Modulation of Filopodial Myosin Function

Figures

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  Figure 1.. The actin-binding HTH region of the myosin motor domain. A. Homology model of the DdMyo7 motor domain based on the Myo10 motor in the pre-power stroke state (ADP-Pi; PDB 510H) showing the helix-turn-helix (HTH) on the lower 50K region of the motor (blue); along with the HCM loop (maroon), Loop 4 (aqua) and Loop 3 (black) regions important for actin binding. The red balls and orange stick represent bound nucleotide, the green helix is the activation loop and the purple ball on the HTH is the ‘jd’ residue at the end of the first helix. B. Alignment of the HTH sequences of Myo15A, DdMyo7 and Myo10. red * denotes the ‘jd’ residue. * actin interacting residues, C. LOGO representation of alignment of the HTH region from 136 different myosin motor domains from 53 different classes plus two divergent orphan myosins across phylogeny. Note that the gap at position 10 is due to the presence of a highly variable or no amino acid at that position. The red * indicates the ‘jd’ residue and the blue * indicate residues on the actin interface with actin.

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  Figure 2.. Mutation of the DdMyo7 ‘jd’ residue significantly inhibits filopodia formation. Quantitative analysis of filopodia formation rescue by mNeon-full-length wild type (WT), E456G (E-G) or E456D (E-D) DdMyo7-expressing myo7 null cells using filoTips. A. Representative confocal micrographs of each cell line that show differences in the number of bright filopodia tips and cortical enrichment in myo7 null cells expressing the HTH mutant myosins. B. Filopodia per cell. The mean is indicated with a thick bar on the plot for each line and dashed lines indicate the quartiles. C. Cell body to cortex ratio. The mean is indicated with a thick bar on the plot. Shown are the results from the analysis of two independent cell lines for each DdMyo7. Two independent cell lines were analyzed for each mutant and a minimum total of four independent assays were performed for each mutant. ** p < 0.03 **** p < 0.0001

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  Figure 3.. The DdMyo7 ‘jd’ mutation does not impact filopodia length. Quantitative analysis of filopodia formation in wild type cells expressing either the wild type (WT), E456G (E-G) or E456D (E-D) mNeon-DdMyo7 using filoTips. A. Filopodia length. The mean is indicated with a thick bar on the plot for each line and thinner lines indicate the SEM. B. Filopodia tip intensity. The mean is indicated with a thick bar on the plot for each line. Shown are the results from the analysis of two independent cell lines for each DdMyo7, Two independent cell lines were analyzed and a minimum of four independent assays were performed for each mutant. ** p < 0.002 **** p < 0.0001, ns = not significant.

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  Figure 4.. Mutation of the Myo10 ‘jd’ residue impairs filopodia formation. Quantitative analysis of filopodia formation by full-length eGFP-wild type (WT), E508G (E-G) or E508D (E-D) Myo10-expressing COS-7 cells using filoTips. A. Representative images of COS-7 cells expressing wild type and mutant Myo10. Scale bar = 10 μm. B. Filopodia per cell. The mean is indicated with a thick bar on the plot, smaller dashed lines indicate quartiles. C. Cortex:cell body ratio of Myo10 localization. The mean is indicated with a thick bar on the plot. Shown are the results from the analysis of two independent cell lines for each transfection. Two independent cell lines were analyzed for each mutant and a minimum total of four independent assays were performed for each mutant. * p < 0.004 ** p < 0.03 **** p < 0.0001

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  Figure 5.. Mutation of the Myo10 ‘jd’ residue reduces intrafilopodial motility. Intrafilopodial motility of eGFP-Myo10 punctae of full-length wild type (WT), E508G (E-G) or E508D (E-D), in Hela cell filopodia measured using TIRF microscopy A, C, E. Histogram showing the measured velocities for WT, E508G and E508D, respectively. Data fit to a Gaussian curve, and dashed line indicates the mean velocity, B,D,F. Representative kymographs of movement of wild type (WT), E508G (E-G) or E508D (E-D) punctae moving from filopodia base (left side) to tip (right side).

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  Figure 6.. The Myo10 ‘jd’ mutation does not impact filopodia length in COS-7 cells. Quantitative analysis of filopodia formation in wild type cells expressing either the wild type eGFP-Myo10 (WT), E508G (E-G) or E508D (E-D) using filoTips. A. Filopodia length. The mean is indicated with a thick bar on the plot for each line and thinner lines indicate the quartiles B. Filopodia tip intensity. The mean is indicated with a thick bar on the plot for each line. Shown are the results from the analysis of two independent cell lines for each myosin. Two independent cell lines were analyzed and a minimum of four independent assays were performed for each mutant. * p < 0.02, *** p < 0.005, **** p < 0.0001