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© The Rockefeller University Press,
0021-9525/1998//815 $5.00
The Journal of Cell Biology, Volume 142, Number 3,
, 1998 815-825
Regular Articles |
Overexpression of a Neural-specific Rho Family GTPase, cRac1B, Selectively Induces Enhanced Neuritogenesis and Neurite Branching in Primary Neurons
Institute of Hormone Chemistry, National Research Council, 20133 Milano, Italy
Rho family GTPases have been implicated in cytoskeletal reorganization during neuritogenesis. We have recently identified a new gene of this family, cRac1B, specifically expressed in the chicken developing nervous system. This GTPase was overexpressed in primary neurons to study the role of cRac1B in the development of the neuronal phenotype. Overexpression of cRac1B induced an increment in the number of neurites per neuron, and dramatically increased neurite branching, whereas overexpression of the highly related and ubiquitous cRac1A GTPase did not evidently affect neuronal morphology. Furthermore, expression of an inactive form of cRac1B strikingly inhibited neurite formation. The specificity of cRac1B action observed in neurons was not observed in fibroblasts, where both GTPases produced similar effects on cell morphology and actin organization, indicating the existence of a cell type-dependent specificity of cRac1B function. Molecular dissection of cRac1B function by analysis of the effects of chimeric cRac1A/cRac1B proteins showed that the COOH-terminal portion of cRac1B is essential to induce increased neuritogenesis and neurite branching. Considering the distinctive regulation of cRac1B expression during neural development, our data strongly support an important role of cRac1B during neuritogenesis, and they uncover new mechanisms underlying the functional specificity of distinct Rho family GTPases.
Key Words: small GTPases • neurites • cytoskeleton • actin • development
Abbreviations used in this paper: CEF, chicken embryo fibroblast; E6, embryonic day 6; F-actin, filamentous actin; GST, glutathione-S-transferase; PEI, polyethylenimine; RGM, retinal growth medium.
NEURITOGENESIS is a central event during neuronal development, and it can be considered as a particular form of cell motility, in which actin dynamics during growth cone navigation evolves into stabilization of the cytoskeleton and neurite elongation (Tanaka and Sabry, 1995). Therefore, the behavior of growth cones can be compared with that of the leading edge of spreading or migrating fibroblasts, where the dynamic adhesive interactions with the substrate are accompanied by a continuous reorganization of the actin cytoskeleton.
In cells, the organization of the actin cytoskeleton is regulated by several actin-binding proteins that contribute to its dynamic properties. Rho family GTPases belonging to the Ras superfamily of small GTPases have been shown to contribute to the organization of the actin cytoskeleton and of the associated sites of cell adhesion to the extracellular matrix (Hall, 1994). Recently, evidence has accumulated for a role of Rho proteins in neuronal development and in the regulation of neuritogenesis (Luo et al., 1997). In N1E-115 neuroblastoma cells, the regulation of Rho by activating (e.g., lysophosphatidic acid) or inhibitory (Clostridium botulinum C3 exoenzyme) factors can affect growth cone behavior (Jalink et al., 1994; Postma et al., 1996). Furthermore, analysis in Drosophila and Caenorhabditis elegans have shown that Rho family GTPases regulate neuritogenesis and axonal guidance in these organisms (Luo et al., 1994; Zipkin et al., 1997), while perturbation of Rac1 activity in mice Purkinje cells leads to modifications of the axonal and dendritic structures of these cells (Luo et al., 1996). More recently, Rac1 has been implicated in the regulation of growth cone behavior (Lamoureux et al., 1997), in collapsin-1–induced growth cone collapse (Jin et al., 1997), and in the regulation of dendritic growth (Threadgill et al., 1997).
The hypothesis that Rho GTPases play an important role during vertebrate neuronal development has been reinforced by our recent finding that four members of the family are highly expressed in the chick embryonic nervous system (Malosio et al., 1997). Interestingly, one of these GTPases is the newly identified cRac1B, which is highly homologous to the chicken Rac1 (that we will refer to as cRac1A in this paper), and whose expression in the developing nervous system is specific.
With the aim of investigating the hypothesized role of the neural-specific cRac1B protein in the development of the neuronal phenotype, functionally active and inactive forms of the GTPases have now been expressed in neuronal cells. The data presented in this paper show that cRac1B has specific effects on neuritogenesis, since it increases the number of neurites per cell, and dramatically increases neurite branching in primary retinal neurons cultured on laminin. These effects are not observed upon cRac1A overexpression. Expression studies in non-neuronal cells show that the specificity of cRac1B-induced cytoskeletal rearrangements is lost in chicken embryo fibroblasts (CEFs),1 where both GTPases induce dramatic changes in cell shape. Moreover, expression of cRac1A/ cRac1B chimeras has allowed us to identify the COOH-terminal portion of the cRac1B polypeptide as the region sufficient to induce the specific effects observed in neurons. Our data indicate that cRac1B plays an important role in the maturation of the neuronal phenotype, and identify a region of the GTPase essential to confer functional specificity.
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Materials and Methods |
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35S-labeled dATP, [32P]dCTP, and 125I-protein A were from Amersham Intl. (Buckinghamshire, United Kingdom). Other chemicals were purchased from Sigma- Aldrich (Milan, Italy). Laminin was purified from Engelbreth-Holm Swarm sarcoma as published (Timpl et al., 1979).
Cell Culture
Neural retinal cells were prepared from embryonic day 6 (E6) chick retinas. Neural retinas were dissected and trypsinized, and cultures of retinal neurons were obtained under serum-free conditions on substrates coated with poly-D-lysine and laminin-1 as described (de Curtis et al., 1991; de Curtis and Malanchini, 1997). After 18 h in culture, neuronal cells were used for transfection experiments, as described below. CEFs were isolated from E10 embryos and cultured at 37°C, 5% CO2 in DME containing 5% FCS, 1% chicken serum, 100 U/ml penicillin and streptomycin, 2 mM glutamine. CEFs up to the fifth passage were used for transfections.
Northern Blot Analysis
Total RNA was prepared from E4, E6, E8, E10, E12, E15, E18, and adult chicken brains, or from different organs from E10 chick embryos, by a single-step RNA isolation method (Chomczynski and Sacchi, 1987). Northern blot analysis of total RNA (20 µg/lane) was performed as previously described (Lehrach et al., 1977). Hybridization took place in hybridization buffer supplemented with 32P-labeled probes (0.5–1 x 106 cpm/ml) for 12– 16 h at 65°C. After high stringency washes (twice with SSC at 65°C), x-ray films were exposed for 1–3 d to the hybridized filters.
Antibodies
The polyclonal antibody specific for the cRac1B GTPase was obtained by injecting rabbits with a peptide corresponding to the COOH-terminal portion of this GTPase (peptide CPPPVKKPGKKCTVF) conjugated to keyhole limpet hemocyanin. The anti–Flag-M5 mAb recognizing the FLAG peptide sequence MDYKDDDDK was from Kodak (New Haven, CT); the anti–β-galactosidase mAb was from Boehringer Mannheim GmbH.
Production of Glutathione-S-Transferase (GST) Fusion Proteins for Rho Family GTPases
Full-length cDNAs coding for chicken cRac1A, cRac1B, cRhoA, and cRhoB polypeptides were obtained by PCR from the corresponding clones in pBluescript KS– vector (Malosio et al., 1997), and subcloned into the pGEX-4T-1 vector (Pharmacia Biotech, Uppsala, Sweden). The resulting pGEX-cRac1A, pGEX-cRac1B, pGEX-cRhoA, and pGEX-cRhoB plasmids were used to obtain GST fusion proteins by expression into Escherichia coli BL21 cells. After induction of the expression with isopropyl-β-D-thiogalactopyranoside (Sigma-Aldrich) for 18 h at 23°C, cells were resuspended in PBS, and then lysed by sonication. Lysates were centrifuged for 10 min at 11,000 g, and GST fusion proteins were purified from the supernatants on glutathione–agarose beads (Sigma-Aldrich).
Preparation of the Constructs for the Expression of the GTPases in Eukaryotic Cells
The cDNAs coding for chicken cRac1A, cRac1B, and cRhoB (obtained from pGEX-cRac1A, pGEX-cRac1B, and pGEX-cRhoB, respectively), and for β-galactosidase (obtained from the pInd-LacZ plasmid; Invitrogen, Carlsbad, CA) were subcloned into the pFLAG-CMV-2 expression vector (Kodak) containing the sequence coding for the FLAG peptide recognized by the anti–Flag-M5 mAb (Kodak). The resulting pFLAG-cRac1A, pFLAG-cRac1B, pFLAG-cRhoB, and pFLAG-LacZ plasmids code for fusion proteins containing the FLAG sequence at the NH2 terminus. The plasmids pFLAG-N17-cRac1A, pFLAG-N17-cRac1B, and pFLAG-V12-cRac1B were obtained by mutation of T17 into N17 of cRac1A and cRac1B, and of G12 into V12 of cRac1B, using degenerate oligonucleotides in combination with the QuickChangeTM site-directed mutagenesis kit (Stratagene GmbH, Heidelberg, Germany).
Constructs coding for the cRac1A/cRac1B chimeric polypeptides were obtained as follows. For the cRac1-AAB and cRac1-BBA chimeras, a XhoI site at position 463 of the cDNA sequences of cRac1A and cRac1B was created by using degenerate oligonucleotides in combination with the QuickChangeTM site-directed mutagenesis kit (Stratagene GmbH, Heidelberg, Germany), starting from the pFLAG-cRac1A and pFLAG-cRac1B plasmids, respectively. The creation of this site did not affect the amino acid composition of the encoded polypeptides. The resulting pFLAG-cRac1A/XhoI and pFLAG-cRac1B/XhoI plasmids were digested with XhoI. Each digestion resulted in the production of a 4.1-kb and a 1.15-kb fragment. Ligation of the 4.1-kb fragment from pFLAG-cRac1A/XhoI with the 1.15-kb fragment from pFLAG-cRac1B/XhoI resulted in the pFLAG-cRac1-AAB plasmid, while the pFLAG-cRac1-BBA plasmid was obtained by ligation of the other two fragments.
The pFLAG-cRac1-BAB plasmid was obtained by using a pair of degenerate oligonucleotides encompassing the nucleotide sequence corresponding to positions 425 to 463 of cRac1A. These oligonucleotides were used in combination with the QuickChangeTM site-directed mutagenesis kit (Stratagene GmbH), starting from the pFLAG-cRac1B plasmid, to obtain the pFLAG-cRac1-BAB plasmid.
For the cRac1-ABB and cRac1-BAA chimeras we have used the unique NcoI site found at position 431 of the nucleotide sequences coding for cRac1A and cRac1B, which had been previously cloned into the BamHI + EcoRI–digested pRSET-A vector (Invitrogen). The pRSET-cRac1A and pRSET-cRac1B plasmids were digested with BamHI and NcoI. Each digestion resulted in the production of a 3-kb and a 0.44-kb fragment. Ligation of the 3-kb fragment from pRSET-cRac1A with the 0.44-kb fragment from pRSET-cRac1B resulted in the chimeric pRSET-cRac1-BAA plasmid, while the pRSET-cRac1-ABB plasmid was obtained by ligation of the other two fragments. The sequences coding for the two chimeric proteins were excised from the two pRSET chimeric constructs by digestion with BamHI and EcoRI, and inserted into the BglII site of the pFLAG vector by blunt ligation, to obtain the pFLAG-cRac1-BAA and pFLAG-cRac1-ABB plasmids, respectively.
The correctness of all the constructs was checked by sequencing, and the plasmids were used for transfection of primary retinal neural cells and CEFs.
Expression of the GTPases in Eukaryotic Cells
For transfections of primary retinal neural cells, we used a protocol modified from Boussif et al. (1995). About 300,000 retinal cells obtained from E6 chick neural retinas were plated in each 1.5-cm-diam well containing a glass coverslip coated with 200 µg/ml poly-D-lysine and 40 µg/ml laminin. Cells were cultured overnight at 37°C, 5% CO2 as described (de Curtis et al., 1991), to induce neurite extension. Cells were then incubated with 200 µl/ well of 150 mM NaCl containing 150 nmoles of polyethylenimine (PEI) 50 kD (Sigma-Aldrich), and 5 µg of plasmid DNA, in 0.5 ml of transfection medium (50% retinal growth medium [RGM] and 50% DME, 5% FCS). After 3 h of culture, cells were washed once with serum-free RGM, and cultured for further 15–24 h in fresh serum-free RGM, before fixation for immunofluorescence. Quantitation of the effects of the expression of N17-cRac1A, N17-cRac1B, V12-cRac1B, and of the chimeric cRac1A/cRac1B constructs in retinal neurons were made by examining transfected, neurofilament-positive neurons in cultures. Neurites longer than three cell diameters were considered for quantitation. The values shown were obtained by analysis of several experiments.
For transfections of CEFs, cells were plated in 1.5-cm-diam wells containing an uncoated glass coverslip, and cultured at 37°C overnight. Cells were then incubated with new culture medium to which 3 µg of plasmid DNA and 6 µg of Dosper (Boehringer Mannheim GmbH) were added. The Dosper and the DNA were diluted separately in 20 mM Hepes, 150 mM NaCl, pH 7.4, to a final volume of 25 µl each. The Dosper was added to the DNA dropwise, and incubated 15 min at room temperature. The mixture was added to the wells containing cells with 1 ml of fresh medium with 5% FCS, 1% chicken serum. After 6 h cells were washed once with medium, and cultured for up to 24 h in fresh medium before further treatment.
To check for cell viability after expression of the N17-cRac1A and N17-cRac1B polypeptides, after transfection for 18 h with the respective constructs retinal cells were resuspended in fresh RGM containing 2.5 µg/ml of propidium iodide (Belloc et al., 1994; Zamai et al., 1996; Darzynkiewicz et al., 1997), and incubated for 40 min at 37°C. After two washes in RGM, cells were fixed and processed for immunofluorescence, as described below. Transfected cells were identified by staining with the anti-cRac1B polyclonal antibody detected by FITC-conjugated sheep anti–rabbit IgG (Boehringer Mannheim GmbH), while the presence of propidium iodide in the same cells was identified by fluorescence in the rhodamine channel.
After transfection, the levels of expression of the cRac1A, cRac1B, N17-cRac1A, and N17-cRac1B polypeptides were evaluated by morphometric analysis. For each construct, 10 transfected cells (either CEFs, or neurofilament-positive retinal neurons) were selected and analyzed. The analysis was performed by analyzing optical sections including the cell body of neurons, excluding the neurites. Optical sections obtained under identical conditions using a DVC-250 confocal microscope (Bio-Rad Laboratories, Hercules, CA) were analyzed using the NIH Image software.
Immunofluorescence
Transfected cells were fixed with 3% PFA and processed for indirect immunofluorescence, as described (Cattelino et al., 1995). Fixed cells were then incubated for 1 h at room temperature with the following dilution of primary antibodies: 1:500 anti-cRac1B polyclonal serum; 4 µg/ml anti– Flag-M5 mAb; 1:200 anti–200-kD neurofilament protein polyclonal antibody; and 1:300 anti–β-galactosidase mAb. Cells were subsequently incubated for 40 min with TRITC-conjugated sheep anti–mouse IgG together with FITC-conjugated sheep anti–rabbit IgG (Boehringer Mannheim GmbH), and observed using an Axiophot microscope (Carl Zeiss Inc., Thornwood, NY). When used, FITC-conjugated phalloidin (Sigma- Aldrich) was added during the incubation with a TRITC-conjugated secondary antibody.
Western Blotting
Aliquots of the GST fusion proteins for the different GTPases were separated by SDS-PAGE (Laemmli, 1970), transferred to nitrocellulose filters, and probed with the polyclonal antibody against cRac1B. For the detection of the primary antibody, filters were incubated with 0.2 µCi/ml of 125I–protein A (Amersham Corp., Arlington Heights, IL), washed, and then exposed to Amersham Hyperfilm-MP.
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The morphology of retinal neurons cultured on laminin was not affected by the expression of the β-galactosidase (Fig. 3, a and d). Transfected cells showed in general one long, poorly branched neurite, identified by staining with an antibody against the 200-kD neurofilament protein, similarly to non-transfected cells in the same preparations (Fig. 3 d). Overexpression of cRac1B by transfection of retinal neurons with the pFLAG-cRac1B plasmid induced a dramatic effect on neuritogenesis (Fig. 3, b, c, e, and f). Very often neurons showed an increased number of neurites per cell, and neurites were frequently highly branched compared with non-transfected cells. Furthermore, localization of filamentous actin (F-actin) and microtubules by incubation of transfected cells with fluorescent phalloidin, and with a mAb against tubulin, respectively, showed presence of F-actin in neurites and in all neuritic branches, and the presence of microtubules in all neurites and major neuritic branches (not shown). Transfection of retinal neurons with pFLAG-cRhoB– induced retraction of neurites and rounding up of the neurons (not shown). On the other hand, when retinal neurons were transfected with the pFLAG-cRac1A plasmid, no dramatic effect on neuronal morphology could be detected. cRac1A-transfected neurons showed a morphology similar to that of non-transfected neurons, characterized by the presence of one long, poorly branched neurite per neuron (Fig. 3, g–i). The different effects observed upon overexpression of the two wild-type Rac proteins in retinal neurons was not due to differences in the levels of expression of the two polypeptides, since these effects were observed when neurons expressing similar levels of the two proteins were compared, as detected by immunofluorescence with the anti–Flag-M5 mAb (see examples in Figs. 3 and 10). Moreover, quantitation in neurons (and in non-neuronal cells) by morphometric analysis performed as described in the Materials and Methods, did not show significant differences between the levels of expression of the two wild-type polypeptides, as well as of the two dominant-negative N17-cRac1A and N17-cRac1B polypeptides (Table I).
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To test for potential toxic effects of N17-cRac1B on neurons, transfected cells were stained with propidium iodide, which can only permeate through the membrane of damaged cells (see Materials and Methods). Retinal cells expressing N17-cRac1B and showing very short of no neurites (Fig. 6, a and d) were impermeable to propidium iodide (Fig. 6, b and e). On the other hand, in the same cultures a number of non-transfected, damaged cells could be detected by positive staining with propidium iodide (Fig. 6, b, c, e, and f, arrows). Also N17-cRac1A–transfected cells were not accessible to propidium iodide (not shown). These data support the idea that inhibition of neurite extension by dominant-negative N17-cRac1B was specific, and not due to a more general toxic effect on neurons.
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The overexpression of cRac1A in CEFs produced effects similar to those obtained by the expression of cRac1B. It induced the reduction of stress fibers in cells that were still keeping a flat morphology (not shown), and caused deep morphological alterations in several cells, which were characterized by the presence of numerous plasma membrane protrusions (Fig. 8 i) rich in F-actin (not shown).
The COOH-Terminal Portion of the cRac1B GTPase Is Sufficient to Confer Functional Specificity
With the aim of identifying the portion(s) of the polypeptide responsible for the specific effects of cRac1B on neuritogenesis, we have prepared five different constructs corresponding to the cRac1A/cRac1B chimeras obtained by different combinations of the three polypeptide stretches that contain all 12 amino acid differences existing between the cRac1A and cRac1B polypeptides. The cRac1A and cRac1B GTPases differ in four amino acid residues included in the Rho's insert region (Fig. 9 A, IR), in two amino acid residues of a region (Fig. 9 A, E) known to be required for the interaction of Rac with some effectors (Diekmann et al., 1995), and in six of the last eight COOH-terminal amino acid residues that contain the CAAX sequence involved in membrane anchoring (Fig. 9 A, M). The five chimeras indicated in Fig. 8 B were obtained as described in the Materials and Methods.
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Fig. 11 shows the quantitative analysis of the effects of the overexpression of wild-type and chimeric constructs on neuritogenesis from E6 retinal neurons. The average number of neurites per neuron in cells expressing either the wild-type cRac1B, or one of the three chimeric constructs with the COOH-terminal portion from cRac1B (cRac1-ABB, cRac1-BAB, and cRac1-AAB) was significantly higher when compared with neurons expressing either the wild-type cRac1A, or one of the two chimeric cRac1-BBA and cRac1-BAA GTPases. Over 50% of the neurons expressing one of the constructs including the M region of cRac1B showed branched neurites, which in the majority of cases included highly branched neurites. In contrast, poor neuritic branching was present in <15% of the neurons expressing any of the constructs containing the M region of cRac1A.
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The striking differences on neuritogenesis observed by separately overexpressing the two Rac proteins in primary neurons was not due to different levels of expression of the two wild-type GTPases, since these differences could be reproducibly observed between neurons showing similar levels of either exogenous protein, as detected by immunofluorescence analysis. Similar levels of expression of cRac1B and cRac1A after transfection with the corresponding constructs were indicated also by the quantitative analysis shown in Table I. Moreover, the analysis of the effects of the chimeric GTPases clearly showed that neuritogenesis, including neurite branching, was significantly enhanced whenever the COOH-terminal eight amino acid residues from cRac1B were present, whereas these effects were not observed with any of the chimeras containing the COOH-terminal portion of cRac1A. Interestingly, the expression of the wild-type cRac1B GTPase was essential to induce increased neuritogenesis, since expression of the constitutively active V12-cRac1B GTPase, as well as the expression of the inactive N17-cRac1B GTPase had drastic inhibitory effects on neuritogenesis. These results strongly suggest that a cycling, wild-type GTPase is required for cRac1B-mediated neuritogenesis.
