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.