Visualisation and quantification of actin responses in grapevine cells
The responses of actin filaments were followed in living grapevine cells of the actin marker line V. vinifera ‘Chardonnay’GFP-AtFABD2 by spinning disc confocal microscopy on a AxioObserver Z1 (Zeiss, Jena, Germany) inverted microscope equipped with a laser dual spinning disc scan head from Yokogawa (Yokogawa CSU-X1 Spinning Disk Unit, Yokogawa Electric Corporation, Tokyo, Japan), and a cooled digital CCD camera (AxioCamMRm; Zeiss) as described in Akaberi et al. (2018). To quantify the degree of actin aggregation, a strategy modified from Schwarzerová et al. (2002) was used. Intensity profiles were collected along a grid of equally spaced lines (four lines oriented perpendicular to the cell axis) using a line width of 10 pixels and the spline averaging option (ImageJ, https://imagej.nih.gov/ij/). The profile shows peaks and troughs, corresponding to actin bundles and non-bundled actin (either G-actin or fine filaments that are not optically resolved). Aggregation of actin will deplete this non-bundled actin, such that the troughs are accentuated, while the peaks will turn more prominent. This phenomenon can be quantified by calculating the standard error over the profile. This standard error can therefore be used as readout for the degree of actin aggregation: Although this strategy is robust against variations in exposure parameters such as laser power, exposure time, or exposure gain, it was made sure that all images were recorded under the same magnification and exposure time by inactivating the automatic image acquisition routine of the software (ZEN, Zeiss, Jena). Around 3-5 cells per data point were used for the quantification.
To estimate the reorganisation of actin into foci (a phenomenon that was observed after treatment with the MAPK inhibitor PD98059), a different strategy had to be used: The mean coverage of actin foci was estimated using the ”analyze particle” tool of ImageJ. Images were first changed into the 8-bit BW format and then transformed into a binary image using the thresholding tool. The value for the threshold was adjusted such that the dots (that were much brighter) remained, while the filaments (that were dimmer) disappeared. To ensure that no residual filaments were selected for quantification, the circularity of the particle selection tool was set to 0.9-1 (i.e. only circular or ovoid structures were selected, while filamentous structures were excluded). To avoid that noise signals were picked up, the threshold for size selection was set to a minimum of 10 square pixels. Then, the readout ”area” [in square pixels] was activated in the ”set measurement” tool. After application of the ”analyze particle” tool to the binary image, the results were exported into a Excel spreadsheet to determine the total area of actin organised in foci. Here, 20-25 cells per data point were used for quantification.