In contrast to what observed in neurons, we have found that cRac1A and cRac1B have similar effects on the cytoskeletal organization of fibroblasts, implicating a cell type– dependent specificity of Rac proteins action. It has been recently found that the activity of Rac3 (the human orthologue of cRac1B) can be regulated in vitro by Bcr (Haataja et al., 1997), a GTPase-activating protein highly expressed in the brain (Heisterkamp et al., 1993). These data, together with the finding that Tiam1, a guanine nucleotide exchange factor highly expressed in the brain (Habets et al., 1995), affects neurite outgrowth in neuroblastoma cells in a Rac-dependent fashion, suggest that neuritogenesis may be modulated by specific Rac regulators during development (van Leeuwen et al., 1997). Moreover, the differences existing between the cRac1A and cRac1B polypeptides may be sufficient to allow the interaction with distinct sets of neuronal effectors and/or regulators, which may be responsible for the different effects observed on the neuronal cytoskeleton. We have preliminary biochemical evidence supporting this hypothesis, since distinct sets of polypeptides from brain lysates interact specifically with the two activated Rac proteins in vitro (Di Cesare, A., S. Paris, and I. de Curtis, unpublished results).
The striking difference observed in this study by overexpressing the two Rac proteins into primary neurons is particularly surprising considering the high degree of homology between the cRac1A and cRac1B polypeptides, which only differ in 12 amino acid residues. Interestingly, none of these residues are included in the "effector loop" (residues 22–45 of Rac). Instead, they are contained in three distinct regions that have been implicated together with the classical effector loop in the recognition of downstream effectors (Kreck et al., 1994; Joseph and Pick, 1995; Diekmann et al., 1995; Westwick et al., 1997). One of the three regions corresponds to a stretch of amino acid residues at the COOH terminus of the polypeptides, which is important also for targeting of the GTPases to the membrane; 6 of the 12 amino acid differences between cRac1A and cRac1B are clustered in this region. The results obtained by expressing distinct cRac1A/cRac1B chimeras in primary neurons have clearly established that this COOH-terminal region is required for cRac1B-specific action, since its substitution with the corresponding sequence from cRac1A completely abolishes the effects on neuritogenesis. Furthermore, this sequence is sufficient to mediate enhanced neuritogenesis when exchanged with the corresponding region onto the cRac1A GTPase. These results point for the first time to an important role of the COOH-terminal region in the determination of the specificity of Rac proteins action during neuritogenesis. Although the manner in which this region mediates cRac1B-enhanced neuritogenesis remains to be established, one possibility is that it may be involved in the interaction with cRac1B neuronal effectors and/or regulators. Alternatively, a different subcellular localization of the GTPases may be responsible for the differences observed between cRac1A and cRac1B action in neurons, although studies in transfected fibroblasts have not shown differences in the subcellular distribution of the two polypeptides (Albertinazzi, C., and I. de Curtis, unpublished data).
