ipla-1 mutants disrupt the orientation and polarity of the S4 divisions. (A) A schematic drawing of seam cell lineages. The V lineage seam cells are shown. Lineage branches expressing scm::gfp are shown in green. The dotted box marks the lineages analysed in this study; V5.pppp lineage (a), V6.papp lineage (b) and V6.pppp lineage (c). Grey circles, anterior daughters that fuse with the hyp7; green squares, posterior daughters that assume the seam cell fate again. Red and blue lines indicate the developmental stages corresponding to those of (B, C), and (D, E), respectively. (B, C) Fluorescent images of seam cells during or just after the S4 divisions visualized by scm::gfp. Three pairs of daughter cells are shown with brackets for each panel. (B) Wild-type. The three seam cells divide parallel to the A–P axis. (C) ipla-1(xh13). Seam cells, as shown by asterisks, divide in various directions. (D, E) Seam cells of late L4 hermaphrodite visualized by scm::gfp. (D) Wild-type. The posterior daughters assume the seam cell fate in all the three lineages. (E) ipla-1(xh13). The seam cell fates are adopted by either or both of the anterior and posterior daughters and the mitotic spindle is misoriented. The seam cell lineages are shown in the images. The letters (a), (b) and (c) correspond to those of (A). Results are summarized in (I). The characters indicate the directions of the S4 divisions; a, anterior; p, posterior; av, anterior-ventral; ad, anterior-dorsal; pv, posterior-ventral and pd, posterior-dorsal. (F) Schematic for measurement of spindle orientation. A, anterior; P, posterior; D, dorsal; and V, ventral. (G, H) Quantification of spindle orientation of the S4 divisions. The percentages are determined from a random sample of angles from wild-type (G) or ipla-1(xh13) (H) seam cells measured as depicted in (F). (I) Abnormal V-cell lineages in ipla-1(xh13) after the S4 divisions. The numbers of seam cells that showed the lineages are indicated below the diagrams. The letters (a), (b) and (c) correspond to those of (A). Spindle orientation is defective in types IV–VI (indicated with diagonal lines). Additional cell divisions occur just after the first divisions in types III and VI. The S4 divisions do not occur in types VII and VIII. The dotted line in type VIII indicates that the scm::gfp fluorescence disappears during the L4 stage. Anterior is oriented towards the left. Scale bars in (B–E) are 20 μm.

Structure of the ipla-1 suppressor locus, gene and protein. (A) xh23 locus. xh23 mutation maps to LG IV between snip-SNPs markers pkP4080 and dbP3. Boxes, exons; lines, introns; red, an exon specific for tbc-3a; blue, exons specific for tbc-3b. xh23 is a G-to-A base change that generates a missense mutation predicted to change an aspartic acid-234 to asparagine. (B) Domain structure, mutation and multiple sequence alignment of TBC-3b and TBC1D22 homologues. TBC domains, light blue. The overall amino-acid identities between TBC-3b and its homologues in human, Drosophila and yeast are shown as well as the identities between the TBC domains. C. e., C. elegans F32B6.8b (NP_001023165); H. s., Homo sapiens TBC1D22B (NP_060242); D. m., Drosophila melanogaster CG5745-PA (NP_650941); S. c., Saccharomyces cerevisiae Gyp1p (NP_014713). (C) Multiple sequence alignment of Rab GAP proteins. The aspartic acid residue, which is mutated in xh23, is completely conserved across the Rab GAP family and indicated in red (Pan et al, 2006). (D) xh22 locus. xh22 mutation maps to LG IV between snipSNP markers snp_K08E4[2] and pkP4087. Boxes, exons; lines, introns. xh22 is a G-to-A base change that predicts translation of a stop codon instead of tryptophan-787. (E) Domain structure, mutation and multiple sequence alignment of MON-2 and Mon2 homologues. DCB domain, green; HUS domain, light blue; HDS1-3 domain, red (Mouratou et al, 2005). The overall amino-acid identities between MON-2 and its homologues in human, Drosophila and yeast are shown as well as the identities between the conserved domains. C. e., C. elegans F11A10.4 (NP_502295); H. s., Homo sapiens MON2 homologue (NP_055841); D. m., Drosophila melanogaster CG8683-PA (NP_001033884); S. c., Saccharomyces cerevisiae Mon2p (NP_014102). (F) Quantification of spindle orientation in tbc-3(xh23), ipla-1(xh13);tbc-3(xh23), mon-2(xh22) and ipla-1(xh13);mon-2(xh22). The angles are measured as depicted in Figure 2F.

