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First published online 31 January 2006
doi: 10.1242/jcs.02742

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Abp1 regulates pseudopodium number in chemotaxing Dictyostelium cells

Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA

View larger version (9K): [in a new window]   Fig. 1. Domain structures of Abp1 orthologs in different species. M.m., Mus musculus; S.c., Saccharomyces cerevisiae; D.d., Dictyostelium discoideum; ADFH, actin depolymerizing factor homology domain; PPP, proline-rich region; SH3, Src homology 3 domain. Numbers indicate amino acid identity shared between domains of abp1 orthologs.

View larger version (89K): [in a new window]   Fig. 2. Knockout and overexpression of Dabp1. A western blot of whole cell lysates from 2.5x105 wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) was stained with affinity-purified anti-Dabp1 antibodies.

View larger version (60K): [in a new window]   Fig. 3. Dabp1 is enriched in the cortex and the leading edge of chemotaxing cells. Growing cells (top panels) and developing cells (bottom panels) were fixed and stained with affinity-purified anti-Dabp1 antibodies followed by BODIPY-FL-labeled secondary antibodies (to detect Dabp1) and TXRED-labeled phalloidin (to detect actin). Arrows and arrowhead show areas where Dabp1 and actin colocalize (in the merged images). Bar, 10 µm.

View larger version (77K): [in a new window]   Fig. 4. (A) The cortical localization of Dabp1 requires an organized actin cytoskeleton. Wild-type cells were treated with 10 µM cytochalasin A for 40 minutes and then stained with affinity-purified anti-Dabp1 antibodies (Dabp1) and TXRED-labeled phalloidin (actin). (B) Neither the absence of, nor an increase in, Dabp1 protein influences the actin cortex. Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were fixed and stained with TXRED-labeled phalloidin. Bar, 10 µm.

View larger version (137K): [in a new window]   Fig. 5. Overexpression of Dabp1 delays early development. Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were submerged under starvation buffer to induce chemotaxis, and photographed at various times as indicated. Bar, 100 µm.

View larger version (41K): [in a new window]   Fig. 6. Overexpression of Dabp1 affects early development. Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were placed on nonnutrient agar plates. Images were taken of cells after the aggregation stage (Aggregates) and after the final fruiting body stage (Fruiting bodies). Bar, 100 µm.

View larger version (84K): [in a new window]   Fig. 7. Overexpression of Dabp1 causes morphological changes during chemotaxis. Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were submerged under starvation buffer until they were actively streaming and photographed to study the morphology. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 8. Actin distribution in supernumerary pseudopodia in Dabp1+ cells is normal. Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were grown in medium (Growing cells) or submerged under starvation buffer until they were actively streaming (Developing cells), and then fixed and stained with TXRED-labeled phalloidin to image the actin cytoskeleton. Bar, 10 µm.

View larger version (18K): [in a new window]   Fig. 9. Dabp1 affects cell motility during chemotaxis in early development. (A) Wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) were submerged under starvation buffer until the cells were actively streaming. Cells were photographed every 12 seconds using DIC optics. Their positions at 15 time points are shown for a typical cell from each cell line. (B) Histogram showing velocities of wild-type cells (WT), Dabp1 null cells (Dabp1-) and cells overexpressing Dabp1 (Dabp1+) during chemotaxis in early development.

View larger version (54K): [in a new window]   Fig. 10. The SH3 domain is important for the function of Dabp1. (A) Diagram of constructs for ADFH and SH3 domain of Dabp1. (B) Morphology of wild-type cells (WT) and cells overexpressing the ADFH domain (ADFH+) and the SH3 domain (SH3+). (B) Cells were placed in the starvation buffer until they were actively streaming (about 9 hours) and then imaged under DIC optics. Bars, 10 µm.

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