<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> 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 <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

<i>mei-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>mei-1(or642 </i>ts<i>) and mei-1(or646 </i>ts<i>)</i> embryos. In the <i>mei-1</i> mutants the polar bodies were large and misshapen and embryos contained multiple [top <i>mei-1(or642 </i>ts<i>)</i> embryo and <i>mei-1(or646 </i>ts<i>)</i>] or zero maternal pronuclei (second <i>mei-1(or642 </i>ts<i>)</i> embryo). The two <i>mei-1(or642 </i>ts<i>)</i> embryos were obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes, the <i>mei-1(or646 </i>ts<i>)</i> 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 <i>C. elegans</i> protein.</p

<i>zyg-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or297 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos. In the <i>zyg-1</i> mutants the two cell stage blastomeres assembled monopolar spindles, cytokinesis failed, and there were multiple nuclei present at the four cell equivilent stage. The <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The <i>zyg-1(or297 </i>ts<i>)</i> 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 <i>C. elegans</i> protein. C. Defect maps for the <i>zyg-1</i> 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.</p

Summary of the TS mutant loci and comparison of previously available alleles.

1<p>Information obtained from: <a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>.</p

The phenotypes of the TS mutants when grown at the restrictive temperature from the L1 larval stage.

1<p>We determined the L1 upshift phenotype by plating hypochlorite-synchronized L1 larvae and incubating them at 26°C until they reached adulthood. Abbreviations: Egl: egg laying-defective, Emb: embryonic lethal, Pvl: protruding vulva, Sb: small broods, Ste: sterile.</p

Determination if the TS mutations are potentially fast-acting.

1<p>We determined if an allele was potentially fast-acting in the following manner: We mounted embryos produced at 15°C on microscope slides and immediately made time-lapse videomicrographs at a room maintained at 24°C. If defects similar to those observed after long temperature shifts were found in at least 20% of the embryos and if there was little embryonic lethality at 15°C, we conclude that the allele may be fast-acting. We have labeled these cases as “Yes”. However, if there was significant embryonic lethality at 15°C, we cannot conclude that the presence of cellular defects after short upshifts is due to the upshift or to defects that occur even at 15°C. We have labeled these cases as “Unclear”.</p>2<p>For <i>mei-1, par-2,</i> and <i>zyg-1</i>, we incubated mutant worms at 26°C for 30 minutes prior to imaging (instead of the usual∼1 min. upshift) because the gene products appeared to be required prior to when we started our imaging (pronuclear migration).</p>3<p>High lethality at the permissive temperature precludes making a determination.</p>4<p>The low penetrance of severe defects precludes making a determination.</p

A <i>plk-1</i> mutant.

<p>A. DIC time-lapse images of wild-type and <i>plk-1(or683 </i>ts<i>)</i> embryos. In the <i>plk-1</i> mutant the nuclear centrosomal complex (NCC) failed to rotate, a transverse P₀ spindle assembled, and the daughter blastomeres were binucleate. The <i>plk-1(or683 </i>ts<i>)</i> 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 <i>C. elegans</i> 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.</p

<i>spd-2</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>spd-2(or293 </i>ts<i>)</i>, <i>spd-2(or454 </i>ts<i>)</i>, <i>spd-2(or493 </i>ts<i>)</i>, and <i>spd-2(or655 </i>ts<i>)</i> embryos. In the <i>spd-2</i> 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 <i>spd-2(or293 </i>ts<i>), spd-2(or454 </i>ts<i>), and spd-2(or493 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The <i>spd-2(or655 </i>ts<i>)</i> 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 <i>spd-2(or454 </i>ts<i>)</i>. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect maps for the <i>spd-2</i> 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.</p

Dynactin mutants.

<p>A. DIC time-lapse images of wild-type, <i>dnc-1(or404 </i>ts<i>), dnc-1(or676 </i>ts<i>), dnc-4(or618 </i>ts<i>), and dnc-4(or633 </i>ts<i>)</i> 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 <i>dnc-1(or404 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 8 hours, the <i>dnc-1(or676 </i>ts<i>)</i> and <i>dnc-4(or633 </i>ts<i>)</i> embryos were shifted to the restrictive temperature for ∼1 min. prior to imaging, and the <i>dnc-4(or618 </i>ts<i>)</i> 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 <i>dnc-4(or633 </i>ts). Homologous proteins are aligned below the <i>C. elegans</i> proteins. <i>dnc-4(or633 </i>ts<i>)</i> 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.</p

A <i>sur-6</i> mutant.

<p>A. DIC time-lapse images of wild-type and <i>sur-6(or550 </i>ts<i>)</i> embryos. In the <i>sur-6</i> mutant the male pronucleus is small, the AB cell contains two nuclei, and the P₁ cell begins mitosis before the AB cell. The <i>sur-6(or550 </i>ts<i>)</i> 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 <i>C. elegans</i> 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.</p