Rac GTPases have been implicated in axonal outgrowth during neuronal development in Drosophila (Luo et al., 1994). Moreover, perturbation of Rac1 activity in mouse Purkinje cells leads to severe loss of presynaptic terminals (Luo et al., 1996). Our finding that cRac1B has a dramatic positive effect on neurite branching suggests that this GTPase may be involved in the development of the mature neuronal phenotype in vivo. This hypothesis is supported by our previous data showing that the expression of cRac1B is restricted to the nervous system during embryonic development (Malosio et al., 1997), and by the finding that the recently identified human Rac3 GTPase (Haataja et al., 1997), which shares 99% amino acid identity with cRac1B, shows the highest expression in the brain. Furthermore, the expression of cRac1A and cRac1B is differentially regulated during retina (Malosio et al., 1997), and brain (this paper) development. In the retina, at E6 cRac1B shows levels of expression up to sevenfold lower than at later stages (E10–E12; Malosio et al., 1997), whereas the expression levels of cRac1A remain quite constant between E6 and E12. We have observed a similar situation in the brain, where cRac1A expression is constantly high at the stages analyzed, between E4 and adulthood, whereas the expression of cRac1B is developmentally regulated, with low levels of transcript up to E6, highest expression around E15, and low levels again in the adult. It is interesting to note that in the chick retino-tectal system, ganglion cell neurites reach the surface of the tectum at E8, and start to arbor through the tectum layers to reach their target around E12. At E18, arbors are relatively mature, and they continue to increase in complexity at least until postnatal day 2. From E11 on, once they have reached the final target, retinal ganglion cell axons form organized synapses (Acheson et al., 1980; McLoon, 1985; Thanos and Bonhoeffer, 1987; Nakamura and O'Leary, 1989; Yamagata and Sanes, 1995). In chicken, many other neuronal types are branching their neurites and forming synapses during the same period. Considering the correlation between the high levels of expression of cRac1B after E8, and the timing of neurite branching during neuronal development, a dose-dependent effect of the cRac1B GTPase on neurite branching in vivo may be envisaged. According to this hypothesis, the increase in neurite branching observed in transfected E6 retinal neurons, which normally express low levels of endogenous cRac1B (Malosio et al., 1997), may be explained by the induced increase of cRac1B activity.
In conclusion, our finding that the neural-specific cRac1B GTPase has dramatic effects on the development of the neuronal phenotype strengthens the idea that Rho GTPases play important roles during neuronal development, and raises interesting questions on the nature of the molecular mechanisms that mediate the effects of this GTPase on the neuronal cytoskeleton. The finding that a short COOH- terminal sequence is essential for cRac1B-induced reorganization of the neuronal cytoskeleton represents an important new indication towards the understanding of the mechanisms underlying Rho proteins' functional specificity. Future work will be aimed at the identification of the molecular machinery responsible for the specificity of action of Rac GTPases during neuritogenesis and neuronal maturation.
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Acknowledgments |
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This work was supported by Telethon-Italy (grant No. 1028 to I. de Curtis), and by a grant from the "Multiple Sclerosis Project" of the Istituto Superiore di Sanità.
Submitted: 20 January 1998
Revised: 17 March 1998
Address all correspondence to Ivan de Curtis, Cell Adhesion Unit, DIBIT, S. Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy. Tel.: 39-02-2643-4828. Fax: 39-02-2643-4813. E-mail: decurtis.ivan @hsr.it
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References |
|---|
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|---|
Acheson DWK, Kemplay SK & Webster KE. Quantitative analysis of optic terminal profile distribution within the pigeon optic tectum, Neuroscience, 1980, 5, 1067–1084.[Medline]
Belloc F, Dumain P, Boisseau MR, Jalloustre C, Reiffers J, Bernard P & Lacombe F. A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells, Cytometry, 1994, 17, 59–65.[Medline]
Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B & Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc Natl Acad Sci USA, 1995, 92, 7297–7301.
Cattelino A, Longhi R & de Curtis I. Differential distribution of two cytoplasmic variants of the 6β1 integrin laminin receptor in the ventral plasma membrane of embryonic fibroblasts, J Cell Sci, 1995, 108, 3067–3078.[Abstract]
Chomczynski P & Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem, 1987, 162, 156–159.[Medline]
Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T & Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis), Cytometry, 1997, 27, 1–20.[Medline]
de Curtis I & Malanchini B. Integrin-mediated tyrosine phosphorylation and redistribution of paxillin during neuronal adhesion, Exp Cell Res, 1997, 230, 233–243.[Medline]
de Curtis I, Quaranta V, Tamura RN & Reichardt LF. Laminin receptors in the retina: Sequence analysis of the chick integrin 6 subunit, J Cell Biol, 1991, 113, 405–416.
Diekmann D, Nobes CD, Burbelo PD, Abo A & Hall A. Rac GTPase interacts with GAPs and target proteins through multiple effector sites, EMBO (Eur Mol Biol Organ) J, 1995, 14, 5297–5305.[Medline]
Haataja L, Groffen J & Heisterkamp N. Characterization of Rac3, a novel member of the Rho family, J Biol Chem, 1997, 272, 20384–20388.
Habets GGM, van der Kammen RA, Stam JC, Michiels F & Collard JC. Sequence of the human invasion-inducing Tiam 1 gene, its conservation in evolution and its expression in tumor cell lines of different tissue origin, Oncogene, 1995, 10, 1371–1376.[Medline]
Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton, Annu Rev Cell Biol, 1994, 10, 31–54.
