Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system - PubMed (original) (raw)

Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system

R E Kohn et al. Mol Biol Cell. 2000 Oct.

Free PMC article

Abstract

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

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Figures

Figure 1

Figure 1

Genomic region around unc-13, with predicted unc-13 transcripts and protein products. (A) Genomic sequence indicating the locations of the major_unc-13_ transcript and the extents of cosmids C44E1 and ZK524. (B) Genomic sequence with introns and exons indicating the production of three predicted unc-13 transcripts. Region_L_ (left) corresponds to the less conserved 5′ end of_unc-13_, and region R (right) corresponds to the more highly conserved 3′ end of unc-13. Region_M_ (middle) corresponds to an exon that was not originally identified in the published sequence of_unc-13_. Asterisks mark positions of alternative splicing. (C) Proteins expressed from the predicted transcripts are shown. cDNA library screens and RACE products indicate the existence of_unc-13_ products. The cDNAs yk50c3 (GenBank accession number D67349), yk16b7 (GenBank accession numbers D26913 and D35032), and yk8h12 (GenBank accession number D37721) were isolated by Yuji Kohara from a dT-primed C. elegans library. The remaining cDNAs (RM#418p, RM#384p, RM#419p, RM#385p, and RM#386p) were isolated in our laboratory from a random primed C. elegans library. 5′ RACE and cRACE (Maruyama et al., 1995) were used to produce RACE products.

Figure 2

Figure 2

UNC-13 has several mammalian homologues. The UNC-13L-R structure is shown. This form is similar to the published form of UNC-13 (Maruyama and Brenner, 1991). Additional N-terminal regions, including a third C2 domain, were identified in cDNA library screens (GenBank accession number M62830). C1 and C2 represent PKC homology domains. Munc13-1, Munc13-2, and Munc13-3 (GenBank accession numbers U24070, U24071, and U75261, respectively) are rat homologues of_C. elegans_ UNC-13 (Brose et al., 1995). Hmunc13 (GenBank accession number AF020202) is a human homologue of UNC-13 (Song et al., 1999). The UNC-13L region is 30% identical to the N-terminal region of Munc13-1.

Figure 3

Figure 3

Northern and Western transfers show products of the unc-13 gene. (A) Northern transfers indicate the presence of unc-13L-R and unc-13M-R transcripts. L, M, and R indicate lanes incubated with32P-labeled probes that hybridize specifically to the_unc-13L_, unc-13M, and_unc-13R_ regions, respectively. (B) Western transfers indicate the presence of several high-molecular-mass protein products from the unc-13 gene. Proteins from N2,unc-13(e1091), and unc-13(s69) bound to nitrocellulose membrane were labeled with anti-UNC-13L antibody. The band present at ∼130 kDa is nonspecific.

Figure 4

Figure 4

Sequenced unc-13 alleles. The positions of mutations are indicated on a diagram of an UNC-13L-M-R protein. Alleles with names shown in bold encode in-frame (i.e., missense) mutations. The unc-13L region (L) includes exons 1–13; unc-13M (M) consists of exon 14; and_unc-13R_ (R) includes exons 15–31.

Figure 5

Figure 5

Appearance of unc-13 mutants. (A) N2 (wild-type) young adult. (B) Young adult homozygous for the severe_L_-region mutation e1091. (C) Young adult homozygous for the severe R_-region mutation_s69. (D) Recently hatched larva homozygous for the lethal mutation md2415. These animals do not grow; they barely move, and they die within a few days. Bars, 100 μm.

Figure 6

Figure 6

unc-13 behavioral phenotypes are recessive, and L and R mutants fail to complement one another. Thrashing values are body bends per minute measured during a 2-min interval; values are means of 10–20 nematodes ± SDs. The wild-type data are representative of tests on a single day. Probabilities were calculated with the use of Student's t test for each strain versus a matched set of wild-type nematodes. *Significantly different from wild-type (p < 0.001). The homozygous unc-13 alleles (e51, e1091, md1325, and_s69_) are not significantly different from each other or from e1091/md2415 or s69/md2415.

Figure 7

Figure 7

Behavioral phenotypes of transgenic strains. Transgenic arrays including the cosmid ZK524 (mdEx42) or the cosmid C44E1 (mdEx43) were generated by injection of wild-type nematodes. The arrays were crossed into the_unc-13_ mutants e1091, s69, and md2415. All strains were assayed for thrashing in liquid. Values given are normalized to N2 values measured on the same day. *Significantly different from N2 plus ZK524 (p < 0.01).

Figure 8

Figure 8

Anti-UNC-13L antibody stains a form of UNC-13 protein that is synaptic but is unlikely to be on synaptic vesicles. C. elegans were double stained with anti-UNC-13L (left) and anti-UNC-17/VAChT (right). (A) Anti-UNC-13L in an adult wild-type head stains the major synaptic regions, including the nerve ring (NR), ventral nerve cord (VNC), dorsal nerve cord (DNC), and sublateral nerve cords (SNC). (B) Anti-UNC-17/VAChT stains a subset of the region stained by anti-UNC-13L. (C and D) A slightly higher magnification shows punctate staining with both antibodies in the dorsal nerve cord and sublateral nerve cords in the body. (E and F) A higher magnification shows the colocalization of anti-UNC-13L (E) and anti-UNC-17/VAChT (F) in synaptic regions (arrowheads) in one of the sublateral nerve cords in the head. (G and H) In an adult_unc-104_ mutant, staining of anti-UNC-13L is somewhat less intense but remains synaptic, whereas anti-UNC-17/VAChT is now localized in neuronal cell bodies (CB). Anterior is left; bars, 10 μ m.

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