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First published online 27 March 2007
doi: 10.1242/jcs.001461

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The microtubule destabilizer stathmin mediates the development of dendritic arbors in neuronal cells

¹ Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan
² Laboratory of Metabolic Regulation Research, Kyushu University Bio-Architecture Center, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan
³ Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan
⁴ CREST, JST, 4-1-8 Honcho, Kawaguchi 332-0012, Japan

View larger version (58K): [in this window] [in a new window]   Fig. 1. Developmental fluctuation of stathmin expression in Purkinje cells (PCs) and phosphorylation of stathmin at Ser16 in cerebellum. (A) Schematic representation of domain organization of stathmin. N-terminal regulatory domain contains four serine residues as phosphorylation sites. The C-terminal interaction domain has two helical structures and interacts with two /-tubulin heterodimers. (B) PC collection from P12 or P15 frozen cerebellar sections (30 µm thick, stained with Toluidine Blue) using the Laser Capture Microdissection System, LM2000. (–) PC (left photo) shows a section after laser treatment, with the PCs removed. PC (right photo) shows captured PCs. (C) PC-specific differential display (PC-DD) showing downregulation of stathmin expression between P12 and P15. Total cellular RNAs were prepared independently from the PCs of four animals (two at P12 and two at P15) and subjected to PCR-differential display. Arrowhead indicates stathmin cDNA, corresponding to nucleotides 782 to 963 of mouse stathmin 1 (GenBank accession number: NM019641). (D) In situ hybridization was carried out with a stathmin antisense probe and frozen cerebellar sections (10-µm thick). Brains were dissected at P12, P15 and P18. Lobule 4-5 (L4-5) is shown. (E) The average signal intensity of stathmin mRNA in PC somata of L4-5 was quantified. Data from each time point were obtained from 114-128 cells from two animals. Data are shown as mean ± s.e.m. P values of Student's t-test are given in the graph. (F) Immunohistochemical analysis of stathmin level in cerebellar cortex at P12 and P18. Micrographs of brain sections from P12 and P18 mice mounted on the same slide were photographed on a laser-scanning confocal microscope under the same conditions. Rightmost panels are higher-magnification images of merged micrographs. Thus, the intensities of the stathmin signals from P12 and P18 can be compared quantitatively. ML, molecular layer; PCL, PC layer; EGL, external granule cell layer; IGL, internal granule cell layer. Bars, 50 µm (D,F). (G,I) Immunoblotting of whole cell lysate prepared from P12, P15 and P18 cerebellum. Anti-stathmin antibody and anti-phospho-stathmin (Ser16) antibody were used in G and I, respectively. An almost equal amount protein was loaded in each lane. Alpha-tubulin provided a loading control. (H,J) Signal level normalized to -tubulin level was quantified from three independent samples of different animals at each time point. Error bars indicate mean ± s.e.m.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Extension of stabilized MTs in PC dendrites is inversely correlated with the stathmin mRNA level. (A) Immunohistochemistry of MAP2 and acetylated -tubulin. Brains were dissected at P9, P12, P15, or P18. Lobule 4-5 (L4-5) is shown. Abbreviations are the same as in Fig. 1. Bar, 50 µm. (B) `Signal length' of MAP2 and acetylated (Ac) -tubulin along the PC dendrites was quantified per area of the molecular layer of L4-5 using a total of 18 images from three animals for each time point. Images used for quantification were two views from three serial and sagittal sections (10 µm thick each) per an animal. Purkinje cells flatly extend the dendrites along the sagittal plane, and it is predicted that the whole dendrites of each Purkinje cells are included in the three serial sections. The signal intensity of stathmin mRNA in PC somata is the same as that shown in Fig. 1E. Error bars indicate mean ± s.d.

