Still life, a Protein in Synaptic Terminals of Drosophila Homologous to GDP-GTP Exchangers

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Science  24 Jan 1997:
Vol. 275, Issue 5299, pp. 543-547
DOI: 10.1126/science.275.5299.543


The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases.

The morphology of synaptic terminals changes during synaptogenesis and in response to environmental cues or neural activity (1). Rho-like GTPases, which include Rac1, Cdc42, and Rho, regulate cell motility, morphology, and adhesion through interaction with the actin cytoskeleton (2), and they are also implicated in the extension or elaboration of axons and dendrites in the nervous system (3, 4). Here we describe the Still life (SIF) proteins of Drosophila that participate in the signaling cascade of the Rho-like GTPases in the synaptic terminals.

To identify factors involved in the formation of neural circuits, we created Drosophila mutants by the insertion of an enhancer trap transposon and then screened them for reduced locomotor behavior, an approach used to identify factors that function during synapse formation (5, 6). We isolated a mutant, sif98.1, that carries a single P element insertion at the cytological location 64E. Flies homozygous for this insertion demonstrated reduced locomotion (Fig. 1A) and were male sterile.

Fig. 1.

(A) Reduced locomotor activity of the sif98.1 mutant flies and its recovery by the elav-sif (type 1) transgene. The wild-type (Canton-S), sif98.1/sif98.1, and w; P[elav-sif]/P[elav-sif]; sif98.1/sif98.1 flies were examined for their ability to climb the wall of a plastic cylinder (6). The error bars represent standard deviation. (B) Genomic organization of the sif locus. Two types of sif transcripts are aligned beneath the genomic structure. The P element insertion of sif98.1 is located 44 base pairs (bp) upstream of the first exon of the type 2 transcript. The boxes represent the exons or the genomic DNA fragments that hybridized to the sif cDNAs. The regions not aligned are shown by broken lines. E, Eco RI; S, Sal I.

We isolated genomic DNA surrounding the P element (Fig. 1B) and analyzed RNA isolated from adult heads and identified a transcript near the P element insertion. From several rounds of screening of adult head cDNA libraries we isolated two cDNA series, 7.9 and 9.2 kb long, corresponding to distinct start sites. A transgene prepared from the 7.9-kb cDNA, under the control of the neuron-specific elav promoter (7), rescued the behavioral phenotypes (Fig. 1A) and fertility of sif98.1 flies. We conclude that the observed sif phenotypes are caused by a defect in the gene encoding the identified transcripts.

The sequences of the 7.9- and 9.2-kb cDNAs contain long open reading frames that predict proteins of 2064 and 2044 amino acids, respectively (Fig. 2, A and B) (8). In the NH2-terminal portion of the 2044-amino acid protein, there is a repeated sequence and a region in which cysteines are regularly spaced (Fig. 2, B and C). These structures do not appear in the 2064-amino acid protein. The regions in common between the two sequences are homologous to the mouse TIAM-1 protein, which induces invasion of T lymphoma and is predominantly expressed in the normal brain and testis (9). The regions of similarity include a pleckstrin homology (PH) domain (10) with its COOH-terminal flanking region (55.0% identity) and a Dbl homology (DH) and another PH domain (51.2%). A potential myristoylation site, PEST sequences (11), and a PDZ domain (12) were also found in SIF and TIAM-1 (Fig. 2, A and C). DH domains are the conserved catalytic domains of guanine nucleotide exchange factors (GEFs) that act on the Rho-like small guanosine triphosphate (GTP)-binding proteins, converting them from the guanosine diphosphate-bound inactive state to the GTP-bound active state (13). TIAM-1 acts as a GEF for Rac1 and Cdc42 in vitro and for Rac1 in vivo (14).

Fig. 2.

The structure of the SIF proteins. (A) The amino acid sequence of the type 1 SIF protein (26). The PH domains are double-underlined, the DH domain is indicated by a bold line, and the surrounding sequences with similarity between SIF and TIAM-1 are single-underlined. The PDZ domain is marked by a wave line. In these underlined sequences, the conserved residues between SIF and TIAM-1 are indicated by blue backgrounds, and the evolutionarily well-conserved residues among several proteins (9, 12) are indicated by red letters. The PEST sequence is boxed (27). The glycine residue at position 2 shown by a green letter is a potential myristoylation site. In one cDNA clone, a nine-amino acid insertion (VTGFCRSPQ) was observed at the position indicated by an arrowhead. The opa repeat is marked by a double-wave line. (B) The amino acid sequence of the type 2-specific region. The internally repeated sequences are marked by broken lines (amino acids 62 to 86, 94 to 118, 154 to 178, and 225 to 249). The regions in which cysteines are regularly spaced are noted by a chained line (395 to 421 and 496 to 225). The PEST sequences are boxed (27). (C) Schematic domain structures of SIF and TIAM-1. The potential myristoylation site is indicated by the arrowhead, and PEST sequences are marked with small black boxes beneath each domain structure. The dark blue bars and pink bars indicate the internally repeated structures and the cysteine-rich regions, respectively. The PH, PDZ, and DH domains and opa repeat are shown by yellow, light blue, red, and black, respectively. The COOH-terminal flanking region of the first PH domain has a strong homology to that of TIAM-1 and is marked by light green.

