The C. elegans evl-20 Gene Is a Homolog of the Small GTPase ARL2 and Regulates Cytoskeleton Dynamics during Cytokinesis and Morphogenesis  Igor Antoshechkin, Min Han  Developmental Cell  Volume 2, Issue 5, Pages 579-591 (May 2002) DOI: 10.1016/S1534-5807(02)00146-6

Figure 1 evl-20 Mutant Phenotypes (A and B) Nomarski images of wild-type vulva (A) and vulva of homozygous evl-20(ar103) mutant animals (B) in the mid L4 stage. Arrowheads in (B) show two nucleoli within an enlarged nucleus. Here and in figures below, anterior is to the left, and dorsal is to the top, unless indicated otherwise. Scale bar in (A) is 5 μm. (C and D) Nomarski images of the anterior arm of the gonad of a wild-type (C) and homozygous evl-20 (D) hermaphrodite in the late L4 stage. Arrowheads in (D) show two nucleoli within an enlarged nucleus. Scale bar in (C) is 10 μm. (E and F) Nomarski images of the tail of a wild-type (E) and homozygous evl-20 (F) male at the end of L4 stage. Images are at the same magnification as (C) and (D). (G) Lineage analysis of evl-20 mutant vulva. Each line represents either wild-type (first line) or evl-20 mutant pattern of final divisions of P5.p–P7.p (Horvitz and Sternberg, 1991). T, cell divided along the left-right (transverse) axis; L, cell divided along the anterior-posterior (longtitudinal) axis; O, cell divided along an oblique axis; N, cell did not divide; SS, Pn.p cell divided once, and daughters fused with the hypodermal syncytium; U, daughter of Pn.p (after first round of divisions) did not divide. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 2 evl-20 Mutant Animals Display Cytokinesis Defects (A–F) Expression patterns of egl-17::gfp reporter construct were examined in the vulva of homozygous animals. Each panel represents Nomarski and corresponding fluorescence images that have been merged. (A and D) Wild-type and evl-20 mutant vulvae after one round of VPC divisions in early L3 stage, respectively. Arrowheads indicate GFP-expressing P6.pa and P6.pp. Arrow indicates anchor cell (AC). (B and E) Wild-type and evl-20 mutant vulva in late L3 stage. Arrowhead indicates GFP-expressing progeny of P6.p. (C and F) Wild-type and evl-20 mutant vulva in mid L4 stage. Arrowheads indicate GFP-expressing P5.p and P7.p progeny. (G–L) Live evl-20 homozygous animals were stained with a fluorescent lipophilic dye, FM 1-43 (Molecular Probes). Panels (G) and (J) show Nomarski images of the gonad in late L4 stage. (H) and (K) show corresponding fluorescent images, and (I) and (L) show merged images. Arrowheads point to nuclei of multinucleated cells. Scale bar in (A) is 5 μm. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 3 evl-20 Encodes an ARF-like Protein (A) Genetic and physical map of evl-20 region. evl-20 maps to LGII next to rol-6. Cosmids corresponding to this physical region as well as subclones of the rescuing cosmid F14E5 are shown. The minimal rescuing fragment carries an operon with two predicted ORFs indicated by arrows. (B) Gene structure of F22B5.1 determined by 3′ and 5′ RACE. Position of the amber stop codon created by mutation in ar103 is indicated. (C) Protein sequence alignment of EVL-20, human ARL2, Drosophila ARL2, S. pombe ALP41, and human ARF1. Gln → Stop at position 140 is marked by asterisk. Alignment was performed using ClustalW algorithm of MacVector software package (Oxford Molecular Group). Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 4 Expression Patterns of evl-20 Expression patterns were analyzed in stable unc-119-rescuing lines with an extrachromosomal array carrying evl-20::gfp transcriptional reporter construct and a wild-type unc-119 genomic fragment. (A–C) Nomarski, fluorescence, and merged images of a wild-type embryo during hypodermal enclosure, respectively (ventral view). Expression of evl-20::gfp reporter is seen in migrating hypodermal cells. In these panels, anterior is to the left, and right is to the top. Scale bar in (A) is 5 μm. (D–F) Nomarski, fluorescence, and merged images of the mid region of a wild-type hermaphrodite in mid L4 stage, respectively (lateral view). Expression of evl-20::gfp reporter is seen in the vulva and the uterus. Scale bar in (D) is 10 μm. (G–I) Nomarski, fluorescence, and merged images of a wild-type male tail in early L4 stage, respectively (lateral view). Expression of evl-20::gfp reporter is seen in the proctodeum and some other cells. Scale bar in (G) is 10 μm. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 5 evl-20 Is Essential for Embryogenesis Progeny of wild-type animals injected with double-stranded evl-20 RNA were analyzed under Nomarski optics. Scale bar in (A) is 5 μm. (A) Embryo arrested during the proliferative stage. Cells that display cytokinesis defects are indicated by arrowheads. (B) Embryo arrested at the lima bean stage. (C) Partially elongated embryo. Pharynx structures are indicated by arrowheads. (D) Embryo ruptured in the anterior during elongation. jam-1::gfp-marked pharynx structures are shown by arrowheads. (E–H) Nomarski and corresponding jam-1::gfp images of wild-type (E and F) and evl-20(RNAi) (G and H) embryos at the beginning of elongation. jam-1::gfp marks adherens junctions between hypodermal, pharynx (marked with arrowheads in [F]), and gut cells. evl-20(RNAi) embryo failed to properly undergo hypodermal enclosure and ruptured during elongation. Arrowheads in (G) and (H) indicate leaking content of the embryo, including GFP-expressing pharynx structures. (I) Escaped hatched L1 larva. Posterior part of the animal is severely disorganized. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 6 evl-20 Regulates Microtubule Cytoskeleton Microtubule and actin cytoskeletons were examined in wild-type and evl-20(RNAi) embryos. Panels (A)–(H) show Nomarski and corresponding fluorescent images of microtubule cytoskeleton visualized in live animals using an integrated β-tubulin tagged with GFP. Images in panels (D) and (H) were taken with a four times-longer exposure than those in (B) and (H) and brightened by 50% using Adobe Photoshop. Arrowheads in (B) and (D) point to mitotic spindles. Panels (K), (L), and (N) show embryos stained with anti-α-tubulin antibody. Filamentous actin was visualized by staining with Alexa Fluor 488 phalloidin (Molecular Probes). Scale bars in (A) and (K) are 5 μm. (A and B) Wild-type embryo during the proliferative stage. (C and D) RNAi-treated embryo during the proliferative stage. (E and F) Wild-type embryo during hypodermal enclosure. (G and H) RNAi-treated embryo during hypodermal enclosure. (I and J) Actin cytoskeleton in wild-type and RNAi-treated embryos during the proliferative stage, respectively. (K and L) Wild-type and RNAi-treated embryos, respectively, stained with anti-α-tubulin antibody. Images taken with the same exposure. (N) Same image as in (L) brightened by 80% using Adobe Photoshop shows that spindle microtubules are still present, although at significantly reduced levels. (M) Western blot using anti-α-tubulin antibody shows no significant difference in total tubulin content. As a control, blot was reprobed with anti-actin antibody. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)

Figure 7 Subcellular Localization of EVL-20 EVL-20 subcellular localization was determined by staining with anti-EVL-20 monoclonal antibody. EVL-20, red; tubulin, green; DNA, blue. Scale bar in (A) is 5 μm. (A and B) EVL-20 localizes to the cortical plasma membrane of embryonic blastomeres. (C) EVL-20 is associated with astral microtubules. (D) RNAi control taken with five times-longer exposure shows no specific EVL-20 or tubulin-GFP staining. Developmental Cell 2002 2, 579-591DOI: (10.1016/S1534-5807(02)00146-6)