Basic fibroblast growth factor elicits formation of interstitial axonal branches via enhanced severing of microtubules.

Cell Culture and Transfection

Cultures of rat hippocampal neurons were prepared as previously described ( Qiang et al. , 2006 ; Yu et al. , 2008 ). DNA constructs were transfected into the dissociated neurons before plating, using the Amaxa Nucleofector (Amaxa Biosystems, Köln, Germany). After nucleofection, the neurons were plated onto poly- l -lysine–treated glass coverslips as previously described ( Qiang et al. , 2006 ; Yu et al. , 2008 ). For some experiments, the coverslips were also coated with laminin after the polylysine treatment because this was found to promote faster and straighter growth of axons. bFGF (Promega, Madison, WI, Cat. no. 9PIG507) was applied to some of the neuronal cultures at a concentration of 20 ng/ml ( Szebenyi et al. , 2001 ). DNA constructs included enhanced green fluorescent protein (EGFP)-α-tubulin (Clontech, Palo Alto, CA), EGFP-EB3 (provided by Dr. Niels Galjart, Erasmus Medical Center), and mCherry-spastin (prepared in our laboratory). For the spastin construct, we used the M85 isoform of spastin, which is the predominant isoform expressed in developing neurons ( Claudiani et al. , 2005 ; Connell et al. , 2008 ; Mancuso and Rugarli, 2008 ; Solowska et al. , 2008 ; Yu et al. , 2008 ). For controls, we used the appropriate constructs for EGFP or/and mCherry. For studies involving siRNA, neurons were transfected with a pool of four sequences specific to rat spastin, p60-katanin, or p80-katanin (purchased as “smartpools” from Dharmacon, Boulder, CO, Accession numbers: XM-343018 , L-080249-01 , and M-092761-00 , respectively) or with a nonspecific control sequence (Dharmacon, Cat. no. D-001206-03-20). Transfections with the siRNA smartpools were performed using the Nucleofector as previously described ( Yu et al. , 2005 , 2008 ; Qiang et al. , 2006 ). After 2 d in culture to permit protein depletion, the neurons treated with siRNA were replated so that neurites could grow anew in the near absence of the relevant protein targeted by the siRNA.

Microtubule Transport Assay

To image the transport of microtubules in axons, the neurons were transfected with EGFP-α-tubulin. In some experiments, control neurons (not treated with bFGF) were compared with neurons treated with bFGF. In other cases, control neurons (expressing mCherry) were compared with neurons expressing mCherry-spastin. The latter was to ascertain any potential changes elicited by spastin overexpression. In other experiments, neurons were transfected with control or spastin siRNA and then transfected with the EGFP-α-tubulin construct 2 d later and then replated. To assist in monitoring transport of microtubules over relatively long and straight trajectories, the neurons were plated onto poly- l -lysine with laminin (see above). Imaging was conducted 24–48 h after plating or replating. Individual unfasciculated axons with clearly identifiable directionality of growth from the cell body were chosen for analysis so that anterograde and retrograde movements could be identified with certainty. We used the Zeiss inverted Observer Z1 microscope (Carl Zeiss, Jena, Germany), with a Zeiss incubation chamber, interfaced with the MicroPoint Mosaic Digital Diaphragm system from Photonics Instruments (St. Charles, IL), coupled to an argon ion laser. The Zeiss incubation chamber provides optimal culture conditions of 37°C and 5% CO 2 . The photonic laser Mosaic system was used to photobleach regions of the axons in similar manner to our earlier studies on microtubule transport ( Hasaka et al. , 2004 ; He et al. , 2005 ; Ahmad et al. , 2006 ; Myers and Baas, 2007 ). Only one photobleached region was made on each axon analyzed. This region was always 40–50 μm in length and always was located roughly equidistant from the cell body and the tip of the axon. A 100× 1.3 NA Plan Apo oil immersion objective and a GFP filter set (Chroma Technology, Brattleboro, VT) were used for acquiring fluorescence images. The CCD camera and most aspects of the microscope were controlled by Axiovision 4.1 software (Zeiss) running on an Intel Xeon processor-based computer (Fujitsu Siemens, Berlin, Germany) with the Windows XP Professional operating system (Microsoft, Seattle, WA). The intensity of epifluorescence illumination was attenuated to 30% to reduce photobleaching and photodamage during acquisition; 2 × 2 binning was applied to increase signal intensity. Images were acquired at 1.8-s intervals, with ∼280–320-ms exposure time. Time-lapse images were saved as “zvi” files and analyzed using Axiovision 4.1 software. Motion analysis was performed only on axons that displayed at least one microtubule that was visible without digital processing. Additional analysis was conducted using digital contrast adjustments within the “Image Properties” application of Axiovision 4.1. Statistics were performed using the Student's t test.