We examined the expression pattern of sif in Drosophila embryos by in situ hybridization (15). At stage 14, several cells expressed sif in each segment of the central nervous system. As development proceeded, cells expressing sif increased in number. At stage 17, the sif transcripts were found only in cells in the brain and ventral nerve cord (Fig. 3A).

Fig. 3.

Distribution of the sif gene products. (A) A lateral view of a stage 17 embryo showing the localization of sif transcripts. sif is exclusively expressed in the brain (B) and ventral nerve cord (Vc). Anterior is to the left. Panels (B) to (G) show localization of SIF proteins revealed by AbI3 staining. (B) Ventral view of a stage 17 embryo. Staining is observed in the neuropils (arrow), where neurites form synapses (16). Anterior is to the left. (C) The NMJ of a third instar larva. The synaptic boutons (arrow) on muscles 6 and 7 are stained. (D) A section of the optic lobe in the adult brain. The neuropils of the lamina (La), medulla (Me), lobula (Lo), and lobula plate (Lp) are specifically stained. The arrow indicates axon bundles of the optic chiasma. Re, retina. (E and F) Immunoelectron micrograph of the lamina. The limited regions near the plasma membrane (arrows) are stained. Note that the stained regions protrude outward. The arrowheads indicate the synaptic ribbon or density that marks the active zone. The thick arrow in (F) points to the capitate projection, a characteristic structure of the photoreceptor axon terminals (19). Many synaptic vesicles are visible. (G) is an immunoelectron micrograph of the larval NMJ. There is staining in the restricted submembranous region (arrow) next to the active zones, which are marked by synaptic ribbons (arrowheads). Many synaptic vesicles appear in the terminal; m, mitochondria; SSR, subsynaptic reticulum. Scale bars, 200 nm.

An antibody (AbI3) to the bacterially expressed SIF fusion protein labeled the neuropils, where neurites form synapses (16), in the ventral nerve cord of a stage 17 embryo (Fig. 3B) (17). In the body wall muscles of the third instar larva, boutons of the neuromuscular junctions (NMJs) were stained (Fig. 3C). The antibody also stained the neuropil in the adult brain (Fig. 3D). Neither the cell bodies in the cortical regions surrounding the neuropils nor the axonal processes were stained. Similar staining patterns were found in the adult brain with another antibody (AbH3) to a distinct region of SIF protein (18). Thus, SIF proteins were exclusively detected in the synaptic regions and predominantly at the stages when synapses had undergone maturation.

SIF proteins are localized to certain limited cytoplasmic regions of the photoreceptor axon terminals, which are mainly presynaptic (19), in the adult optic lobe (Fig. 3, E and F). SIF proteins are closely associated with the plasma membrane and the membrane protruding toward surrounding cells. In the larval NMJ, staining was also detected adjacent to the plasma membrane of presynaptic nerve terminals (Fig. 3G). Lateral sides of active zones of both the lamina and the NMJ were stained for SIF. Thus, SIF is localized to the submembranous region, which may affect synaptic events.

To assess SIF function, we expressed transgenes that were under the control of the elav promoter (20). We detected no abnormalities in lines carrying either of two types of intact SIF cDNAs. Because many DH domain-containing proteins express their oncogenic or invasive abilities when their NH2-terminal portions are truncated (9, 13), we constructed a truncated SIF protein (SIFΔN) lacking the NH2-terminal sequences before the first PH domain, but containing in its place a part of the kinesin protein to ensure that the truncated protein would be localized to synapses (21). Lines carrying this transgene demonstrated various levels of viability, possibly correlating to the amount of expression of the transgene. In embryonic and first-larval lethal strains, the nervous system was disrupted. In the strain SIFΔN4.1, the axons of SNb, a motor neuron bundle (22), did not reach their target muscles in 81% of the segments examined (arrow in Fig. 4C) (23). In the remaining 19%, the SNb axons reached the muscles but did not develop terminal arbors, even at stage 17 when wild-type axons have normally formed arbors (arrowheads in Fig. 4, A and C). It has been reported that the constitutively active mutants of the Drosophila Rho-like GTPases, Drac1 and Dcdc42, display blocked axon outgrowth of peripheral neurons (4). These mutant proteins also interfere with the elongation of SNb axons (Fig. 4B), as observed in the truncated SIF transgenic lines. Thus, SIF protein is likely a factor in the cascade of Drac1 or Dcdc42 in the neurons.

