DEV01938 Supplementary Material

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• Supplemental Figure 1 - Fig.S1. Extracts were obtained using a protocol adapted from Miller et al. (Miller et al., 1991). Zero to 3 hour embryos were dechorionated, dried, washed and weighed then homogenized at a 1:10 (g/ml) ratio in buffer containing 50 mM HEPES-KOH (pH 7.6), 250 or 500 mM KCl, 1 mM Na3EGTA, 1 mMMgCl₂, 10% glycerol, 1 mM PMSF and protease inhibitor cocktail, and centrifuged for 10 minutes to pellet membrane and yolk. The supernatants were then centrifuged in a Beckman TLA-100 rotor at 150,000 g to pellet F-actin. Pellets and supernatants were then resuspended and run on a western or the supernatant was used in the actin-binding assay. At 250 mMKCl, much of the mYFP-myosin IIDN remained in the supernatant but most of the wild-type myosin was associated with the actin pellet. A salt concentration of 500 mM KCl therefore had to be used to prepare extracts for the actin-binding spin-down assay because at this concentration both wild-type and mYFP-myosin IIDN could be obtained from the supernatant for the assay. For the actin-binding spin-down assay, F-actin was made from a 1 mg/ml solution of G-actin in 5 mM Tris-HCl (pH 8), 0.2 mMCaCl2, left on ice for 30 minutes before adding polymerization buffer (final concentration 50 mM KCl, 1 mM MgCl₂ and 1m MATP) and incubating at 24°C for 1 hour. The F-actin was then pelleted at 150,000 g for 1.5 hours and resuspended in 5 mM Tris-HCl (pH 8), 50 mMKCl, 0.2 mM CaCl₂, 1 mM AMP-PNP (Sigma). Binding assays were then carried out using different dilutions of the F-actin and the same spin down conditions as used for preparing extracts. The protocol was tested with controls (a-actinin as an actin-binding control and BSA as a non-binding control) before being used for the test samples. mYFP-myosin IIDN binds to F-actin with lower affinity than wild-type myosin. Western blots to detect myosin heavy chain and alpha-tubulin. (A) Extracts made in 250 mMKCl buffer from embryos expressing mYFP-myosin IIDN were centrifuged to pellet F-actin. The pellets (p) and supernatants (s) were run on the western blot. The antimyosin antibody detects both the wild-type myosin heavy chain (MHC, upper band, 210 kDa) and the mYFP-myosin IIDN fusion lacking the head domain (DN-YFP, lower band, 180 kDa). Although most of the wild-type myosin pellets with the F-actin, a significant proportion of the mYFP-myosin IIDN remains unbound in the supernatant. (B) Actin-binding spin-down assays performed on extracts obtained at a salt concentration of 500 mM KCl from mYFP-myosin IIDN expressing embryos. When no actin is added to the assay, both the wild-type and mYFP-myosin IIDN remain in the supernatant (–actin lanes). At actin concentrations of 0.1 mg/ml, most the wild-type myosin pellets with the actin, whereas a significant proportion of the mYFP-myosin IIDN remains unbound in the supernatant. At lower actin concentration (4 mg/ml) both wild-type and mYFP-myosin IIDN remain mostly in the supernatant, while at higher concentrations (1 mg/ml, not shown) most of both myosins now pellet with the actin. Given the different behavior of wild-type and mYFP-myosin IIDN in these assays, we conclude that the mYFP-myosin IIDN binds to F-actin with a reduced affinity.

  Reference

  Miller, K. G., Field, C. M., Alberts, B. M. and Kellogg, D. R. (1991). Use of actin filament and microtubule affinity chromatography to identify proteins that bind to the cytoskeleton. Methods Enzymol. 196, 303-319.