Attachment toan Endogenous Laminin-like Protein Initiates Sprouting by Leech Neurons

Leech neurons in culture sprout rapidly when attached to extracts from connective tissue surrounding the nervous system. Laminin-like molecules that promote sprouting have now been isolated from this extracellular matrix. Two mAbs have been prepared that react on immunoblots with a =220and a =340-kD polypeptide, respectively. These antibodies have been used to purify molecules with cross-shaped structures in the electron microscope. The molecules, of =103 kD on nonreducing SDS gels, have subunits of =340, 220, and 160-180 kD. Attachment to the laminin-like molecules was sufficient to initiate sprouting by single isolated leech neurons in defined medium. This demonstrates directly a function for a laminin-related invertebrate protein. The mAbs directed against the =220-kD chains of the lamininlike leech molecule labeled basement membrane extracellular matrix in leech ganglia and nerves. A polyclonal antiserum against the =220-kD polypeptide inhibited neurite outgrowth. Vertebrate laminin did not mediate the sprouting of leech neurons; similarly, the leech molecule was an inert substrate for vertebrate neurons. Although some traits of structure, function, and distribution are conserved between vertebrate laminin and the invertebrate molecule, our results suggest that the functional domains differ. N "EURONS from the central nervous system of the leech, Hirudo medicinalis, grow to regenerate after injury and form synapses in the animal as well as in tissue culture (for reviews see references 26, 28). From this simple nervous system, single cells can be isolated and cultured for several weeks (7). Leech neurons are especially useful because they can grow neurites on certain substrates and form synapses in defined media (1, 3, 7). This allows us to dissect the mechanism of regeneration into distinct steps that can be studied separately (5). To isolate physiologically relevant molecules from leech tissues, functional assays were established for single cultured neurons. Focusing on the mechanism of neurite outgrowth, it was found that only a few substrates, including leech extracellular matrix (ECM), t mediate sprouting by identified leech neurons in culture; no other cells or soluble growth factors are needed (3). We have recently demonstrated that neurite-promoting activity is concentrated in high molecular mass fractions isolated from leech ECM (3). Substrates coated with the vertebrate basement membrane protein, laminin, are known to promote neurite formation by vertebrate neurons (8). Recently, molecules that are related to laminin have been described in some invertebrates (14, Dr. Beck's present address is the Institute for Biophysics, Johannes Kepler University, 4040 Linz, Austria. 1. Abbreviations used in this paper: ECM, extracellular matrix; EHS, Englebreth-Holm swarm; NGF, nerve growth factor; TES buffer, TrisHCI/EDTA/saline. 25). We therefore suspected that a laminin-like component from our leech ECM extracts (5) could be at least in part responsible for the observed activity. In this paper, we report the isolation of laminin-like leech molecules using mAbs. This enabled us to determine their neurite-promoting activity. We asked whether attachment to these molecules was sufficient to initiate the sprouting of isolated leech neurons, and whether they were the major or merely one of several neurite-promoting components in leech ECM. We also tested whether vertebrate laminin and the leech molecules would promote the sprouting of neurons across phyla. Finally, the mAb allowed us to study the distribution of laminin-like molecules within the leech central nervous system. Materials and Methods Preparation of ECM Extracts Ganglion capsule ECM was prepared from leeches (H. medicinalis; Ricarimpex, Audenge, France) as described (3). Briefly, chains of ganglia that make up the central nervous system of adult leeches were dissected in Ringer solution (26) and transferred to 10 mM Tris-HCl buffer (pH 7.4) conmining 2% Triton X-100 (E. Merck GmbH, Darmstadt, Federal Republic of Germany) and protease inhibitors (3). Ganglia were crushed first with forceps and then with a Dounce homogenizer and extracted overnight at 4°C in an excess of buffer. The insoluble material was sedimented at 10,000 g (5 rain) and washed three times with distilled water. The pellet was extracted for 24 h at 4°C with 4 M urea (100 Ixl per ganglion chain) as described (3), or alternatively with 150 mM NaCI, 10 mM EDTA, 10 mM Tris-HC1, pH 7.4 (TES buffer; 50 I.tl per ganglion chain). No protease inhibitors were included in the extraction buffers because of toxic effects in subsequent bioassays. The amount of protein extracted from one ganglion chain © The Rockefeller University Press, 0021-9525/88/0911189/10 $2.00 