A binding protein regulates myosin-7a dimerization and actin bundle assembly

Figures

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  Fig. 1. M7BP is a bona fide binding partner for myosin-7a that relieves its state of autoinhibition. A Domain organizations of myosin-7a and the identified binding protein M7BP (Schematics are in scale with the length of their primary sequences). Numbers indicate the positions of amino acid residues. B Immunofluorescence staining of eye imaginal discs of third instar larvae with anti-myosin-7a (green) and anti-M7BP (red) antibodies. Scale bar = 5 μm in the left panel and 2 μm in the right panels of magnified images. C SDS-PAGE analysis of actin co-sedimentation assays. Fractions of supernatant (S) and pellet (P) isolated from mixtures of actin, myosin-7a, M7BP, or myosin-5 after ultracentrifugation were indicated. D Class averages from negative-stain EM images of M7BP in 150 mM salt buffer, showing an asymmetric globular structure. Scale bars = 10 nm. E, F Class averages from EM images of myosin-7a alone (E) and myosin-7a in complex with M7BP (F) in 150 mM salt buffer with ATP. Myosin-7a alone exhibits a compact, autoinhibited conformation in which the distal end of the folded tail contacts the motor domain (see orange arrow in the global average image). In the presence of equimolar…

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  Fig. 2. M7BP enables highly processive motility of myosin-7a along actin filaments. A Left panel: A single time frame of GFP-myosin-7a alone on immobilized actin filaments (labeled with Alexa-Fluor 647 phalloidin, magenta). Right panel: Kymograph analysis of the box region in A showing that no processive movement was detected. Note the transient appearance of GFP-myosin-7a, which is indistinguishable from its binding to the background surface. Scale bar = 5 μm. B Left panel: A single frame taken from the time-lapse recordings of GFP-myosin-7a (green) with unlabeled M7BP. Actin is labeled with Rhodamine phalloidin (red). Right panel: Example kymographs (box region in B) show clear processive movements along actin filaments. Scale bar = 5 μm. C–E Frequency distribution histograms of the velocity (mean ± SD) (C), characteristic run length (D), and characteristic run duration (E), respectively for the GFP-myosin-7a in the presence of unlabeled M7BP. N = 231 processive tracks.

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  Fig. 3. Processive myosin-7a complexes are predominantly dimeric. A TIRF view of single-molecule motility assays with mCherry-tagged M7BP demonstrates that GFP-myosin-7a (green) and M7BP-mCherry (red) are colocalized in the majority of the puncta moving along actin filaments. Actin filaments were labeled with Alexa-Fluor 647 phalloidin (gray). Scale bar = 5 μm. B Kymograph analysis of box in A shows that the processive movements (diagonal lines) of GFP-myosin-7a and M7BP-mCherry are overlaid. C The histograms of fluorophore numbers (GFP for myosin-7a and mCherry for M7BP) determined by fluorescence intensity analysis. N = 661 quantified complexes. D Schematic of the three-color labeling experiments to test the hypothesis that the motile complex is composed of dimerized myosin-7a. Halo-tagged myosin-7a was labeled with TMR (red), Alexa-Fluor 488 (green), or Alexa-Fluor 660 (blue) and equal quantities of all three labeled myosins were simultaneously introduced into the flow chamber. E Movie frames show that myosin-7a labeled with three different colors moves processively along actin with low motor unit numbers (as most puncta exhibit the original or a mixture of two colors). Actin is…

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  Fig. 4. M7BP tethers actin filaments and enables myosin-7a-M7BP complex to drive actin-filament alignment in vitro. A Speed distribution of actin movement by ensembles of surface-immobilized myosin-7a in the absence or presence of M7BP. N = 6044 actin filaments for myosin alone over four independent experiments. N = 7467 actin filaments for myosin with M7BP over four independent experiments. B Cartoon diagrams how infused M7BP tethers actin filaments and reduces the actin gliding speed. C Motilities of GFP-myosin-7a zippered dimer (green) and the myosin-7a-M7BP complex (shown in blue and red for myosin-7a and M7BP respectively) on the same actin filaments (unlabeled). Note that the zippered dimer of myosin-7a moves faster. D–G Myosin-7a-M7BP complexes tether actin filaments and actively drive them into alignment. Myosin-7a-M7BP complex is visualized by a GFP tag on myosin-7a (green). Immobilized actin filaments are labeled with Rhodamine-phalloidin (red) and free actin filaments labeled with Alexa-647 phalloidin (cyan). D A TIRF field view shows free actin filaments were bound to the surface by myosin-7a complexes. Scale bar = 5 μm. E Free actin filaments were transported by myosin…

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  Fig. 5. Expressing myosin-7a-M7BP complexes in cells produces substantial filamentous networks and filopodial protrusions. A S2 cells transfected with M7BP-mCherry or GFP-myosin-7a alone show no discernable difference in cell morphology compared to that of GFP transfection. Scale bar = 5 μm. (representative of five independent experiments) B Co-transfection of GFP-myosin-7a (green) with M7BP-mCherry (red) results in the induction of an extensive filamentous structure throughout the cell body and the extension of filopodial protrusions. Scale bar = 5 μm. Images are representative of 12 independent transfection experiments. C Co-transfection a monomeric tailless myosin-7a (GFP-M7aS1SAH, green) with M7BP-mCherry (red) does not induce the filopodia or the filamentous network phenotypes observed in B. Scale bar = 5 μm. (representative of four independent experiments) D Magnified images of box 1 in B (top panel) and box 3 in C (bottom panel) show that full-length myosin-7a and M7BP are colocalized on the filamentous structures, whereas M7aS1SAH and M7BP are diffusive and non-colocalized within the cell. Scale bars = 2 μm. E Colocalization analysis of regions from box 1 and box 3 yielded high…

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  Fig. 6. Myosin-7a-M7BP complexes drive actin filament bundling and filopodia extension. A TIRF-SIM live-cell imaging reveals that expression of myosin-7a-M7BP complexes (visualized by a GFP tag to myosin-7a, green) produces substantial actin bundles in cell body and filopodial protrusions (actin was labeled with F-tractin-mCherry, red). Scale bars = 5 μm. Fluorescent Images are representative of three independent experiments. B Magnified insets of box 1 in A and line scans showing that actin networks is composed of densely packed actin filaments and bundles. Myosin-7a-M7BP complexes are primarily colocalized with actin bundles that thus results in a “filamentous appearance” of the complexes. C Kymograph analysis (cyan dash sample line in A) showing that myosin-7a complexes are associated with actin bundles during entire imaging period. Note a more vigorous actin retrograde flow was detected at one side of the cell edge (a), compared to the regions in cell body and in the opposite side (b) where actomyosin bundles are relatively static. Newly polymerized actin filaments on the cell edge (a) were associated with less myosin-7a complex. D Magnified images of box 2 in A showing single…