Figure 1.
tba-1 and tbb-2 tubulin mutants.
A. Differential interference contrast (DIC) time-lapse images of wild-type, tba-1(or594 ts), and tbb-2(or600 ts) embryos. In the two tubulin mutants the P0 spindle often is positioned transverse to the anterior posterior axis, and daughter cells contained multiple nuclei. The tba-1(or594 ts) embryo was from a∼1 min. upshift and the tbb-2(or600 ts) embryo was shifted to the restrictive temperature for 8 hours. Black dots represent centrosomes/spindle poles, asterisks denote multiple nuclei per cell, and the arrowhead indicates a second maternal pronucleus. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Amino acid alterations in the two mutants. Asterisks indicates the changed residues. Homologous proteins are aligned below the C. elegans protein. C. Defect maps for individual embryos observed during time-lapse recordings; embryos are listed on the left and phenotypes are listed on the top: 1; pronuclei meet prior to NEBD, 2; Nuclear centrosomal complex centration, 3; Nuclear centrosomal complex rotation, 4; spindle alignment, 5; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took about 1 minute. Red color indicates a defective trait, black color represents the lack of a defect.
Table 1.
Embryonic lethality of the TS mutants.
Table 2.
Sequence alterations in the TS mutants.
Table 3.
Determination if the TS mutations are potentially fast-acting.
Table 4.
The phenotypes of the TS mutants when grown at the restrictive temperature from the L1 larval stage.
Figure 2.
A. DIC time-lapse images of wild-type and sur-6(or550 ts) embryos. In the sur-6 mutant the male pronucleus is small, the AB cell contains two nuclei, and the P1 cell begins mitosis before the AB cell. The sur-6(or550 ts) embryo was shifted to the restrictive temperature for ∼1 min. prior to imaging. White arrowheads denote multiple nuclei per cell, and the arrows in the last panels indicate the first mitotic spindle at the two cell stage. Times in min:sec are given relative to pronuclear meeting. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the C. elegans protein. C. Defect maps for individual embryos observed during time-lapse recordings. In this and all subsequent figures, embryos are listed on the left and phenotypes are listed on the top: 1; Male pronuleus normal size, 2; Nuclear centrosomal complex centration, 3; spindle alignment, 4; successful cytokinesis, 5; one nucleus per cell at two cell stage, 6; AB divides first at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min. In this and all subsequent figures, red color indicates a defective trait, black color represents the lack of a defect, and white indicates that the trait was not visible in the recording.
Figure 3.
A. DIC time-lapse images of wild-type and rsa-1(or598 ts) embryos. In the rsa-1 mutant the nuclear centrosomal complex (NCC) failed to rotate and a small transverse P0 spindle assembled, cytokinesis failed, and multiple nuclei were present at the two cell equivilent stage. The rsa-1(or598 ts) embryo was shifted to the restrictive temperature for∼1 min. prior to imaging. Black dots represent centrosomes/ spindle poles and asterisks denote multiple nuclei per cell at the two cell stage. Times in min:sec are given relative to NEBD. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the C. elegans protein. C. Defect map for individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; pronuclei meet prior to NEBD, 2; Nuclear centrosomal complex centration, 3; Nuclear centrosomal complex rotation, 4; spindle size, 5; successful cytokinesis, 6; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.
Figure 4.
A. DIC time-lapse images of wild-type, dnc-1(or404 ts), dnc-1(or676 ts), dnc-4(or618 ts), and dnc-4(or633 ts) embryos. In the dynactin mutants the NCC often failed to centrate and rotate, the P0 spindle was oriented transverse to the anterior posterior axis, and multiple nuclei were present per cell at the two cell stage. The dnc-1(or404 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 8 hours, the dnc-1(or676 ts) and dnc-4(or633 ts) embryos were shifted to the restrictive temperature for ∼1 min. prior to imaging, and the dnc-4(or618 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 7 hours. Black dots represent centrosomes/spindle poles, asterisks denote multiple nuclei per cell, and the “m” denotes the maternal pronucleus that did not meet the male pronucleus prior to NEBD. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Sequence alterations in the mutants. Asterisks indicate the changed residues (or nucleotide for dnc-4(or633 ts). Homologous proteins are aligned below the C. elegans proteins. dnc-4(or633 ts) contains a mutation in an intron that may affect RNA splicing. C. Individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; pronuclei meet prior to NEBD, 2; Nuclear centrosomal complex centration, 3; Nuclear centrosomal complex rotation, 4; spindle alignment, 5 one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.
