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. 2013 Sep 2;202(5):793–806. doi: 10.1083/jcb.201303005

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© 2013 Worth et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Drebrin contains two F-actin–binding domains responsible for filopodia formation in heterologous cells. (A) Domain diagrams of drebrin and drebrin deletion constructs used for the functional domain analysis of drebrin. Drebrin has five potential functional domains identified by an in silico analysis using InterPro, Pfam, SMART, and PROSITE. Constructs are YFP-tagged at the C terminus. ADFH, actin-depolymerizing factor homology domain (red, residues 1–135); CC, coiled-coil domain (turquoise, residues 176–256); Hel, helical domain (orange, residues 256–355); PP, proline-rich region (green, residues 364–417); BB, blue box, drebrin C-terminal region (blue, residues 431–649). Numbering is for human drebrin E. (B–E) Immunofluorescence confocal images of COS-7 cells transfected with cDNA encoding YFP-tagged drebrin and drebrin deletion constructs (green) and labeled with phalloidin for F-actin (red). Bars, 5 µm. (B) COS-7 cells expressing YFP alone are rounded and have few processes. The YFP protein is diffusely distributed throughout the cell. (C) Drebrin-YFP expression in COS-7 cells induces F-actin–rich filopodia containing drebrin-YFP (arrows). Drebrin-YFP also localizes to stress fibers (arrowheads). (D and E) CC-YFP (D) or CC-Hel-YFP (E) expression in COS-7 cells also induces F-actin rich filopodia (arrows). The CC-YFP (D) and the CC-Hel-YFP (E) protein localize to filopodia (arrows) and to stress fibers (arrowheads). (F) The CC and Hel domains of drebrin induce filopodia to the same extent as the full-length protein. Shown is a quantification of the number of filopodia per unit length of cell perimeter (filopodia density) in COS-7 cells. Expression of drebrin deletion constructs that contain the CC, the Hel, or both domains, including full-length drebrin, significantly increases the density of filopodia. Surprisingly, filopodia formation induced by the Hel domain is suppressed in the Hel-PP-BB deletion construct. Mock, mock transfection; YFP, YFP alone transfection. Values are mean ± SEM (error bars) of at least 10 cells per transfection from three independent experiments. Significant differences (unpaired Student’s t test): *, P < 0.05; **, P < 0.01). (G) Drebrin and the CC and Hel domains increase F-actin levels in transfected COS-7 cells. Shown is a quantification of the F-actin levels in COS-7 cells transfected with various constructs. F-actin levels were quantified by measuring the intensity of phalloidin fluorescence in single confocal optical planes of COS-7 cells at the level of stress fibers and dividing this by the area of the cell. Values are mean ± SEM (error bars) of at least 10 cells per transfection from two or three independent experiments. Significant differences (unpaired Student’s t test): *, P < 0.001; **, P < 0.0001.