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First published online 1 April 2003
doi: 10.1242/jcs.00421

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Specific requirement for two ADF/cofilin isoforms in distinct actin-dependent processes in Caenorhabditis elegans

Department of Pathology, Emory University, Whitehead IO5N, Atlanta, Georgia 30322, USA

View larger version (77K): [in a new window]   Fig. 1. Expression of mRNAs for UNC-60A and UNC-60B during C. elegans development. 25 µg of total RNA from the indicated stages was loaded on each lane and probed with radiolabeled cDNAs for unc-60A, unc-60B or 18S rRNA. Samples are embryos (lane 1), L1 larvae (lane 2), L2 larvae (lane 3), L3 larvae (lane 4), L4 larvae (lane 5), young adults (lane 6) and gravid adults (lane 7).

View larger version (96K): [in a new window]   Fig. 2. Localization of UNC-60A in early C. elegans embryos. Wild-type embryos at the one-cell stage (a-c), transition from the one-cell to two-cell stage (d-f), two-cell stage (g-i), and three-fold stage (j-l) were stained by anti-UNC-60A (a,d,g,j) and anti-actin (b,e,h,k) antibodies. Merged images of double-staining of UNC-60A (red) and actin (green) are shown (c,f,i,l). Staining of DNA by DAPI (blue) is shown in c, f and i to indicate the phases in the cell cycle. The nerve ring is indicated by an arrow in j. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 3. Localization of UNC-60A and UNC-60B in adult hermaphrodite gonads. Dissected gonads were stained with anti-UNC-60A (a) or anti-UNC-60B (b), anti-myoA (c,d) and DAPI (e,f). Merged images are shown in g and h: red, UNC-60A; green, myoA; blue, DAPI. Both UNC-60A and UNC-60B were expressed in the spermatheca (sp) (a,b). UNC-60A is also abundant in the germ cells in the distal gonad (dis) and maturing oocytes (o). Bar, 20 µm.

View larger version (47K): [in a new window]   Fig. 4. Reduction of the UNC-60A protein by RNA interference. Wild-type C. elegans strain was fed by E. coli HT115 (DE3) with the control plasmid (a-c) or the expression vector for the unc-60A dsRNA (d-f). The F1 embryos were stained with anti-UNC-60A (a,d), anti-tubulin (b,e), and DAPI (c,f). Micrographs of control and unc-60A (RNAi) were taken at the same exposure settings. Staining of UNC-60A was greatly reduced by the RNAi treatment (compare a with d), whereas weak UNC-60A staining was still observed in some structures (d, arrowheads). Staining with the anti-tubulin antibody (b,e) ensured that these embryos were properly permeabilized. Bar, 40 µm.

View larger version (141K): [in a new window]   Fig. 5. Effects of unc-60A (RNAi) on early development. Developmental processes of the control or unc-60A (RNAi) embryos from pronuclear meeting (a-c) to the four-cell stage (m-o) were observed by time-lapse DIC microscopy. A control embryo is shown in a, d, g, j, m and Movie 1. A representative cytokinesis defect resulting from unc-60A (RNAi) is shown in b, e, h, k, n and Movie 2. A cleavage furrow appeared after nuclear division (h) but regressed before completing the cleavage (k, arrows). The attempts at cleavage continued and resulted in irregular compartmentalization (n). A representative patterning defect is shown in c, f, i, l, o and Movie 3. Spindle orientation was normal at the second division (l), but the subsequent positioning of the four blastomeres was abnormal (o) compared with the control embryo (m). Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 6. Effects of unc-60A (RNAi) on polar body extrusion, cortical activity and anterior-posterior polarity. The unc-60A (RNAi) treatment resulted in the appearance of an extra pronucleus (a, arrow) owing to the lack of polar body extrusion and formation of abnormal membrane protrusions (b, arrowheads). (c-e) Measurements of markers for the anterior-posterior polarity in the one-cell embryo. (c) The position of pronuclear meeting was measured as x/Lx100, where x is the distance from the anterior pole (A) to the site of pronuclear meeting and L is the length of the egg between the anterior and posterior (P) poles. (d) Similarly, the positions of the anterior spindle pole (y/Lx100) and the posterior spindle pole (z/Lx100) were measured. Bars, 20 µm. (e) Data from control or unc-60A (RNAi) embryos (n=17) are presented by a box plot generated by SigmaPlot 2000 (SPSS Science). Boxes indicate the ranges between 25th and 75th percentiles, and bars on both sides indicate ranges between 10th and 90th percentiles. Bars in the boxes indicate medians.

View larger version (108K): [in a new window]   Fig. 7. Effects of unc-60A (RNAi) on cortical actin in early embryos. Staining of actin (a,d,g), tubulin (b,e,h), and DNA (c,f,i) in the control (a-c) and unc-60A (RNAi) embryos (four- and eight-cell stage) (d-f and g-i) are shown. Premature cleavage furrow (arrow in g) is indicated. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 8. Characterization of the unc-60B-null mutants. (A) Exon-intron structure of the unc-60 gene (McKim et al., 1994) and the locations of deletions in unc-60 (s1586) and unc-60 (su158). (B) Motility of wild-type, unc-60 (su158), or unc-60 (e677) homozygous adults. Data are means±s.d. n=10. (C) Expression of UNC-60A and UNC-60B in unc-60B mutants. Total lysates (10 µg proteins) from wildtype (lane 1), unc-60 (su158) (lane 2) and unc-60 (e677) (lane 3) were resolved by SDS-PAGE and visualized by Coomassie blue (a) or subjected to western blot with anti-actin (b), anti-UNC-60A (c) or anti-UNC-60B (d). The strong loss-of-function mutant unc-60 (e677) has a greatly reduced amount of UNC-60B (d, lane 3), whereas the unc-60 (su158) mutant has no detectable UNC-60B (d, lane 2). Molecular mass markers in kDa (lane M) are indicated on the left of a.

View larger version (81K): [in a new window]   Fig. 9. Assembly of myofibrillar components in the unc-60B-null mutant embryos. Wild-type (a-c) or unc-60 (su158) (d-f) embryos at the 1.75-fold stage (430 minutes after first cleavage) (A), two-fold stage (450 minutes) (B) and three-fold stage (520 minutes) (C) were stained with anti-actin (a,d), anti-myoA (b,e) and anti-vinculin (c,f) antibodies. Actin and myoA were stained by double-labeling of the same embryo. In Bd and Cd, arrows indicate the regions where actin is markedly disorganized. The age of the embryos was determined from their morphology as described previously (Epstein et al., 1993; Hresko et al., 1994). Bar, 20 µm.

View larger version (125K): [in a new window]   Fig. 10. Organization of myofibrillar components in the adult body wall muscle of the unc-60B-null mutant. Wild-type (a-c) or unc-60 (su158) (d-f) adult body wall muscles were stained with anti-myoA (Aa,d), anti--actinin (Ba,d) and anti-vinculin (Ca,d) and double-labeled with anti-actin (b,e). Merged images are shown (c,f). Bar, 20 µm.