View larger version (80K): [in this window] [in a new window]   Fig. 3. Stathmin overexpression dramatically limits dendritic growth of cultured PCs. (A) Schematic representation of GFP and stathmin expression constructs for PC-specific overexpression. GFP, expression of Gfp is controlled by partial L7 promoter sequences downstream of the CMV promoter (see Materials and Methods). Sta, a stathmin-Myc cDNA was inserted into the L7 gene cassette (Ichise et al., 2000; Oberdick et al., 1990). The expression constructs for the stathmin mutants have the same structure except for the mutation points. Note that the drawings are not to scale. CMV promoter, 0.6 kb; stathmin-Myc, 0.5 kb; L7 gene cassette, 3.0 kb; GFP construct contains 1.3 kb of the L7 gene sequence upstream of the initiation codon located in exon 2. (B) Fluorescence micrographs of GFP- and stathmin-overexpressing PCs in primary culture. Cells were transfected at DIV-0 and fixed at DIV-14. KN-93 (5 µM) was supplied from DIV-7 to -14. Calbindin (red) was used as a PC marker. Expression of GFP or Myc-tagged stathmin is shown in green. Lower-magnification images are shown to the right of the red line, with arrowheads indicating axons. Bars, 50 µm. (C) KN-93 affects dendritic arborization. Cells were treated with 5 µM KN-93 from DIV-7 to -14. Immunohistochemistry was performed with an anti-calbindin antibody to detect PCs. Arrowheads indicate an axon originating from a PC soma (asterisk). Bar, 50 µm. (D,E) Quantification of total dendritic branch length of GFP- and stathmin-expressing PCs in primary culture (D), and number of primary dendrites of cells transfected with GFP and various forms of stathmin (E). Number of PCs: GFP no-treatment, n=39; GFP KN-93, n=19; Sta, n=52; 4A, n=36; S16A, n=21; S16E, n=21. Error bars indicate mean ± s.e.m. *P<0.05; ***P<0.001 versus GFP no-treatment. #P<0.05; ##P<0.01 versus Sta. +P<0.05; +++P<0.001 versus S16E. Student's t-test.

View larger version (86K): [in this window] [in a new window]   Fig. 4. Phosphorylation of stathmin at Ser16 is regulated by neural activity in PCs. Immunocytochemistry of primary cultured PCs at DIV-9 was carried out with anti-calbindin and anti-stathmin-phospho-Ser16 antibodies. (A) KN-93 (5 µM) or TTX (1 µM) was supplied from DIV-7 to -9. (C,E) PC cultures were treated with DMSO, 10 µM nifedipine (C), or 30 µM CPCCOEt (E) from DIV-7 to -9. Bars, 20 µm. (B) Quantification of stathmin-phospho-Ser16 signal intensity. The average signal intensity of phospho-Ser16 was normalized to that of calbindin. (D,F) Signal intensity of stathmin-phospho-Ser16 was quantified as in B. Data were obtained from three sister cultures, and P values derived from Student's t-test are shown in the graphs. n, number of PCs measured. In addition to PCs, our culture contained granule cells and glial cells. We therefore employed immunocytochemistry, rather than western blot, to measure the level of phospho-Ser16 in PCs. (G) Immunocytochemical analyses of primarily cultured PCs at DIV-9 were performed with anti-calbindin and anti-stathmin antibodies. Treatment with each reagent was performed as in A, C and E. There are no obvious differences in the stathmin signal level in PCs. Bars, 20 µm. (H) Signal intensity of stathmin was quantified as in B. n, number of PCs measured.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Knockdown of endogenous stathmin reduces dendritic growth of PCs. (A,B) Efficacy of stathmin knockdown by siRNAs in rat PC12 cells. (A) Immunoblotting of whole cell lysate from PC12 cells transfected with the indicated siRNAs. Alpha-tubulin provided a loading control. (B) Knockdown efficacy was quantified from three independent experiments. (C) Typical images of PCs in primary culture transfected with control siRNA and stathmin siRNA-2. Alexa Fluor-546-conjugated siRNAs were used in this experiment. Arrowheads indicate soma of transfectant. Bars, 50 µm. (D) Quantification of total dendritic branch length of PCs transfected with control siRNA or stathmin siRNA-2. Error bars indicate mean ± s.e.m. A P value from Student's t-test is shown in the graph.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Overexpression of wild-type SCG10 or an S50A mutant similarly reduced dendritic growth of cultured PCs. (A) Subcellular distribution of stathmin and SCG10 in cultured PCs at DIV-9. (B) High-magnification micrographs of SCG10- or S50A-overexpressing PCs in primary culture. The micrographs are merged images of calbindin (red) and Myc (green) signals. Both endogenously and ectopically expressed SCG10 localize mainly in the perinuclear region (arrows). (C) Immunocytochemistry of GFP-, SCG10-, or S50A-overexpressing PCs in culture at DIV-14 as described in Fig. 3B. Arrowheads demarcate the axons of transfectants. (D) Quantification of total dendritic branch length of GFP-, SCG10-, and S50A-overexpressing PCs in primary culture. Number of cells measured: GFP, n=26; SCG10, n=20; S50A, n=23. Data are presented as mean ± s.e.m. Bars, 20 µm (A,B), 50 µm (C).