The culture supernatant of stably transfected human embryonic kidney (HEK)293 cells secreting Sema3A (generous gift from Dr. M. Tessier-Lavigne, Genentech, Inc., South San Francisco, CA) was concentrated by ultrafiltration (Centriplus membrane, 50,000-mol. wt. cut-off; Millipore, Billerica, MA). Sema3A concentration was calibrated by comparing the degree of collapse response to that of a known standard. Special reagents and their sources were 12(S)-HETE (BIOMOL Research Laboratories, Plymouth Meeting, PA); bisindolylmaleimide I (Bis) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (EMD Biosciences/Calbiochem, San Diego, CA); culture media, media supplements, and TRIzol reagent (Invitrogen, Carlsbad, CA); GC-Melt, EGFP-N1 vector, and pIRES-GFP vector (Clontech, Mountain View, CA); and small interfering RNAs (siRNAs) (Dharmacon RNA Technologies, Lafayette, CO). Antibody specificities and their sources were α3-integrin (developed by L. Reichardt, University of California, San Francisco, CA) (Developmental Studies Hybridoma Bank, maintained under the auspices of the National Institute of Child Health and Human Development by Department of Biological Sciences at the University of Iowa, Iowa City, IA); MARCKS (antibody used for Western blots) and lamin A and C (Santa Cruz Biotechnology, Santa Cruz, CA); MARCKS (antibody used for immunofluorescence and blot in Supplemental Figure 1) (Proteintech Group, Chicago, IL); β1-integrin and PKCε (BD Biosciences, Franklin Lakes, NJ); MARCKS phosphorylated in the ED (P-MARCKS) (Cell Signaling Technology, Beverly, MA); and tubulin β III (Abcam, Cambridge, MA). Additional chemicals, unless stated otherwise, were from Sigma-Aldrich (St. Louis, MO) and of the highest quality available.
Growth Cone Isolation
Growth cone particles (GCPs) were prepared as described previously (Pfenninger et al., 1983
; Lohse et al., 1996
). Briefly, whole brains from fetal rats (18-d gestation) were homogenized in 0.32 M sucrose containing 1 mM MgCl2
, 2 mM TES buffer, pH 7.3, and 2 μM aprotinin. Low-speed (1660 × g
for 15 min) supernatant of the homogenate was layered onto a discontinuous density gradient (0.83 and 2.66 M sucrose containing 1 mM MgCl2
and 2 mM TES) and spun to equilibrium at 242,000 × g
at 4°C for 40 min in a vertical rotor (VTi50; Beckman Coulter, Fullerton, CA). GCPs at the 0.32/0.83 M sucrose interface were collected, diluted with ~5–10 volumes 0.32 M sucrose buffer, pelleted (40,000 × g
for 30 min), and then resuspended in an appropriate buffer depending upon subsequent experimentation.
MARCKS Phosphorylation and Translocation Assays
Pelleted GCPs (60–100 μg of protein per reaction) were resuspended in 100 μl of ice-cold kinase buffer (20 mM HEPES, pH 7.0, 10 mM MgCl2
, and 1 mM EGTA; Mikule et al., 2003
). Effectors were added; after 10 min on ice, reaction mixtures were incubated at 30°C for 2 min and then chilled on ice. In experiments with PKC inhibitor, 10 nM Bis was added 10 min before the effector. Samples were homogenized (Teflon-glass) and separated into particulate (membrane/cytoskeleton) and cytosolic fractions by centrifugation at 100,000 × g
for 30 min at 4°C. The resulting pellets were solubilized in 20 μl of 5% SDS. Protein in supernatant fractions was precipitated with chloroform/methanol, and these pellets also were solubilized in 5% SDS. After addition of Laemmli sample buffer, polypeptides of all samples were resolved by SDS-PAGE, blotted, and probed with antibody to MARCKS or to P-MARCKS (see below).
Gel Electrophoresis and Western Analysis
Polypeptides were resolved by SDS-PAGE along side with dual-colored Precision Plus Protein standards (Bio-Rad, Hercules, CA). Blots were prepared by wet electrotransfer (Towbin et al., 1979
) to a polyvinylidene difluoride membrane (Immobilon P; Millipore). They were blocked in Tris-buffered saline (TBS) with 5% nonfat evaporated milk and 0.1% Tween 20 for at least 2 h at room temperature. After quenching, blots were incubated in blocking buffer containing primary antibodies for 1 h, rinsed (three times) in the same buffer, and incubated in blocking buffer containing Cy5-conjugated secondary antibody (Invitrogen), rinsed (three times) in TBS/Tween, and then scanned using a Typhoon 9400 multi-mode imager (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).
