Myosin 2 drives actin contractility in fast-crawling species outside of the amorphean lineage

Figures

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  Figure 1:. Myosin 2 exists outside of Amorphea, in the fast-crawling amoeba Naegleria gruberi. (A) A simplified species tree of representative eukaryotes from major lineages, and their maximum reported cell crawling speeds (bar graph, right).75–85. The Amorphea (teal) are the only lineage with functionally characterized Myosin 2. Naegleria gruberi and Acrasis kona are heterolobosean amoebae from the Discoba lineage (maroon) with top crawling speeds of 120 and 160 μm/minute.79 The species without values are not capable of crawling motility or lack published data on speed (Reticulomyxa filosa). (B) Naegleria amoebae crawl quickly across surfaces. A montage of a representative amoeba crawling on glass, imaged over 20 seconds using DIC. (C) Naegleria amoebae express three Myosin 2 genes. A histogram shows relative transcript abundance of genes expressed in amoebae. Frequency of values is shown for each bin of calculated abundance, calculated as log2-transformed average transcripts per million (TPM+1) for 5 replicates. The bins containing NgMyo2s (Myo2A, Myo2B, Myo2C) and the major actin gene (jgi_56107) are noted.

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  Figure 2:. Naegleria expresses all three Myosin 2 genes. (A) Naegleria undergoes mitosis as an amoeba, but transiently differentiates into a swimming flagellate stage under stress (diagram, left). Expression of Myosin 2 varies during mitosis (top graph) and differentiation (bottom graph), shown as an adjusted log2 fold-change of each gene over 80 minutes, relative to 0 minutes post-induction. Cytokinesis occurs at 40 minutes after mitotic induction. The lines are color coded according to the genes expressed; green = actin, dark pink = Myo2A, pink = Myo2B, light pink = Myo2C. Mitotic tubulins (top graph, dark gray α-tubulin = AKG_0016; light gray β-tubulin = jgi_53354) are shown as a reference for mitotic progression. Flagellar tubulins (bottom graph, dark gray α-tubulin = jgi_39221; light gray β-tubulin = AKG_0052) are shown as an indicator for differentiation. (B) A cartoon of the domain architecture of Naegleria’s Myosin 2s indicates where the custom polyclonal antibodies bind; black arrows mark names and sites of peptide sequences used. Myo2A = dark pink, Myo2B = medium pink, Myo2C = light pink. (C) Naegleria’s Myosin 2s are expressed at the protein level. Western blot validation of…

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  Figure 3:. Naegleria’s Myosin 2s bind actin networks. (A) Naegleria amoebae were fixed and stained with Myo2 antibodies (pink) to detect Myosin 2 and Alexa Fluor™ 488 Phalloidin to label actin filaments (green). Single z slices through the midsection of cells are shown. The Region of Interest (ROI) for each example depicts the area used for colocalization analyses; dotted squares were used in 90° rotation tests, and dotted lines through the peripheral actin cortex were used for line scans (far right). The colocalization values are plotted as Pearson’s correlation coefficients, with the representative cells shown on the left indicated by pink circles. The Myo2 and actin line scan fluorescence intensity was normalized to the dimmest (0%) and brightest (100%) signal for regions within the cortex boundary. (n ≥ 6 cells for each antibody; scale bar = 10μm). (B) Myosin 2 remains bound to Naegleria’s extracted actin cytoskeleton (ghosts). Following cytoskeletal extraction (see Methods), ghosts were fixed, stained and analyzed as in (A). (n ≥ 21 ghosts per antibody; scale bar = 10μm). (C) Myosin 2 is retained in the insoluble fraction of protein lysates. Cells were lysed and protein samples were…

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  Figure 4:. Naegleria actin networks are contractile in an ATP dependent manner. (A) Naegleria’s actin networks are contractile, like Dictyostelium. Images show representative Dictyostelium and Naegleria ghosts before (teal, pink) and after (black) the addition of 1mM Mg-ATP to activate Myosin 2. The graph shows ghost surface area contraction, calculated as the percent change in pixel area after addition of buffer (−) or 1mM Mg-ATP (+). (N = 5, scale bars = 10 μm). (B) Ghost contractility is abolished by salt concentrations that disrupt Myosin 2 filaments. Dictyostelium and Naegleria ghosts were treated with 200mM KCl and imaged before (teal, pink) or after the addition of ATP (black). Quantification was completed as in (A) for ghosts in wells with (+) and without (−) prior KCl treatment. (N = 3, scale bars = 10 μm)

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  Figure 5:. Naegleria’s Myosin 2s release actin under high salt with ATP. (A) Right: A schematic representation of the expected states of Myo2 filaments in each experimental condition. Left: Images of ghosts treated in the conditions as shown above with buffer alone (control), or buffer supplemented with: 200mM KCl (‘KCl’), 1mM Mg-ATP (‘ATP’), or sequentially with KCl 10 minutes, then Mg-ATP for 10 minutes (‘KCl+ATP’). The ghosts were then fixed and immunostained with Myo2A-1 antibody (pink) and labeled with phalloidin-488 (green). Scale bar = 10 μm. (B) Average fluorescence intensity quantified per ghost from maximum intensity-projections across 3 replicates.

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  Figure 6:. Other heterolobosean species also have Myosin 2. (A) A modified jelly bean plot depicts subdomain motifs in a multiple sequence alignment of heterolobosean Myosin 2s, aligned with ClustalO from the N-terminus through the second IQ motif, and organized phylogenetically with a tree built in Geneious using RAxML v 8.2.12, with a scale bar indicating substitutions per site. Highlighted subdomain motifs within (gray box) and proximal to the motor domain were colorized by pairwise positive scores via Blosum62 matrix, as compared to the Dictyostelium Myosin 2 sequence (shown in black). Scores above 80% similarity are shaded in dark teal, 30–80% in teal, and <30% in light teal. Missing regions of proteins due to partial or incomplete datasets are noted as “Not Found” (striped). (B) Another Heterolobosean, Vahlkampfia avara, also crawls quickly. A montage of typical Vahlkampfia amoebae crawling on an untreated glass surface (top left panel) shows morphology and representative speed. Individual cell tracks are shown for a 90-second timelapse (bottom left panel), normalized so time=0 is at the origin. Average crawling speed (top right panel) and directional persistence (maximum displacement…