The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction
Introduction
Small G proteins switch between an active, GTP-bound conformation and an inactive, GDP-bound form, thereby allowing downstream signaling pathways to be quickly turned on or off. Activation of small G proteins is facilitated by guanine nucleotide exchange factors (GEFs), which promote the binding of GTP. Specific effectors are then activated to launch the downstream signaling pathway [1]. Gef26 contains all of the conserved domains of mammalian and nematode PDZ-GEFs, including the cyclic nucleotide monophosphate-binding domain (cNMP), PDZ domain, Ras association (RA) domain and N-terminal catalytic GEF domain. It also contains a Ras exchange motif (REM), a proline-rich region (P), and a PDZ-binding motif (PBM) (Fig. S1A). There are two mammalian homologues of Gef26, RapGEF2, which has been reported to be involved in neuronal migration [2], and RapGEF6. Previous research has shown that Gef26 regulates cell mobility via integrins in the embryo [3] and cell adhesion via DE-cadherin in the reproductive system [4]. However, much less information has been reported so far on the function of Gef26 in nervous system development. Given that synapses represent a form of cell adhesion between neurons or between a neuron and another cell type, Gef26 may play an important role in synapse development as well.
The Drosophila NMJ consists of 30 muscles per hemi-segment repeated in each abdominal segment and 36 motor neurons (MNs) innervating these muscles accurately, and therefore, is an ideal system to study synapse development and to understand disorders of neurotransmission in mammals [5], [6], [7]. These 36 MNs bundle together in three main branches containing the transverse nerve (TN), intersegmental nerve (ISN), and segmental nerve (SN) [8]. Pioneer neurons of the ISN project away from the central nervous system and navigate through the muscle field to target specific muscles during NMJ development. The transcription factor Even-skipped (Eve) plays a critical role in determining the specific guidance characteristics of these ISN pioneer neurons [9]. Increasing studies have dissected that Eve regulated the cell adhesion molecule (CAM) FasII and Neuroglian (Nrg) expression levels in fasciculation of the MNs [10].
FasII, the Drosophila ortholog of mammalian neural cell adhesion molecule (NCAM), has been shown to play pivotal roles in NMJ growth and maintenance during nerve development [11]. In the absence of FasII, synapses are formed but fail to be maintained [12], [13], whereas decreased levels of synaptic FasII and overexpression of FasII in both pre-neuron and post-muscle can lead to supernormal NMJ expansion. However, overexpression of FasII in either preneurons or post-muscle decreased NMJ size, which indicates that excess FasII on either side of the NMJ is essential for constraining synaptic growth [11]. Thus, there exists a linear relationship between presynaptic FasII and NMJ size, and modification—rather than elimination—of FasII levels results in a significant difference in the final size of the NMJ [13], [14]. In addition to regulation of expression, local FasII level is regulated through multiple mechanisms, including integrin and mitogen-activated protein kinase (MAPK) signaling pathways [11], [15], [16], [17], [18], [19].
Rap1, also known as Roughened in Drosophila, is reported to be activated by six GEFs, including Gef26 [20]. Rap1 is classified as a Ras-like small G protein because its structure is highly analogous to that of Ras [21]. Previous research has reported that Rap1 blocks mitogenic activity in cells by silencing downstream MAPK [22], [23], but that it can also activate MAPK independent of Ras during eye development and embryogenesis in Drosophila [8], [24]. However, it remains controversial how Rap1 interacts with Ras to mediate signaling downstream of receptor tyrosine kinases. Previous studies demonstrated that PDZ-GEFs function as Rap1 activators in different tissues and organisms [25], [26], [27]. Genetic analyses showed that, in migrating macrophages, Gef26 acts upstream of Rap1 to regulate cell adhesion and cell shape via a pathway that requires the function of βPS integrins in the Drosophila embryo [3], but the underlying mechanism and effect of the Gef26-Rap1 pathway on integrin remain unknown.
In the present study, significant defects of NMJ morphology were detected in Gef26 mutants, and the downstream signaling pathways responsible for this process were analyzed. Our study highlights Gef26 as a novel factor participating in NMJ growth, and shows that FasII is an essential molecule responsible for the function of Gef26 in NMJ growth. Moreover, our results indicate that Gef26 functions upstream of small G protein Rap1 to regulate local FasII level, likely through an integrin-mediated mechanism, during NMJ growth.
Section snippets
Materials and methods
Fly stocks-All stocks were grown at 25 °C on standard medium. The wild-type (WT) Drosophila melanogaster strain used in this study was w1118. Elav-Gal4 (C155), UAS-rlRNAi (36058), and UAS-rl (36270) were obtained from the Bloomington Drosophila Stock Center (bdsc.indiana.edu). The C57-Gal4 line was provided by V. Budnik (University of Massachusetts School of Medicine, Worcester, MA). gef263, gef266, UAS-gef26ΔN1, and P[Gef26+] were generated in Steven X. Hou's lab (National Institutes of Health
Results
Loss of Gef26 results in impaired NMJ morphology - To investigate the function of Gef26 in vivo, two Gef26 mutants were utilized. In gef263, the P element l(2)SH1450 was inserted into the 5′ promoter sequence of the gene encoding Gef26, 846 bp away from the ATG translation start site. The phenotype and lethality associated with l(2)SH1450 was reverted to WT by the mobilization of the P element. The gef266 allele contains a 3-kb deletion surrounding the l(2)SH1450 insertion site, including the
Discussion
Previous studies demonstrated that the Drosophila PDZ-GEF Gef26 is involved in cell morphogenesis and migration. Subsequent studies showed that the Ras-like small G protein Rap1 functions as a target of Gef26, and that Rap1 was the well-studied Gef26 specific small GTPase. In this study we discovered that Gef26 plays a pivotal role in larval NMJ morphogenesis via activating Rap1, and showed that FasII, a well-known CAM participating in NMJ development, is responsible for this process. This is
Acknowledgments
We thank Drs. Zhengping Jia and Junhai Han for useful discussions and critical comments on the manuscript; Dr. Rolf Reuter of the Bloomington Stock Center for providing Drosophila stocks; and the members of the Xie laboratory for their critical comments on the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (30928014 to SH, 31430035 to WX), and National Basic Research Program of China (973 Program) (2012CB517903 to WX).
Conflict of interest
The authors declare that they have no conflicts of interest.
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These authors contributed equally to this work.