Cloning, Vector Construction, and Small Interfering RNA (siRNA)
Total RNA was isolated from fetal rat brain (18-d gestation) by using TRIzol reagent. cDNA encoding rat MARCKS was generated by reverse transcriptase-polymerase chain reaction (RT-PCR) by using the purified RNA, Ready-To-Go RT-PCR beads (PerkinElmer Life and Analytical Sciences, Boston, MA), and the following primers: forward, 5′-ccgctcgagatgggtgcattctcc-3′ and reverse, 5′-cccaagcttctcggccaccggcgcgg-3′.
Due to the high gc-content of these primers and of MARCKS, we added GC-Melt during the PCR cycles. The RT-PCR product was then ligated into the EGFP-N1 vector. Plasmid containing the MARCKS-ED mutant was a generous gift from Dr. Alan Aderem (Institute for Systems Biology, Seattle, WA). The coding sequence for MARCKS-ED was excised and subcloned into the bicistronic pIRES-GFP vector, which expresses both the inserted protein and green fluorescent protein (GFP) (for the identification of neurons expressing mutant MARCKS) under the control of the cytomegalovirus promoter. Coexpression was confirmed in transfected HEK293 cells by indirect immunofluorescence microscopy (our unpublished data). Correctness of all constructs was established using restriction digests and Big Dye sequencing (Barbara Davis Center for Childhood Diabetes, DNA Sequencing Core, University of Colorado at Denver and Health Sciences Center, Aurora, CO).
Rat MARCKS siRNA was custom synthesized by the SMARTPool siRNA design service of Dharmacon RNA Technologies. The lamin A and C control siRNA also was purchased from Dharmacon RNA Technologies. The pmaxGFP plasmid used for cotransfection was purchased with the Nucleofector kit (Amaxa Biosystems, Gaithersburg, MD).
Neuron Culture and Transfection
For explant cultures, dorsal root ganglia (DRGs) were dissected from 15-d gestation Sprague-Dawley rat fetus and cultured on laminin-coated coverslips (Assistent brand) in B27/Neurobasal medium supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 100 ng/ml nerve growth factor (NGF). For some of the collapse assays shown in , we also used poly-d-lysine (polylysine)-coated coverslips for culture. After 24-h incubation at 37°C, 4% CO2 in air, this medium was replaced with fresh B27 medium without other supplementation. After a second day in culture, neurites with spread growth cones were used for turning assays and indirect immunofluorescence experiments as described below.
Figure 1. Role of adhesion in growth cone repulsion. (A) Effect of the substrate on Sema3A-mediated growth cone collapse. Growth cones of DRG neurons were cultured either on laminin- or polylysine-coated surfaces. Phase contrast micrographs were taken just before, (more ...)
For experiments requiring transfection, excised DRGs from 10 to 12 fetal rats were dissociated. In some cases, 5 mg/ml dispase and 1 mg/ml collagenase in modified Hank's balanced salt solution were used first (25 min at 37°C). The partially digested or the fresh ganglia were pelleted, and the supernatant was replaced with trypsin/EDTA. After 15 min, ganglia were washed in B27 medium with serum, triturated, and cells were counted. Dissociated cells (2–3 × 106) were pelleted and resuspended in 100 μl of Nucleofector solution (Amaxa) with 5 μg of DNA or 0.4 μM siRNA plus 2.5 μg of pmaxGFP (Amaxa) and electroporated using the Amaxa Nucleofector device per the manufacturer's instructions (setting O-003). Transfected neurons were cultured on laminin in B27 medium with FBS and NGF, replaced every 24 h. Experimentation was conducted in the presence of both FBS and NGF.
Knockdown of MARCKS expression in neurons could not be assessed by Western blot because the transfection efficiency was only ~30% in these cultures. However, growth cone MARCKS immunofluorescence shown in is represented at exactly the same enhancement and contrast levels so that direct comparisons are possible.
Figure 6. Silencing MARCKS expression in DRG neurons. All experiments included a GFP-encoding plasmid to identify transfected cells. Cells were immunostained for MARCKS (red). (A and B) siMARCKS, 12 h. Transfected neurons contain reduced but variable amounts of (more ...)
Growth Cone Turning Assays
Sema3A gradients were generated in the proximity of cultured nerve growth cones by repetitive pulse application (Lohof et al., 1992
). Micropipettes (inner tip diameter consistently 1–2 μm) were connected to a Picospritzer (set at 6 psi; General Valve, Fairfield, NJ) controlled by a square wave generator (2 Hz, duration 10 ms; Astro-Med, West Warwick, RI). The system was calibrated by generating a model gradient of fluorescein-conjugated dextran. Such gradients proved reproducible and stable over time.