Fig. 4.

The effects of the truncated SIF protein and constitutively active mutants of Rho-like GTPases on cell morphology or actin localization. In (A) to (C) the motor axons of early stage 17 embryos were detected with monoclonal antibody 1D4 (22). Anterior is to the left. (A) Wild-type (Canton-S) embryo. The SNb axon bundle is present and extends the terminal arbor between muscles 6 and 7 (arrowhead). (B) elav-Gal4; UAS-Dcdc42V12.2 embryo. Dcdc42V12 encodes a constitutively active Dcdc42 protein (4). The SNb axon bundle is missing (arrow). A similar phenotype was observed in the embryos that express the constitutively active Drac1 protein, Drac1V12 (4). (C) elav-Gal4; UAS-SIFΔN4.1 embryo. In 81% of the segments (A2 to A7, n = 59) examined, the SNb axon bundle did not reach the target muscles 6 and 7 (arrow). In the remaining 19%, the SNb reached the muscles, but did not form terminal arbors (arrowhead). (D and E) Motor terminal arborization on the third instar larval muscles. Muscles 12 and 13 of the A3 segment are indicated. The motor axons and boutons are stained with antibody to horseradish peroxidase (28). Large type 1 and small type 2 boutons are indicated by arrows and arrowheads, respectively. Anterior is to the left. Panel (D) shows a wild-type embryo and (E) an elav-Gal4; UAS-SIFΔN1.1 embryo. The total number of boutons on muscles 12 and 13 of the A3 segment is reduced [129 ± 34 (SD), n = 5, P < 0.005] when compared with the wild type (260 ± 64, n = 8). (F to H) Human KB cells expressing the truncated SIF protein (29). The same view was visualized with phase-contrast optics (F), fluorescein isothiocyanate (green) for the FLAG-tagged truncated SIF (G), and rhodamine (red)-phalloidin for filamentous actin (H). Note that only the cell expressing the truncated SIF shows membrane ruffles (arrows) and altered actin localization. The truncated SIF colocalizes with actin.

One line with the truncated SIF, SIFΔN1.1, occasionally survived to the adult stage and was used to examine the NMJ of the third instar larva. In this line, the SNb axon bundles reached the target muscles 12 and 13 in 74% of the segments (n = 47) examined, but the patterns of their terminal arborization on the muscles were abnormal. Typically, both the terminal processes that form large (type 1) or small (type 2) synaptic boutons (24) were short, and the number of boutons was reduced (Fig. 4, D and E). Thus, SIFΔN disturbs the formation of synaptic arbors on target muscles.

Because Rho-like GTPases regulate cell morphology through actin fiber formation, we examined whether SIF affects the organization of the actin cytoskeleton. When expressed in human KB cells, the truncated SIF protein induced membrane ruffles (Fig. 4F) and led to the localization of actin fibers at the altered structures (Fig. 4H). This cellular phenotype resembled that induced by the constitutively active mutant of Rac1 (25). Furthermore, the truncated SIF protein colocalized with actin fibers (Fig. 4, F through H). Therefore, SIF protein locally affects actin cytoskeleton, possibly by activating Rac1 or its closely related GTPase in KB cells. Similar mechanisms that evoke membrane ruffling may also occur in synaptic terminals.

Our findings that the truncated SIF protein disturbs axonal extension and motor terminal arborization and induces membrane ruffling with altered actin localization imply a relevant function of intact SIF in neuronal morphology. Our data also suggest that SIF protein acts as a GEF for the Rho-like GTPases.

The subcellular localization of SIF in synapses indicates its site of function and may represent the presence of local machinery activating Rho-like GTPases that control the actin cytoskeleton. The PH and PDZ domains possibly serve SIF function in a signal cascade transduced through the plasma membrane (10, 12). Thus, SIF likely regulates the organization of actin-based cytoskeleton in the synaptic terminals by linking the Rho-like GTPases to the extracellular signals or to neural activity.


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