Figure 5.
A. DIC time-lapse images of wild-type, mei-1(or642 ts) and mei-1(or646 ts) embryos. In the mei-1 mutants the polar bodies were large and misshapen and embryos contained multiple [top mei-1(or642 ts) embryo and mei-1(or646 ts)] or zero maternal pronuclei (second mei-1(or642 ts) embryo). The two mei-1(or642 ts) embryos were obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes, the mei-1(or646 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 7 hours prior to imaging. White arrowheads indicates polar bodies, black arrowheads indicate multiple maternal pronuclei, the black arrow denotes multiple nuclei per cell at the two cell stage, and the “p” refers to the paternal pronucleus in an embryo lacking a maternal pronucleus. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Defect maps of individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal polar body size, 2; normal pronuclear number, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes. C. Amino acid alteration in the mutants. Asterisk indicates the changed residue. Homologous proteins are aligned below the C. elegans protein.
Figure 6.
A. DIC time-lapse images of wild-type, spd-2(or293 ts), spd-2(or454 ts), spd-2(or493 ts), and spd-2(or655 ts) embryos. In the spd-2 mutants the pronuclei often met in the center, NCC rotation failed, a bipolar spindle failed to assemble, cytokinesis failed, and there were aberrent numbers of nuclei present at the two cell stage. The spd-2(or293 ts), spd-2(or454 ts), and spd-2(or493 ts) embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The spd-2(or655 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for ∼1 min prior to imaging. Black arrows indicate instances when pronuclei meet in the center of the embryo, asterisks represent one nucleus present in a two cell stage equivalent embryo, and white arrowheads indicate multiple nuclei. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Sequence alterations in the mutants. Asterisks indicates the changed residues (or nucleotide for spd-2(or454 ts). Homologous proteins are aligned below the C. elegans protein. C. Defect maps for the spd-2 mutants. Individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; nuclear centrosomal complex centration, 2; nuclear centrosomal complex rotation, 3; bipolar spindle, 4; successful cytokinesis, 5; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.
Figure 7.
A. DIC time-lapse images of wild-type, zyg-1(or278 ts), zyg-1(or297 ts), zyg-1(or409 ts), and zyg-1(or1018 ts) embryos. In the zyg-1 mutants the two cell stage blastomeres assembled monopolar spindles, cytokinesis failed, and there were multiple nuclei present at the four cell equivilent stage. The zyg-1(or278 ts), zyg-1(or409 ts), and zyg-1(or1018 ts) embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The zyg-1(or297 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes prior to imaging. Black arrows indicate normal bipolar spindles in the wild-type embryo and white arrowheads indicate multiple nuclei present at the four cell equivalent stage. Times in min:sec are given relative to AB nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Amino acid alterations in the mutants. Asterisks indicate the changed residues. Homologous proteins are aligned below the C. elegans protein. C. Defect maps for the zyg-1 mutants.Individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal two cell embryo, 2; bipolar spindles at two cell stage, 3; one nucleus per cell at four cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes.
Figure 8.
A. DIC time-lapse images of wild-type and plk-1(or683 ts) embryos. In the plk-1 mutant the nuclear centrosomal complex (NCC) failed to rotate, a transverse P0 spindle assembled, and the daughter blastomeres were binucleate. The plk-1(or683 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 6 hours prior to imaging. Black dots represent centrosomes/spindle poles and asterisks denote multiple nuclei per cell at the two cell stage. Times in min:sec are given relative to NEBD. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates changed residue. Homologous proteins are aligned below the C. elegans protein. C. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; nuclear centrosomal complex rotation, 2; spindle alignment, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.
Table 5.
Summary of the TS mutant loci and comparison of previously available alleles.
Figure 9.
A. DIC time-lapse images of wild-type par-2(or373 ts), par-2(or539 ts), and par-2(or640 ts) embryos. The blastomeres in the par-2 mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The par-2(or373 ts) embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The par-2(or539 ts) and par-2(or540 ts) embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the par-2(or373 ts) mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the C. elegans protein.
Figure 10.
Sequence alterations in the lit-1(or393 ts) and mex-1(or286 ts) mutants.
Amino acid alterations in the mutants. Asterisks indicates changed residues. Homologous proteins are aligned below the C. elegans protein for LIT-1. A glutamine codon was changed to a stop codon in the mex-1(or286 ts) allele.