DOI: 10.5061/dryad.bvq83bk6f
title: Calponin-Homology Domain mediated bending of membrane associated actin filaments

description: This dataset contains data from experiments described in 'Palani S,
 Ghosh S, Ivorra-Molla E, Clarke S, Suchenko A, Balasubramanian MK, Köster
 DV. 2021. Calponin-homology domain mediated bending of membrane associated
 actin filaments. Elife 10. doi:10.7554/eLife.61078'. This dataset
 consists of fluorescence microscopy images obtained by total internal
 reflection fluorescence (TIRF) microscopy. Using an in vitro approach, we
 studied the effect of the IQGAP protein fragment Rng2(1-189) on the
 geometry of actin filaments when tethered to supported lipid bilayers all
 reconstituted from purified proteins. The main findings are that
 Rng2(1-189) bends actin filaments into tight rings when tethered to
 supported lipid bilayers.
description: <b>In
 vitro assay and Total Internal Reflection Fluorescence (TIRF) microscopy
 </b> <i>Supported
 Lipid Bilayer and Experimental Chamber
 Preparation:</i> The sample
 preparation, experimental conditions and lipid composition were similar to
 the ones described in previous work [Koester et al, 2016]. Glass
 coverslips (#1.5 borosilicate, Menzel, cat. no. 11348503, Fisher
 Scientific) for SLB formation were cleaned with Hellmanex III (Hellma
 Analytics, cat. No. Z805939, Merck) following the manufacturer's
 instructions followed by thorough rinses with EtOH and MilliQ water and
 blow dried with N2 gas. For the experimental chamber, 0.2 ml PCR tubes
 (cat. no. I1402-8100, Starlab) were cut to remove the lid and conical
 bottom part. The remaining ring was stuck to the cleaned glass using UV
 glue (cat. no. NOA88, Norland Products) and three minutes curing by
 intense UV light at 265 nm (UV Stratalinker 2400, Stratagene). Freshly
 cleaned and assembled chambers were directly used for experiments.
 Supported lipid
 bilayers (SLB) containing 98% DOPC (cat. no. 850375, Avanti Polar Lipids)
 and 2% DGS-NTA(Ni2+) (cat. no. 790404, Avanti Polar Lipids) lipids were
 formed by fusion of small uni-lamellar vesicles (SUV) that were prepared
 by lipid extrusion using a membrane with 100 nm pore size (cat. no.
 610000, Avanti Polar Lipids). SLBs were formed by addition of 10 µl of SUV
 mix (at 4 mM lipid concentration) to chambers filled with 90 µl KMEH (50
 mM KCl, 2 mM MgCl<sub>2</sub>, 1 mM EGTA, 20 mM HEPES, pH 7.2)
 and incubation for 30 min. Prior to addition of other proteins, the SLBs
 were washed 10 times by buffer exchange (always leaving 20 µl on top of
 the SLB to avoid damage by drying). We tested the formation of lipid
 bilayers and the mobility of lipids in control samples by following the
 recovery of fluorescence signal after photobleaching of hexa-histidine
 tagged GFP (His<sub>6</sub>-GFP) as described in (Köster et
 al., 2016). <i>Actin
 filament polymerization and tethering to
 SLBs:</i> Actin was purified
 from muscle acetone powder form rabbit (cat. no. M6890, Merck) and
 labelled with Alexa488-maleimide (cat. no. A10254, Thermo Fisher)
 following standard protocols (Köster et al., 2016; Pardee &
 Spudich, 1982). In a typical
 experiment, actin filaments were polymerized in parallel to SLB formation
 to ensure that all components of the experiment were freshly assembled
 before starting imaging. First 10%<sub>vol</sub> of 10x ME
 buffer (100 mM MgCl<sub>2</sub>, 20 mM EGTA, pH 7.2) were
 mixed with unlabeled and labeled G-actin (to a final label ratio of 20%),
 optionally supplemented with labelled capping protein in G-actin buffer (1
 mM CaCl<sub>2</sub>, 0.2mM ATP, 2mM Tris, 0.5 mM TCEP-HCl, pH
 7.2) to a final G-actin concentration of 10 µM and incubated for 2 min to
 replace G-actin bound Ca<sup>2+</sup> ions with
 Mg<sup>2+</sup> ions. Polymerization of actin filaments was
 induced by addition of an equal amount of 2x KMEH buffer supplemented with
 2 mM Mg-ATP bringing the G-actin concentration to 5 µM. After 30 min
 incubation time, actin filaments were added to the SLBs using blunt-cut
 pipette tips at a corresponding G-actin concentration of 100 nM (to ensure
 a homogenous mix of actin filaments, 2 µl of actin filament solution was
 mixed in 18 µl KMEH and then added to the SLB containing 80 µl KMEH).
