Alexa 488 phalloidin

Cultures were washed in PBS and fixed in 4% (w/v) paraformaldehyde for 15 min at room temperature (RT). Cultures were then placed in 5% (w/v) bovine serum albumin (BSA, Sigma Aldrich) in 0.1 M Triton X-100 in H₂O (PBT) for 1 h at RT and then treated with primary antibodies (YAP, sc101199, Santa Cruz, 1:100; pMLC2, 3671S, Cell Signaling Technology 1:100; collagen type II, ab34712, Abcam 1:200) in 5% (w/v) BSA in PBT overnight at 4 °C. Cultures were washed in 3% (w/v) BSA in PBT with gentle agitation. Primary antibodies against pMLC2 and collagen type II were visualised after incubating with ab150077 (Abcam) for 1 h at RT (1:500 in 5% (w/v) BSA + PBT). Antibodies against YAP were detected using biotin conjugate secondary antibody (ab6788, Abcam) which were detected with a fluorescently labelled streptavidin biotin-binding protein (S11223, Thermo Fisher Scientific). The secondary antibody solution also included 0.1 μg/ml DAPI. Fluorescence signal was imaged on an Axiovert200M microscope (Zeiss). Following imaging, PA gels were re-stained with Alexa488 Phalloidin (Sigma Aldrich) at 1:200 in PBS for visualisation of the actin cytoskeleton.

Rabbit skeletal muscle lyophilized actin (Cytoskeleton, Inc) was dissolved in G-buffer (5 mM Tris-HCl, pH 8.0), 0.2 mM CaCl₂, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN₃) to the concentration of ~8 μM, incubated on ice for 1 h and injected into Superdex 75 10/300 GL (Amersham Biosciences) gel filtration column. Monomer fractions were collected and used for the experiment. Usually, the fractions contained G-actin concentration of 3–5 μM. Then we polymerized 1 μM G-actin by addition of salts (50 mM KCl, 2 mM MgCl₂, 1 mM ATP) in the presence of IPP buffer or IPP protein (1:1 molar ratio) and the filaments were labeled with ~1 μM Alexa 488-phalloidin (Sigma). Prior to the visualization, 1 μl of sample was added to 9 μL of F-buffer (G-buffer above plus salts) and applied to a cover slip coated with poly-L-lysine (0.01%). Widefield images were acquired using a Leica DM5000B upright fluorescence microscope (Leica Microsystems, GmbH, Wetzlar, Germany), equipped with a Retiga SRV camera and QCapture Pro software (QImaging, Surrey, BC Canada). Confocal images were acquired using a Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).

Arabidopsis pollen grains and pollen tubes were subjected to actin staining with Alexa-488 phalloidin (Thermo Fisher Scientific, A12379) as previously reported [58 (link),59 (link)]. To determine the effect of LatB on the actin cytoskeleton, pollen grains or pollen tubes were treated with 150 nM LatB in liquid GM for 30 min before fixation with 300 μM 3-maleimidobenzoic acid N-hydroxysuccinimide ester crystalline (Sigma-Aldrich, M2786) and subsequent staining with Alexa-488 phalloidin. The samples were visualized under an Olympus FluoView FV1200 confocal microscope equipped with a 100× oil immersion objective (1.4 numerical aperture) and were excited under a 488-nm argon laser with the emission wavelength set at 505 to 545 nm. The z-series images were collected with the step size set at 0.5 μm. The amount of F-actin in pollen grains and pollen tubes was quantified by measuring the intensity of the fluorescent pixels with ImageJ software (http://rsbweb.nih.gov/ij/; version 1.48g) as reported previously [10 (link)].