EB3 Live-Cell Imaging

To image the assembly of microtubules from their plus ends, we expressed fluorescently tagged EB3, which is a microtubule “end binding protein” that tracks with the plus ends of microtubules during bouts of rapid assembly ( Stepanova et al. , 2003 ). We have used this approach in a number of our studies on microtubule behaviors in cultured neurons ( Hasaka et al. , 2004 ; Ahmad et al. , 2006 ; Myers et al. , 2006 ; Nadar et al. , 2008 ). To visualize EB3 comets within the axon of the cultured neurons in the experiments involving bFGF, we transfected neurons with EGFP-EB3 and cultured in medium containing bFGF. To image EGFP-EB3 within the axons of spastin-overexpressing neurons, we cotransfected neurons with both EGFP-EB3 and mCherry-spastin. Images were acquired at a rate of 1 per second excluding camera exposure times to time-lapse movies. Depending on the fluorescence intensity of EB3, exposure time ranged from 250 to 350 ms so that the real-time imaging of individual axons ranged from 2 to 4 min. As with the microtubule transport assay described above, we used the Zeiss inverted Observer Z1 microscope with the Zeiss incubation chamber, and we analyzed only one region per axon that always located roughly equidistant from the cell body and the tip of the axon. The region we analyzed was always 50–70 μm in length, a bit longer than the length analyzed in the microtubule transport studies. Details on the objective, camera, software, illumination, and binning were also the same as described above for the microtubule transport studies. EB3 quantification was performed using Axiovision “measure” module. Numbers of EB3 comets, average speeds of the comets and longevities of the comets were analyzed in details. Frames extracted for still images were exported from Axiovision as TIFF files and processed in Photoshop 7 (Adobe Photosystems, San Jose, CA). Statistics were performed using the Student's t test.

Primary Antibodies Used for Western Blotting and Immunofluorescence Analyses

Primary antibodies included the following: monoclonal: tau1 (anti-dephosphorylated tau, obtained from Dr. Lester Binder of Northwest University; Binder et al. , 1985 ), tau5 (anti-total tau, also obtained from Binder; Papasozomenos and Binder, 1987 ), anti-β tubulin directly conjugated with Cy3 (Sigma, St. Louis, MO, Cat. no. C4585), anti-GAPDH (Ambion, Austin, TX, Cat. no. AM4300); Polyclonal: anti-spastin (AAA spastin antibody; Solowska et al. , 2008 ), tauR1 (anti-total tau, obtained from Binder; Berry et al. , 2004 ), anti-P60-katanin ( Yu et al. , 2005 ), anti-P80-katanin ( Yu et al. , 2005 ), and anti-GFP (Abcam, Cambridge, MA, Cat. no. ab6556).

Western Blotting

Western blotting was conducted as in our previous studies for siRNA experiments, to ensure that over 95% of the spastin protein had been depleted before the replating step ( Qiang et al. , 2006 ; Yu et al. , 2008 ). Western blotting was also used for experiments aimed at ascertaining changes in the levels of various proteins during treatment of neurons with bFGF. For the latter, Western blotting was conducted on cultures that had been exposed to bFGF for 4, 24, or 72 h.

Immunofluorescence Analyses

Procedures of the immunofluorescence studies were performed as previously described ( Yu et al. , 2005 , 2008 ; Qiang et al. , 2006 ). For the experiments on microtubule distribution after experimental manipulations, the cultures were simultaneously fixed and extracted 24–48 h after plating. For the experiments relevant to the ratio images of tau1/tauR1, the cultures were fixed and then extracted after fixation 24–72 h after plating. Primary antibodies against proteins of interest (same as used for the Western blotting, see above; except that we used the polyclonal antibody tauR1 for staining total tau) were applied followed by appropriate secondary antibodies. Images (except those using tau1 and tauR1 antibodies) were obtained on an Axiovert 200M microscope (Carl Zeiss) equipped with a high-resolution CCD (Orca, Hamamatsu, Japan). The images were obtained using identical camera, microscope, and imaging criteria such as gain, brightness and contrast, and exposure time. In some cases, fluorescence images were subjected to the “invert” function in Adobe Photoshop (San Jose, CA) that converts blacks to whites and whites to blacks and inverts gray levels proportionally, because we found that fine details were more clearly visualized. The resulting images are referred to as “inverted images.” In one set of studies, ratio images were generated of cultures double-labeled with tau1 and tauR1 antibodies using the Pascal confocal microscope. Statistics were performed using the Student's t test.

Supplementary Material [Supplemental Material]