 After 10 min of incubation, His<sub>6</sub>-Curly or other
 variants of histidine-tagged actin binding proteins at a final
 concentration of 10 nM were added and a short time after (1 - 5 min)
 binding of actin to the SLB could be observed using TIRF microscopy.
 In experiments with
 formin, the SLB was first incubated with 10 nM
 His<sub>6</sub>-SpCdc12(FH1-FH2) and 10 nM
 His<sub>6</sub>-Curly for 20 min, then washed twice with KMEH.
 During the incubation time, 10%<sub>vol</sub> of 10x ME buffer
 was mixed with unlabeled and labeled G-actin at 4 µM (final label ratio of
 20%) together with 5 µM profilin and incubated for 5 min prior to addition
 to the SLB and imaging with TIRF microscopy. In experiments with
 tropomyosin or fimbrin, actin filaments (C<sub>G-actin</sub> =
 1 µM) were incubated with tropomyosin at a 1:3 protein concentration ratio
 or with fimbrin at a 3:2 protein concentration ratio for 15 min prior to
 addition to the SLB (Palani et al., 2019). In experiments with
 muscle myosin II filaments, we prepared muscle myosin II filaments by
 diluting the stock of muscle myosin II proteins (rabbit, m. psoas, cat.
 no. 8326-01, Hypermol) (C<sub>myoII</sub> = 20 µM; 500mM KCl,
 1mM EDTA, 1 mM DTT, 10 mM HEPES, pH 7.0) 10-times with MilliQ water to
 drop the KCl concentration to 50 mM and incubated for 5 min to ensure
 myosin filament formation. Myosin II filaments were further diluted in
 KMEH to 200 nM and added to the actin filaments bound to the SLB by
 His<sub>6</sub>-Curly by replacing 1/10 of the sample buffer
 with the myosin II filament solution and supplemented with 0.1 mM Mg-ATP
 as well as a mix of 1 mM Trolox (cat. no. 648471, Merck), 2 mM
 protocatechuic acid (cat. no. 03930590, Merck) and 0.1 µM protocatechuate
 3,4-dioxygenase (cat. no. P8279, Merck) to minimize photobleaching. To
 summarize, the final buffer composition was 50mM KCl, 2mM
 MgCl<sub>2</sub>, 1mM EGTA, 20mM HEPES, 0.1mM ATP, 1 mM
 Trolox, 2 mM protocatechuic acid and 0.1 µM protocatechuate
 3,4-dioxygenase at pH 7.2 containing actin filaments
 (C<sub>G-actin</sub> = 100 nM) and myosin II filaments
 (C<sub>myoII</sub> = 20 nM). It was important to keep the pH
 at 7.2, as changes in pH would affect motor activity. As reported earlier,
 myosin filaments started to show actin network remodeling activity after
 about 10-15 min of incubation (Köster et al., 2016; Mosby et al., 2020).  
 <i>TIRF
 microscopy:</i> Images were acquired
 using a Nikon Eclipse Ti-E/B microscope equipped with perfect focus
 system, a Ti-E TIRF illuminator (CW laser lines: 488nm, 561nm and 640nm)
 and a Zyla sCMOS 4.2 camera (Andor, Oxford Instruments, UK) controlled by
 Andor iQ3 software
 (https://andor.oxinst.com/products/iq-live-cell-imaging-software/).
 <b>Image
 analysis</b> Images were analyzed
 using ImageJ (http://imagej.nih.gov/ij). Curvature was
 measured by fitting ellipses to match the actin filament contour by hand,
 while measuring first fully formed rings before curved actin filament
 segments and by going from the highest curvatures down to lower curvatures
 in each image with a cut off for measurements at curvatures smaller than
 0.1 µm<sup>-1</sup> or at 30-40 measurements per image (see
 examples in Figure 1 – figure supplement 1D; Figure 1-figure supplement
 2B). To measure the angle
 of kinks in individual actin filaments, cropped images of individual actin
 filaments were processed with a Sobel filter (part of the Mosaic suit for
 ImageJ, http://mosaic.mpi-cbg.de/?q=downloads/imageJ) to highlight the
 actin filament center, and the angles were measured manually with the
 ImageJ angle tool. The actin ring
 contraction rate upon myosin II filament action was measured by generating
 kymographs based on a line (3 pixels width) dividing the ring into two
 equal halves.
description: deposited here are all raw image files used for the curvature
 measurements of different curly constructs and other actin binding
 proteins tethered to lipid bilayers together with the ROIs depicting the
 individual curvature measures using imageJ.