For turning assays, culture coverslips were placed in an open chamber with medium, layered over with inert mineral oil (embryo tested, sterile filtered; Sigma-Aldrich) to avoid evaporation and maintain pH, and observed on the microscope stage under convective heating at a constant 37°C. For interference reflection microscopy (IRM) imaging, we additionally used an objective heater (set at 36°C) in conjunction with the oil immersion lens. At the start of each experiment, the tip of the factor-loaded micropipette was positioned 100 μm away from the selected growth cone, at an angle 45° from the initial direction of growth cone advance (as determined by the orientation of the growth cone's neurite shaft). Initiation of factor expulsion marked the start (time t = 0) for each experiment. Phase-contrast images were captured at 5-min intervals over the course of 1 h. To be scored, growth cones had to advance a minimal distance of at least twice their original length. Once this criterion had been met, growth cones were tracked for 1 h or until they either stopped (i.e., no advancement for ≥10 min) or branched. Statistical significance of final turning angles was determined using Student's t test (assuming equal variances). For sequences involving IRM, micrographs were acquired at 1- or 2-min intervals for 30–45 min.
All images were acquired using a Zeiss Axiovert 200M microscope equipped with Zeiss optics, a Cooke Sensicam digital camera, and Slidebook software (Intelligent Imaging Innovations, Denver, CO). The following objective lenses were used: Zeiss Plan-Apochromat 63×/1.4 oil for epifluorescence and IRM; Zeiss Plan-NeoFluar 63×/1.25 oil for phase contrast. To generate digitally deconvolved images, we applied the nearest neighbor algorithm of Slidebook to images taken at 0.2- to 0.3-μm intervals through the sample. Images shown are from the first optical slice exhibiting fluorescence as the plane of focus moved from the coverslip into the growth cone. Images were adjusted for brightness and contrast with Adobe Photoshop software (Adobe Systems, Mountain View, CA). However, fluorescence levels are shown so they can be compared (see above), unless indicated otherwise. For IRM, optics were calibrated by acquiring color images with a tunable RGB filter (CRI MicroColor, Cambridge, MA) to identify and minimize contributions from higher order interference. To objectively determine growth cone close contact area, mean intensity of a 100 × 100 pixel region near the growth cone was measured as background using MetaMorph software (Molecular Devices, Sunnyvale, CA). IRM images were thresholded to include only pixels with intensities less than 2 SDs below the mean background intensity of the 100 × 100 pixel region. The area of thresholded pixels within the periphery of the growth cone was then determined and reported as adhesive or close contact area.
Growth Cone Collapse Assays
DRG neurons on laminin-coated coverslips were placed under a convective heater on the microscope stage as described above. Factors were introduced into the medium with a syringe and needle, and images were acquired at specific intervals thereafter. The thresholding function in Slidebook was used to measure growth cone areas. Student's t test (assuming equal variances) was used to determine statistical significance of observed differences.
Fixation and Labeling of Growth Cones in Culture
DRG cultures were fixed using slow infusion of 4% (wt/vol) formaldehyde in 0.1 M phosphate buffer, pH 7.4, with 120 mM glucose and 0.4 mM CaCl2
, as developed for electron microscopy (Pfenninger and Maylie-Pfenninger, 1981
). Cultures were rinsed (three times) with phosphate-buffered saline (PBS) containing 1 mM glycine, permeabilized with blocking buffer [PBS, 1% (wt/vol) bovine serum albumin] plus 1% (vol/vol) Brij 98 detergent for 2 min at room temperature, and placed in blocking buffer for 1 h at room temperature. Quenched cultures were incubated with primary antibody at 1:100 dilution in blocking buffer, for 1 h at room temperature, and washed (three times) with blocking buffer. This process was repeated with Alexa Fluor 488- (green), 594- (red), or Marina-blue (blue)-conjugated secondary antibodies (Invitrogen) at the same dilution. In some experiments that required dual fluorescence labeling of antigens in GFP-expressing growth cones, we used secondary antibodies conjugated to Alexa Fluor 594 and 647. Coverslips were mounted on slides by using a polyvinyl alcohol/glycerol medium containing n
-propyl gallate as antifade reagent.
We made significant efforts to generate IRM and immunofluorescence images from the same growth cones. However, even very mild fixation with 1% formaldehyde or 0.1% glutaraldehyde at 4°C for as little as 60 s, followed by immediate quenching of aldehyde groups, resulted in extensive artifactual “close contacts” that covered most of the growth cone area. Thus, it seemed that even minimal protein cross-linking, needed for growth cone preservation and labeling, altered the IRM images significantly so that colocalization of close contacts with integrins and/or MARCKS in the same growth cone was impossible.
Colocalization Analysis and Morphometry
Digitally deconvolved (nearest neighbor) images of growth cones were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). To exclude contributions of background noise in an unbiased manner, we proceeded in two ways: 1) We thresholded all images by limiting the eight-bit display range to 10–255 and calculated Manders' coefficient (R) by using the Manders' coefficient plug-in. 2) Alternatively, we performed automatic threshold calculation in conjunction with overlap analysis (Manders' coefficient RT
) according to Costes et al. (2004)
. For this, we used the “Colocalization Threshold” plug-in (see the ImageJ Web site established by the Wright Cell Imaging Facility, Toronto Western Research Institute, Toronto, Ontario, Canada; www.uhnresearch.ca/wcif
). In both cases, zero/zero pixels were excluded from the quantitation.