Calponin-Homology Domain mediated bending of membrane associated actin filaments
Palani, Saravanan; Balasubramanian, Mohan; Köster, Darius Vasco (2021), Calponin-Homology Domain mediated bending of membrane associated actin filaments, Dryad, Dataset, https://doi.org/10.5061/dryad.bvq83bk6f
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.
In vitro assay and Total Internal Reflection Fluorescence (TIRF) microscopy
Supported Lipid Bilayer and Experimental Chamber Preparation:
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 MgCl2, 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 (His6-GFP) as described in (Köster et al., 2016).
Actin filament polymerization and tethering to SLBs:
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%vol of 10x ME buffer (100 mM MgCl2, 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 CaCl2, 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 Ca2+ ions with Mg2+ 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, His6-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 His6-SpCdc12(FH1-FH2) and 10 nM His6-Curly for 20 min, then washed twice with KMEH. During the incubation time, 10%vol 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 (CG-actin = 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) (CmyoII = 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 His6-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 MgCl2, 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 (CG-actin = 100 nM) and myosin II filaments (CmyoII = 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).
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/).
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-1 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.
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.
European Research Council, Award: ERC-2014-ADG N° 671083
Wellcome Trust, Award: WT 101885MA
Wellcome Trust, Award: 208384/Z/17/Z