Fig. 6: Choanoflagellate SPIRE interacts with myosin 5.
From: Actomyosin organelle functions of SPIRE actin nucleators precede animal evolution
a Domain organisation of human (Hs, Homo sapiens) and Monosiga brevicollis (Mb) myosin-5 (MYO5) proteins. The MYO5 proteins share all characterised functional domains, including the ATPase motor domain, calmodulin-binding IQ motifs, coiled-coil regions and a C-terminal cargo-binding globular tail domain (GTD). Numbers indicate amino acid residues. b Protein structure alignment of the ColabFold/AlphaFold-multimer66,67,68 predicted Mb-MYO5 globular tail domain (GTD, orange) in complex with Mb-SPIRE-GTBM (purple) and the crystal structure of the human MYO5A-GTD in complex with the human SPIRE2-GTBM (PDB-ID: 5JCY, grey/white28). GTD subdomains (SD-1, SD-2) are indicated as well as the helices H3, H4” and H5. c GST-pulldown assay with purified GST-Mb-MYO5-GTD, GST-Mb-FMN-eFSI and GTP-locked GST-Mb-RAB8-Q67L from lysates of HEK 293 cells transiently expressing full-length AcGFP1-tagged Mb-SPIRE (GFP-Mb-SPIRE, input). GST and AcGFP1 (GFP) were used as controls and Ponceau S staining shows equal amounts of GST and GST-tagged proteins. N = 2 experimental repeats. d Localisation of transiently co-expressed tagged C-terminal Mb-SPIRE (AcGFP1; GFP-Mb-SPIRE-ΔKW; green) and Mb-MYO5-GTD (mRuby3; Ruby3-Mb-MYO5-GTD; red) in human HeLa cells was analysed by fluorescence microscopy. AcGFP1 (GFP) and mRuby3 (Ruby3) expressions were used as controls. Deconvoluted images indicate the colocalisation of the mRuby3-Mb-RAB8 and AcGFP1-Mb-SPIRE-ΔKW proteins on vesicular structures. Scale bars represent 5 µm. At least six cells from two distinct experiments were imaged for each condition and the cytoplasmic region of one representative cell is presented here. e GST-pulldown assay with purified GST-Mb-MYO5-GTD from HEK 293 cell lysates transiently expressing different N-terminal and C-terminal AcGFP1(GFP)-tagged Mb-SPIRE protein fragments. Ponceau S staining shows equal amounts of GST-Mb-MYO5-GTD proteins. N = 2 experimental repeats. KW, KIND-WH2. Numbering indicates amino acid residues. f Schematic representation of N-terminal and C-terminal Mb-SPIRE protein fragments as used in (e) and their ability to bind (+) or not to bind (-) to Mb-MYO5-GTD. GTBM, globular-tail-domain-binding motif. g WebLogos108,109 depicting the amino acid conservation within vertebrate (upper panel, 93 sequences) and choanoflagellate (lower panel, 18 sequences) SPIRE GTBM amino acid sequences. Corresponding amino acids of Monosiga brevicollis (Mb) and Salpingoeca rosetta (Sr) SPIRE proteins experimentally shown to be involved in MYO5-GTD binding are depicted below and aligned to the choanoflagellate WebLogo, which is provided with respective amino acid boundaries.
