A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans

Abstract

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

Figure 3

Screen for rapidly reversible, temperature-sensitive, uncoordinated mutants. The F2 progeny of animals treated with the mutagen EMS were placed in the center of a plate cooled to 15°C with E. coli around the edge (see Materials and Methods). Uncoordinated or paralyzed worms tended to remain in the center of the plate whereas worms that moved well at the nonrestrictive temperature migrated toward the bacteria. The mobile animals were recovered and placed in the center of a plate warmed to 25°C with E. coli spread around the edge. Animals that became uncoordinated at the restrictive temperature tended to remain in the center of the plate and were picked for further study.

Figure 1

(A) Nucleotide and predicted amino acid sequences of the dyn-1 3.4-kb cDNA. Nucleotides are numbered beginning at the first nucleotide of the spliced leader 1 trans-spliced leader. Amino acids are numbered from the first predicted methionine. The proline altered in the ky51 mutant is circled. Spliced leader 1 and exon 8, which is present in some dyn-1 transcripts, are underlined. Splice sites are marked by triangles. Polyadenylation sites are as marked. The termination codons are marked by asterisks. (B) dyn-1 genomic structure derived from cDNA and genomic DNA sequences. Coding regions are denoted by solid boxes; the 5′ and 3′ untranslated regions are shown as open boxes. The boundaries of the GTPase, pleckstrin homology and PRDs are marked. SL1, the trans-spliced leader; AAA, sites of polyadenylation; ATG and TAA, the predicted start and stop sites of translation, respectively. (C) dyn-1::lacZ gene fusion. LacZ coding sequences were fused in-frame to an XhoI site in the third exon of dyn-1 (see Materials and Methods).

Figure 2

Alignment of the C. elegans dyn-1 amino acid sequence (C. elegans) with D. shibire (Drosophila) (4) and human dynamin-1 sequences (Homo sapiens) (7). Conserved elements (I, II, and II) within GTPase domain are boxed. The pleckstrin homology and PRDs are indicated by dotted lines. The alternative C-terminal amino acid sequences for each dynamin isoform are boxed.

Figure 4

(A) Genetic and physical maps of the dyn-1 region of the X chromosome. The positions of genes are marked and the extents of deficiencies are denoted by solid lines. The approximate positions of YAC Y53G9 and Y69B5 are shown below. Other than for dyn-1, we have not determined if other genes within this interval are contained within these yeast artificial chromosomes. (B) Alignment of amino acid sequences near element I from dynamin-related GTPases showing the substitution of the invariant proline at position 70 by a serine in the dyn-1(ky51) mutant. C. elegans dyn-1 sequences are from positions 39–78.

Figure 5

Mobility of wild-type and dyn-1(ky51) animals. The rate of movement of a single animal was analyzed by recording the tracks left in a bacterial lawn over time. Animals raised at 15°C were transferred to the center of a bacterial lawn on a warmed plate. Photographs were taken at 1-min intervals, and the distance each animal traveled was calculated. Tracks shown are 5-min time points for wild-type animals at (A) 25°C and (C) 30°C and for dyn-1(ky51) animals at (B) 25°C and (D) 30°C. (Bar = 1 mm.)

Figure 6

Expression pattern of a dyn-1::lacZ gene fusion. (A) Adult animal showing neuronal staining in the head region, along the ventral nerve cord and in preanal and lumbar ganglia. (B and C) Close-ups of heads showing staining in motor neurons around the pharynx. (D) A larva showing staining along the ventral nerve chord.