the exon 4 of the zebrafish spastin gene. (H,I) Distinct subcellular distribution of spastin main isoforms in spinal motor neurons in vivo. (H) Immunolabelling of 28 hpf Tg(mnGFF7;UAS:DrM1-HA) and Tg(mnGFF7;UAS:DrM61-HA) transgenic embryos with an HA antibody. Arrows indicate the punctate distribution of DrM1-spastin along the axon shaft while empty arrowheads indicate its specific enrichment in spinal motor growth cones. Scale bars: 10 µm. (I) Mean fluorescence intensity profile of DrM1 and DrM61 staining along the distal part of SMN axons (n=6 and n=8, respectively). |