ipla-1 mutants have aberrant number and alignment of seam cells. (A–D) Seam cells in adult hermaphrodites were visualized by a seam cell marker::gfp (scm::gfp) fusion. (A) Wild-type. Evenly spaced 16 scm::gfp-positive nuclei are observed. (B–D) ipla-1(xh13). More than 16 seam cell nuclei align unevenly. scm::gfp-positive nuclei are occasionally observed outside the row of seam cells (C, arrow). ipla-1(xh13) mutants show loss of scm::gfp-positive nuclei (D, delimited by arrowheads). (E) The number of scm::gfp-positive nuclei in each developmental stage. On average, more scm::gfp-positive nuclei were observed in ipla-1(xh13) than in wild-type animals at the late L4 stage and the young adult stage (YA). L2 cell counts were often high by one cell because of the comparatively late division of cells of the T lineage, causing both daughters to be scored. The numbers in parentheses indicate the number of animals scored. (F) Stage-specific rescue of seam cell defects of ipla-1(xh13). Synchronized ipla-1(xh13) animals carrying hsp::ipla-1 were heat shocked at various developmental times. Rescue was obtained by heat-shock treatments before the L4 stage. The number above each point is the number of animals scored. Scale bars are 50 μm in (A–D).

ipla-1 regulates polarity of the seam cells in the S4 divisions. (A) Schematic for the seam cells before the S4 divisions. WRM-1 localization is shown in green; WRM-1 localizes to the anterior cortex of the seam cells. (B, C) Confocal images showing the localization of WRM-1::GFP in the V6.papp and V6.pppp cell of wild-type (B) and ipla-1(xh13) (C). (D) Frequency of the localization patterns of WRM-1::GFP. The number of samples is shown above each column. The equality and inequality signs indicate relative intensities of cortical WRM-1::GFP. A, intensity of WRM-1::GFP at the anterior cortex; P, intensity of WRM-1::GFP at the posterior cortex. No cortex means that WRM-1::GFP signal is absent from the cortex of the cells. (E) Schematic for the seam cells just after the S4 divisions. POP-1 localization is shown in green; the anterior daughters contain more nuclear POP-1 than posterior daughters. (F, G) Confocal images showing the localization of GFP::POP-1 in wild-type (F) and ipla-1(xh13) (G). Pairs of daughter nuclei are shown with brackets for each panel. The equality and inequality signs indicate relative intensities of GFP::POP-1 levels between two daughters. (H) Frequency of the localization patterns of GFP::POP-1. The number of samples is shown above each column. A, intensity of nuclear GFP::POP-1 in the anterior daughter; P, intensity of nuclear GFP::POP-1 in the posterior daughter. Scale bars are 10 μm in (B, C), or 20 μm in (F, G). Anterior is oriented towards the left; ventral is oriented towards the bottom.

Retrograde trafficking is involved in cortical asymmetry of WRM-1 before the S4 divisions. (A–C) Confocal images showing the localization of WRM-1::GFP in the V6.pppp cell of ipla-1(xh13);mon-2(RNAi) (A), ipla-1(xh13);tbc-3(RNAi) (B) and ipla-1(xh13);snx-1(RNAi) (C). Scale bars are 5 μm. (D) Frequency of the localization patterns of WRM-1::GFP. The number of samples is shown above each column. (E) Wild-type. The A–P polarity is formed through biosynthetic sorting from the Golgi (black arrow), and is maintained by protein degradation pathway (blue dotted arrow); proteins on plasma membrane (black boxes) are endocytosed and degraded through an ipla-1-dependent mechanism (black dotted arrows). ipla-1 promotes degradation pathway directly, or indirectly by inhibiting the endosome-to-Golgi retrograde pathway that requires the retromer complex, mon-2 and tbc-3b (purple dotted arrow). As a result, β-catenin wrm-1 localizes to the anterior cortex (green ellipses). (F) ipla-1 mutants. The endocytosed proteins are not degraded, but transported to the Golgi by the retromer, mon-2 and tbc-3b, and then missorted to various plasma membrane domains. As a result, cortical asymmetry of β-catenin wrm-1 is disturbed. (G) ipla-1;sup double mutants, or ipla-1;sup (RNAi). The endocytosed proteins are retained in the endosome or progress towards the lysosome through an ipla-1-independent mechanism (shown with an additional blue dotted arrow).