Heisterkamp N, Kaartinen V, van Soest S, Bokoch GM & Groffen J. Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain, J Biol Chem, 1993, 268, 16903–16906.
Jalink K, van Corven EJ, Hengeveld T, Morii N, Narumiya S & Moolenar WH. Inhibition of lysophosphatidate- and trombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho, J Cell Biol, 1994, 126, 801–810.
Jin Z & Strittmatter SM. Rac1 mediates collapsin-1-induced growth cone collapse, J Neurosci, 1997, 17, 6256–6263.
Joseph G & Pick E. Peptide "walking" is a novel method for mapping functional domains in proteins. Its application to the Rac1-dependent activation of NADPH oxidase, J Biol Chem, 1995, 270, 29079–29082.
Kozma R, Sarner S, Ahmed A & Lim L. Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid, Mol Cell Biol, 1997, 17, 1201–1211.[Abstract]
Kreck ML, Uhlinger DJ, Tyagi SR, Inge KL & Lambeth JD. Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase, J Biol Chem, 1994, 269, 4161–4168.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 277, 680–685.
Lamoureux P, Altun-Gultekin ZF, Lin C, Wagner JA & Heidemann SR. Rac is required for growth cone function but not neurite assembly, J Cell Sci, 1997, 110, 635–641.[Abstract]
Lehrach H, Diamond D, Wozney JM & Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 1977, 16, 4743–4751.[Medline]
Luo L, Liao YJ, Jan LY & Jan YN. Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion, Genes Dev, 1994, 8, 1787–1802.
Luo L, Hensch TK, Ackerman L, Barbel S, Jan LY & Jan YN. Differential effects of Rac GTPase on Purkinje cell axons and dendritic trunks and spines, Nature, 1996, 379, 837–840.[Medline]
Luo L, Jan LY & Jan YN. Rho family small GTP-binding proteins in growth cone signalling, Curr Opin Cell Biol, 1997, 7, 81–86.
Malosio ML, Gilardelli D, Paris S, Albertinazzi C & de Curtis I. Differential expression of distinct members of the Rho family of GTP-binding proteins during neuronal development: Identification of cRac1B, a new neural-specific member of the family, J Neurosci, 1997, 17, 6717–6728.
McLoon SC. Evidence for shifting connections during development of the chick retinotectal projection, J Neurosci, 1985, 5, 2570–2580.[Abstract]
Nakamura H & O'Leary DDM. Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axons remodeling to develop topographic order, J Neurosci, 1989, 9, 3776–3795.[Abstract]
Postma FR, Jalink K, Hengeveld T & Moolenar WH. Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor, EMBO (Eur Mol Biol Organ) J, 1996, 15, 2388–2395.[Medline]
Tanaka E & Sabry J. Making the connection: cytoskeletal rearrangements during growth cone guidance, Cell, 1995, 83, 171–176.[Medline]
Thanos S & Bonhoeffer F. Axonal arborization in the developing chick retinotectal system, J Comp Neurol, 1987, 261, 155–164.[Medline]
Threadgill R, Bobb K & Ghosh A. Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42, Neuron, 1997, 19, 625–634.[Medline]
Timpl R, Rhode H, Gehron-Robey P, Rennard S, Foidart J-M & Martin G. Laminin, a glycoprotein from basement membrane, J Biol Chem, 1979, 254, 9933–9937.
van Leeuwen FN, Kain HET, van der Kammen RA, Michiels F, Kranenburg OW & Collard JG. The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho, J Cell Biol, 1997, 139, 797–807.
Westwick JK, Lambert QT, Clark GJ, Symons M, van Aelst L, Pestell RG & Der CJ. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways, Mol Cell Biol, 1997, 17, 1324–1335.[Abstract]
Yamagata M & Sanes JR. Lamina-specific cues guide outgrowth and arborization of retinal axons in the optic tectum, Development (Camb), 1995, 121, 189–200.[Abstract]
Zamai L, Falcieri E, Marhefka G & Vitale M. Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation, Cytometry, 1996, 23, 303–311.[Medline]
Zipkin ID, Kindt RM & Kenyon CJ. Role of a new Rho family member in cell migration and axon guidance in C. elegans, Cell, 1997, 90, 883–894.[Medline]
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