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First published online 12 August 2003
doi: 10.1242/jcs.00705

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Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans

¹ Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA
² Molecular Neurobiology Program, Department of Pharmacology, Skirball Institute, New York University School of Medicine, 540 First Avenue, SKI 5-17, New York, NY 10016, USA

View larger version (61K): [in a new window]   Fig. 1. Talin RNAi results in DTC migration defects. N2 hermaphrodites were grown on E. coli HT115(DE3) carrying empty vector (A) or pPD129.36-talin plasmid (B). Arrow in B points to clearing in the body cavity from displacement of the dark gray intestines. Both animals are anterior to the left, ventral up. DIC images show wild-type (C) and 52-hour talin RNAi (D) hermaphrodite gonad arms. Arrows indicate locations of DTCs. Anterior is to the left, ventral down in both images. Scale bars: 100 µm (A,B), 25 µm (C,D).

View larger version (50K): [in a new window]   Fig. 2. Expression pattern of talin in adult hermaphrodites. The GFP expression pattern was examined in the talin::GFP(zdIs15) line by fluorescence microscopy. In these animals, GFP is expressed under the control of the talin promoter. (A) GFP is seen throughout the body wall and sex muscle cells. Confocal microscopy of dissected gonad arms shows GFP expression in the DTC (B, arrow) and in the gonad sheath cells (C, arrow). Scale bars: 25 µm.

View larger version (78K): [in a new window]   Fig. 3. Ovulation defects in talin RNAi hermaphrodites. Nematodes were grown on HT115(DE3) with empty vector (A,C) or with pPD129.36-talin (B,D) for 52 hours. (A) DIC image shows normal linear arrangement of oocytes in the proximal gonad arm. (B) This arrangement contrasts with stacking of oocytes in a distended gonad arm of talin RNAi animals. (C) Normal DNA organization in the germ cells and oocytes (arrows) was visualized by DAPI staining. (D) Talin RNAi animals had stacked oocytes in the proximal gonad arm (arrows) and germ line nuclei were observed throughout the gonad arm. Brackets in C and D indicate location of sperm. All animals are anterior to the left, ventral down. Scale bars: 25 µm.

View larger version (45K): [in a new window]   Fig. 4. Actin filament organization is disrupted in the gonad sheath cells. Rhodamine-conjugated phalloidin was used to stain actin filaments in dissected gonad arms of (A) animals grown on bacteria carrying the empty RNAi vector or (B) talin RNAi animals for 48 hours. (A) Proximal gonad arm has longitudinal actin filaments in the sheath cells. This pattern is absent from the proximal arm of a talin RNAi gonad (B, left), but actin filaments appear normal in distal gonad arm (B, right). Scale bars: 25 µm.

View larger version (35K): [in a new window]   Fig. 5. Progressive paralysis and loss of muscle filament organization with talin RNAi. (A) Time course of paralysis in talin-depleted animals (circles) or animals grown on bacteria carrying the empty RNAi vector (squares). Paralyzed animals were counted at 24, 48, 52, 58 and 72 hours. The error bars represent the standard deviation of at least 5 independent experiments. (B) At 72 hours, paralyzed animals are immobile except for occasional movement of the head. Staining with MH25 anti-PAT-3 monoclonal antibody was used to visualize dense bodies in body wall muscle cells of animals grown on bacteria with empty vector (C) and with talin RNAi (D) for 72 hours. Scale bars: 100 µm (B), 25 µm (C,D).

View larger version (111K): [in a new window]   Fig. 6. Depletion of PAT-2/ or PAT-3/ß integrins disrupts muscle structure. Rhodamine-conjugated phalloidin was used to stain actin filaments in body wall muscle cells of animals grown on bacteria carrying the empty vector (A), or bacteria expressing dsRNA for talin (B), pat-2 (C) and pat-3 (D) for 72 hours. Compared with wild type, actin filaments were disorganized in all of the RNAi-treated animals. Percentages in each panel represent proportion of paralyzed animals at 72 hours post-hatch (±s.d.). Scale bars: 25 µm.

View larger version (16K): [in a new window]   Fig. 7. Bacterial RNAi decreases mRNA levels for talin, pat-2 and pat-3. Real-time RT-PCR analysis was applied after RNAi treatment. Shown are relative levels of RT-PCR products amplified from total RNA isolated from control animals fed bacteria containing empty vector (-) or bacteria expressing the indicated dsRNA (+) for 48 hours. Data were normalized to GAPDH and (-) cDNA samples were set to 1.

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