QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 November 2005
doi: 10.1242/jcs.02653

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Franz, C. M. Articles by Müller, D. J. Search for Related Content PubMed PubMed Citation Articles by Franz, C. M. Articles by Müller, D. J. Social Bookmarking                         What's this?

Analyzing focal adhesion structure by atomic force microscopy

Center of Biotechnology, University of Technology Dresden, Tatzberg 49, 01307 Dresden, Germany

View larger version (87K): [in a new window]   Fig. 1. Near-confluent cells were de-roofed by a sonication procedure (D-F) or left untreated (A-C), fixed and stained for F-actin using TRITC-phalloidin (A,D). Untreated cells showed an abundance of stress fibers (A) terminating in paxillin-containing plaques (B). The size and shape of paxillin-containing patches was unchanged after the de-roofing step (E), indicating the preservation of FA complexes. FAs also stained positive for F-actin although the link between FAs and stress fibers was generally broken during de-roofing (D). Bar, 10 µm.

View larger version (171K): [in a new window]   Fig. 2. Cells were de-roofed and an AFM image recorded (A, deflection and B, height). Parts of the ventral plasma membrane were preserved during the de-roofing procedure (B, arrows). (C) The localization of paxillin-YFP within an area corresponding to the dashed box in (B) was determined by fluorescence microscopy. (D) Overlaying the AFM height image with the paxillin fluorescence image highlights paxillin-containing FAs. (E) Several paxillin-positive protein complexes (arrows) were subsequently imaged at higher resolution by AFM (E, height and F, deflection signal; scan area corresponding to the dashed box in D). Filamentous structures arranged at slight angles to each other can be distinguished in some of the FAs. The full range of the height scale corresponds to heights of 600 nm (B,D) and 350 nm (E). Bar, 10 µm (A-D) and 1 µm (E,F).

View larger version (142K): [in a new window]   Fig. 3. Fluorescence images of YFP-paxillin (A) and actin filaments (B) in de-roofed cells. AFM topographs (D) and deflection image (E) of the same area. (F) Overlay of the merged fluorescence images (C) with the AFM deflection image. (G) The boxed area in D scanned at higher resolution. Arrows indicate filaments that cross parallel arrays of actin filaments at different angles. (H,J) Boxed area in G scanned at further increased resolution. Microfilaments occasionally branch at angles between 30° and 45° (H, branching points indicated by asterisks). The trace and retrace topographies show predominantly parallel filaments exhibiting apparent diameters ranging from 20 to 90 nm. A merge of the trace (green) and retrace topographs (red) demonstrates good correlation between both scanning directions. (J,K) Filaments are decorated by globular structures with apparent diameters between 50 and 80 nm. A cross section (solid black line) through a group of adjacent 20-nm-wide filaments (arrows) and the corresponding height profile indicates a height difference of 12 nm between two neighboring filaments. (K) Height profiles (black solid lines) taken perpendicular to two filaments (black and white dashed lines) before and after their crossing point demonstrate a height difference of between 10 and 16 nm. The full range of the height scale corresponds to heights of 450 nm (D), 160 nm (G), 60 nm (H) and 50 nm (J,K). Bar, 3 µm (A-F); 500 nm (G); 50 nm (H); 100 nm (J,K).

View larger version (29K): [in a new window]   Fig. 4. FA topographs (A) and 3D-reconstruction (B) showing the wedge-shaped morphology and the finger-like array of microfilaments at the distal end of the SF. (C) Side view of the same FA with Y and Z dimensions at the same scale. The height of the FA increases towards the SF-associated end at an angle of approximately 3°. From the AFM topograph the area (1.9x106 nm2) and the volume (1.2x108 nm3) of the FA was determined. (D) Location of X-Z cross sections taken through the FA topograph every 500 nm in the Y direction. (E) Overlay of the cross section demonstrates that the FA structure is low and wide at the front and high and narrow at the SF-associated end. (F) Graphs showing the relative changes in FA height, width and cross-section area moving from the distal to the proximal end of the FA. (G) Schematic representation of the 3D-array of microfilaments in FAs. Height scale in A, 200 nm.

View larger version (50K): [in a new window]   Fig. 5. (A) Topograph and 3D-reconstruction of a cluster of four FAs. Cross sections along the indicated lines (bisecting FAs perpendicularly) were taken. (B) Height profiles corresponding to the cross sections taken in (A). The average height of bisecting cross sections corresponds to 84±16 nm (average height), whereas the filamentous structures extended over the substratum by 118±27 nm. The dashed lines indicate the average values generated from analysis of more than 30 FAs. (C) Histogram showing the distribution of distances between neighboring filament/filament bundles (>30 FAs analyzed). The mean spacing between filaments/filament bundles at the FA half-length was 127±50 nm.

View larger version (68K): [in a new window]   Fig. 6. F-actin and paxillin fluorescence images of an FA in a de-roofed cell (A-C). Corresponding topograph (D) and fluorescence/height overlay image (E). The circular area with decreased F-actin staining (A, asterisk) corresponds to an area of decreased height in the AFM topograph (D). This defect may be the result of mechanical disruption of the FA structure during de-roofing. In contrast to the F-actin signal, the paxillin signal in this area is not decreased (B). (F) A line scan through the overlay image (E) demonstrates a correlation between the height and F-actin signal but not the paxillin signal. The shaded area (light green) indicates a higher relative fluorescence intensity of paxillin-YFP compared to the F-actin staining. As the disruption of FA architecture down to a height of less than 50 nm does not decrease the paxillin signal, the paxillin localization must be restricted to a membrane-proximal region of the FA. F-actin-containing structures, on the other hand, must be localized predominantly in the membrane-distal half, as disruption of the FA structure down to about 50 nm reduces F-actin staining to almost background levels. Bar, 500 nm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter