Fig 1.
Zasp52MI02988 and Act88FKM88 interact genetically during myofibril assembly.
Confocal microscopy images of IFM of wild type flies and heterozygotes stained with phalloidin (magenta) to visualize myofibrils, and anti-α-actinin antibody (green) to visualize Z-discs. (A) Wild type myofibrils show no defects with properly formed sarcomeres. (B) Zasp52MI02988/+ heterozygotes look indistinguishable from wild type. (C) In the Act88KM88/+ heterozygote, myofibrils form properly, but some frayed myofibrils and loss of sarcomere structure occurs compared to wild type flies. (D) The Zasp52MI02988/+; Act88FKM88/+ transheterozygotes frequently exhibit frayed myofibrils and fragmented Z-discs, and sometimes complete loss of sarcomere integrity. (E) Box plot of quantification of remaining sarcomeres per image in wild type flies, and various transheterozygous mutants. n = 7 muscle fibers. Scale bar, 5 μm. P-values were calculated using Welch’s two-sample t-test followed by Bonferroni correction.
Fig 2.
Schematic of different Zasp52 proteins used.
Size of proteins is indicated in amino acids (a.a.). The full-length protein Zasp52-PR is shown for comparison.
Fig 3.
Zasp52 binds actin with micromolar affinity.
(A) GST pull-down assay. Zasp52-PK, the N-terminal half of Zasp52, binds actin, while Zasp52-LIM234, the C-terminal half of Zasp52, and GST alone do not bind. Binding results were observed in at least three independent experiments. (B, C) Surface plasmon resonance imaging of the real-time binding to GST-Zasp52-PK tethered to the biosensor chip. (B) Binding of indicated concentrations of G-actin flown into the chamber at 100 s and replaced with buffer at 500 s. (C) Binding of indicated concentrations of α-actinin flown into the chamber at 100 s and replaced with buffer at 500 s. Binding is measured in real time in arbitrary units.
Fig 4.
Zasp52 binds filamentous actin and the extended PDZ domain of Alp/Enigma proteins contributes to actin binding.
(A, B) Coomassie blue-stained SDS-PAGE gel showing the results of high-speed co-sedimentation assay of purified His6-Zasp52-PK-FLAG or pure BSA and F-actin. S and P indicate the supernatant and the pellet after high-speed centrifugation, respectively. (A) A small amount of His6-Zasp52-PK-FLAG precipitates together with F-actin after high-speed centrifugation. (B) The control protein BSA remains in the supernatant. (C, D) GST pull-down assays with PWGFRL motif point mutations. (C) G25W and G26D in Zasp52-PP reduce actin binding. (D) P18DW19F in Zasp52-PK strongly reduces actin binding. (E) GST pull-down assay with representative purified extended PDZ domains of Drosophila Zasp52, and human ZASP, PDLIM7, ALP, and PDLIM2. We observe strong actin binding of PDLIM7 and PDLIM2, and weak or no binding of the other extended PDZ domain proteins. Binding and co-sedimentation results were observed in at least three independent experiments.
Fig 5.
Limited rescue ability of a Zasp52 protein lacking the ZM region.
Confocal microscopy images of IFM of different Zasp52 genetic backgrounds stained with phalloidin (magenta) to visualize myofibrils, and anti-kettin antibody (green) to visualize Z-discs. (A) Wild type myofibrils show no defects with properly formed sarcomeres. (B) In the Zasp52MI02988/Df, myofibrils appear frayed and the Z-disc does not form. (C) In Zasp52MI02988/Df rescued by Zasp52-PP flies, myofibrils look indistinguishable from wild type. (D) In Zasp52MI02988/Df rescued by Zasp52-*143 muscles, myofibrils are frequently frayed and smaller. (E) Box plot quantification of the ratio of frayed myofibrils in different Zasp52 genetic backgrounds. UH3-Gal4 was used for the expression of Zasp52 transgenes. n = 10 muscle fibers. Scale bar, 5 μm. P-values were calculated using Welch’s two-sample t-test followed by Bonferroni correction.