The Journal of Cell Biology, Volume 107, September 1988 1189-1198 1189 on A uust 0, 2017 jcb.rress.org D ow nladed fom was 15-25 I.tg (EDTA extract) or =50 I.tg (urea extract) as estimated from A2s0 assuming an average absorption coefficient of A280 = 1,000 cm 2 g-1. Isolation of mAbs 203 and 206 Urea extracts from leech ganglion capsules (3) were dialyzed against 150 mM NaC1, 20 mM sodium phosphate, pH 7.3 (PBS). BALB/c mice (Madtrin AG Kleintierfarm, Fiillinsdorf, Switzerland) were injected intraperitoneally with 100 I.tl extract (50 I.tg antigen) which was suspended in 100 Ixl complete Freund's adjuvant (Gibco Laboratories, Grand Island, NY). Mice were boosted after 1 mo with 100 lal dialyzed extract injected into the tail vein. This was repeated 2 d later, and after tv~ more days the spleens were removed. Lymphocytes isolated from each spleen were fused (13) with 5 × 107 PAl myeloma cells (33). Resulting hybridoma lines were grown as described (16). Culture supernatants were screened by an ELISA (12) on microtiter wells (No. 3911; Falcon Labware, Oxnard, CA) coated with 200fold diluted antigen extract. 150 hybridoma lines from two fusions yielded 0 . 5 > 2 . 0 0 D (488 rim) above background in this assay; these were rescreened on whole mounts of fixed and permeabilized leech ganglia (1). The supernatants of 70 lines clearly stained capsule ECM. These were tested by immunoblotting for their reactivity with specific polypeptides found in neurite-promoting ECM fractions (3). Selected hybridoma lines were cloned by serial dilution (16) and after rescreening, the clones were injected into pristane-primed (Aldrich Chemical Co., Milwaukee, WI) BALB/c mice to generate ascites tumors. Mice were killed and ascites fluid was collected 10 d later. Immunoglobulins were precipitated with 50% saturated ammonium sulfate, dissolved in the original volume, dialyzed against PBS, Millipore filtered, and stored at -70°C. Single immunoglobulin batches obtained from clones 203-1B5 and 206-1H1 were used. On SDSpolyacrylamide gels, mAbs 203 and 206 revealed one heavy chain (=50 kD) and one light chain; they are IgGs (not shown). Culture supematants from all screened subclones of hybridomas 203 and 206 gave identical staining patterns by immunofluorescence and immunoblotting when compared to the original lines, thus confirming their monoclonal origin. Other lmraunoreagents and Specificity Controls A polyclonal antiserum (No. 99) against a =220-kD polypeptide from neurite-promoting extracts was generated as follows. Ganglion capsule urea extract (3) was run on a preparative SDS-polyacrylamide gel under reducing conditions. The =220-kD region was cut out, washed briefly with distilled water, and minced in a Dounce homogenizer. Two rabbits were injected subcutaneously, each with material obtained from 10 ganglion chains suspended in complete Freund's adjuvant. A boost was made with the same amount of antigen in incomplete adjuvant after 1 mo, and the rabbits were killed and the serum was collected 1 wk later. Several control experiments were done to ascertain the target specificity of mAbs 203 and 206 and antiserum No. 99. To account for effects of nonspecific IgG binding, the unrelated mAb M1 (against chick tenascin [4]) and rabbit preimmune serum were used. These gave very low or no background in all assays described. We also used other immunoreagents as positive controls, mAb 65 reacted broadly with many polypeptides present in leech ECM extract. Antiserum No. 101 was prepared against and strongly bound to the =70-kD bands present in ECM extracts, but did not inhibit sprouting (data not shown). SDS Gel Electrophoresis and Immunoblotting Extract samples and column fractions were analyzed by SDS-PAGE according to Laemmli (20) with reagents from Bio-Rad Laboratories (Richmond, CA). Samples were run with or without reduction by 0.1 M dithiothreitol (DTT; Sigma Chemical Co., St. Louis, MO) on 7.5 % straight or 3-15 % gradient acrylamide gels. Two-dimensional gels (nonreduced vs. reduced) were run by cutting gel lanes from a gradient gel run under nonreducing conditions, turning them by 90 °, and placing them on top of a second gel. Lanes were overlaid with sample buffer containing DTT and run in the second dimension. All gels were stained with 0.2% Coomassie Brilliant Blue (Bio-Rad Laboratories). Molecular mass standards were obtained from Sigma Chemical Co. (6-H); calibrations (x l0 -3 kD) are indicated at left of each figure. Immunoblotting was done as described (36). Briefly, polypeptides were transferred from unfixed SDS gels onto nitrocellulose paper (BA-85; Schleicher & Schuell, Inc., Dassel, FRG). Free protein binding sites were blocked with PBS containing 10% horse serum (North American Biologicals, Miami, FL) and the sheets were incubated serially (each step for 1 h at 37°C) with