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. 1999 Nov 1;13(21):2838-51.
doi: 10.1101/gad.13.21.2838.

The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode caenorhabditis elegans

C A Rappleye  1 , A R Paredez, C W Smith, K L McDonald, R V Aroian
Affiliations

The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode caenorhabditis elegans

C A Rappleye et al. Genes Dev. .

Abstract

Establishment of anterior-posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. Previously, we isolated an F-actin-binding protein that was enriched in the anterior cortex of the one-cell embryo and was hypothesized to link developmental polarity to the actin cytoskeleton. Here, we identify this protein, POD-1, as a new member of the coronin family of actin-binding proteins. We have generated a deletion within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. We conclude that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. We propose models to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis.

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Figures

Figure 1
Figure 1
The sequence of POD-1 contains two regions with significant similarity to coronin. (A) Predicted amino acid sequence of POD-1. The two separate regions showing sequence similarity to coronin are shaded. Predicted WD-motifs are underlined and the proline-rich region is indicated by asterisks. (B) Schematic representation of POD-1 showing the two regions of coronin homology and the respective sequence identity shared with Dictyostelium coronin. Lines above the POD-1 protein indicate residues removed by the deletion allele ye11 and the location of the POD-1 polypeptides used to generate the 11-1 and 11-9 antibodies.
Figure 2
Figure 2
Loss of pod-1 function leads to defects in physical and molecular markers of a-p polarity. All embryos are oriented with left being anterior. (A,B) DIC images. (C–H) Immunofluorescence images. (A) Wild-type one-cell embryo dividing asymmetrically into AB and P1. (Right) Orthogonal orientations of the next round of divisions are indicated by arrows. These divisions will also occur asynchronously with AB dividing before P1. (B) A pod-1(ye11) one-cell embryo dividing symmetrically into equivalently sized blastomeres. The next round of divisions occurs synchronously and in parallel orientations (arrows, right). pod-1(ye11) mutant embryos are slightly smaller (see text) and also fail to extrude polar bodies, which appear as an additional pronucleus (asterisk). (C–H) One-cell embryos between pronuclear meeting and anaphase showing the distribution of P granules, PAR-3, and PAR-1. (C) In pod-1(+) embryos, P granules are segregated to the posterior cytoplasm. (D) pod-1(ye11) mutant embryos fail to properly segregate P granules. (E) PAR-3 is restricted to the anterior cortex of pod-1(+) embryos. (F) Loss of pod-1 causes delocalization of PAR-3 to both the anterior and posterior cortices. (G) Cortical PAR-1 is restricted to the posterior of pod-1(+) embryos. (H) Loss of pod-1 causes delocalization of PAR-1 to both the anterior and posterior cortices.
Figure 3
Figure 3
POD-1 is localized asymmetrically in germ-line blastomeres in a cell cycle-dependent fashion. Immunofluorescence images of POD-1 (green), actin (red), and DNA (blue) staining in early C. elegans embryos. All embryos are oriented with anterior to the left. All embryos are wild type except for G. (A) One-cell embryo in pronuclear migration showing asymmetric POD-1 (extent indicated by arrowheads). (B) One-cell embryo in prometaphase showing asymmetric POD-1 (extent indicated by arrowheads). The difference in POD-1 staining between A and B is indicative of various extents of POD-1 asymmetry. (C) One-cell embryo in telophase. POD-1 stains equally the anterior and posterior cortex and localizes to the cleavage furrow. (D) Early two-cell embryo with P1 in early prometaphase. Cortical POD-1 is present in the posterior of P1. (E) Two-cell embryo with P1 in metaphase. POD-1 staining is depleted from the posterior and is enriched at the anterior–ventral cortex of P1 (arrowhead). (F) A four-cell embryo with P2 in metaphase. POD-1 is enriched on the anterior–dorsal side (arrowhead). (G) A pod-1(ye11) four-cell embryo. There is no detectable POD-1 staining in the mutant, but cortical actin is still present. (a) Anterior; (p) posterior; (d) dorsal; (v) ventral.
Figure 4
Figure 4
Loss of pod-1 function leads to the formation of abnormal cellular structures. (A,C,E–J) DIC images. (B,D) Fluorescent images showing FM 4-64 staining. Anterior is oriented to the left in each image. (A) Dividing one-cell pod-1(ye11) embryo containing a large circular granule-free zone (arrow). (B) The same embryo as in A. The circle stains with FM 4-64, indicating it is endocytic in origin (arrow). (C) One-cell pod-1(ye11) late anaphase embryo with a large hyaline protrusion at the posterior (arrowhead). (D) The same embryo as in C. The hyaline protrusion is contained within the plasma membrane but does not form an intracellular compartment (arrowhead). (E–G) Time-lapse series of images of a pod-1(ye11) mutant embryo with cell division defects at the one- to two-cell stage. (E) The first cleavage furrow has formed and appears to be complete. (F) Within a few minutes, the division vanishes leading to the formation of a multinucleated cell. (G) Subsequent division attempts lead to additional cleavage furrows. (H–J) Osmotic sensitivity of pod-1(ye11) mutant embryos. (H) In 100 mm KCl, pod-1(ye11) mutant embryonic cells swell. (I) In 150 mm KCl, pod-1(ye11) mutant embryonic cells have morphology similar to wild type. (J) In 250 mm KCl, pod-1(ye11) mutant embryos shrink and become highly refractile.
Figure 5
Figure 5
Ultrastructure of wild-type and pod-1 mutant eggshells. In each panel, an embryo (to the right) with its eggshell is shown. (A) Three layers are visible in the wild-type C. elegans eggshells: a thin vitelline layer (black line indicated with lower arrowhead), a chitinous layer (light-staining layer indicated with central arrowhead), and a lipid-rich layer (darker layer indicated by upper arrowhead). (B) All three layers are present and appear normal at this resolution in the pod-1(ye11) eggshell (left pointing arrowheads). However, an extra layer of dark-staining material is evident on the outside of the pod-1 mutant embryos (right pointing arrowhead) that is not evident in wild type.
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