mAb or antiserum (diluted 1:200), goat anti-mouse or anti-rabbit Ig (diluted 1:100, Cappel Laboratories, Cochranville, PA), and mouse or rabbit peroxidase-antiperoxidase complex (diluted 1;3,000; Jackson Immunoresearch Laboratories, Avondale, PA). PBS-horse serum was used to dilute the reagents and to wash between the incubations. The blots were developed with 4-chloro-l-naphthol (E. Merck GmbH) as published (18). lmmunoaffinity Purification About 3 nag mAb 203 or 206, respectively (1 ml of the ammonium sulfate cut of ascites fluid), were coupled to 1 g CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) as recommended by the manufacturer. Typically, 0.5 ml EDTA extract was applied to a column containing 0.5 ml Sepharose coupled with antibody 203. The column was washed with TES buffer, then with a mixed detergent buffer (0.3 M NaCI, 0.1% SDS, 0.05% Triton X-100, 10 mM Tris-HCl, pH 8.4) followed by TES buffer. Bound antigen was eluted either with 0.1 M triethylamine, pH 11.0, or with 4 M urea containing 10 mM EDTA and 10 mM Tris-HC1, pH 7.4. Both types of eluates looked identical upon SDS gel electrophoresis. Neurite-promoting activity could only be restored from the urea eluates after dialysis against TES buffer. Material which did not bind to the antibody 203 column was reapplied to a 0.5-ml column of antibody 206-Sepharose. In this case, adsorbed antigen was released by the mixed detergent buffer (see above). Protein concentration in the column eluates was estimated by comparing Coomassie Blue-stained gel bands to a mouse laminin standard (reference 31; gift of Dr. M. Paulsson, Biocenter, University of Basel, Basel, Switzerland). Electron Microscopy of ECM Fractions Fractions from antibody affinity columns were examined by EM using the rotary shadowing technique as described (32). Samples were dialyzed against 0.2 M ammonium bicarbonate ('AnalaR'-grade; BDH Chemicals Ltd., Poole, England), pH 7.9, mixed with an equal volume of 87 % glycerol (E. Merck GmbH) and sprayed onto freshly cleaved mica sheets (11). Samples were dried in vacuo and rotary shadowed (120 rpm) using an electron beam evaporator in a Bahers BAF 300 or BAE 307 D, respectively, with platinum-carbon at 5-7 ° to a thickness of 0.6-0.7 nm followed by carbon at 90 °. The replicas were floated on distilled water, picked up on 400-mesh copper grids, and examined with an electron microscope (model 109; Carl Zeiss GmbH, Oberkochen, FRG) (80 kV) at a magnification of ,'~50,000. Calibration was performed regularly with negatively stained catalase. The dimensions of all structures were measured at a total magnification of •500,000 using a graphics tablet. Immunohistochemistry For whole-mount preparations, leech ganglia were fixed with 4% paraformaldebyde and permeabilized with 0.4% Triton X-100 as described (1). For sectioning, unfixed ganglia were embedded in Tissue Tek (Miles Laboratories, Inc., Elkhart, IN) and frozen on dry ice. 10-I.tm sections prepared on a cryostat microtome (model 2,000; Reichert Jung, Vienna, Austria) were mounted on gelatin-coated slides and routinely stained with ascites or antiserum diluted 1:200 with PBS containing 10% horse serum (KC Biological Inc., Lenexa, KS), followed by rhodamine-conjugated second antibodies (Cappel Laboratories) (4). Neuronal Cell Cultures Retzius and Anterior Pagoda neurons were isolated from leech (H. medicinalis; Ricarimpex) ganglia and cultured at 20°C as described (3, 7) in either standard medium containing 2% FCS (KC Biological Inc.) (7) or in protein-free Leibovitz-15 medium (Gibco Laboratories) supplemented with 30 mM glucose and 2 mM L-glutamine. For antibody inhibition experiments, serum-containing media were supplemented with heat-inactivated (56°C, 30 min) antiserum No. 99 or preimmune serum at a dilution of 1:50. Dorsal root ganglion neurons were isolated by Dr. H. Vedder (Biocenter, University of Basel) from newborn rats (Madfrin AG Kleintierfarm) as described (2) and cultured at 37°C and 5% CO2 in Ham's F12/DME (1:1) containing N2 supplement (2) with 20 ng/m113-nerve growth factor (NGF) (8). This NGF concentration is high enough to ensure neuronal survival for at least 2 d, but induces very little neurite outgrowth on polylysine-coated control dishes (8), thus allowing detection of substrate-mediated neurite outgrowth by the vertebrate neurons. Cell culture microwe}l dishes (No. 3034; The Journal of Cell Biology, Volume 107, 1988 1190 on A uust 0, 2017 jcb.rress.org D ow nladed fom Falcon Labware) were coated with leech ECM fractions or 100 p.g/ml mouse Englebreth-Holm swarm (EHS) tumor laminin-nidogen complex (reference 29; gift of Dr. M. Paulsson, Biocenter, University of Basel) diluted in TES buffer for 2 h at room temperature (5 p.l per well). After washing the dishes three times with sterile water, dishes were ready for use.