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. 2021 May 27;10:e68082. doi: 10.7554/eLife.68082

Figure 7. DdMyo7 motor activity is required to release autoinhibition.

(A) Schematic of proposed effect of mutations on DdMyo7 function (see alignment in Figure 7—figure supplement 1). (B) Confocal images of myo7 null cells expressing GFP-DdMyo7 fusion proteins, scale bar is 10 µm. (C) Quantification of the cortical band intensity variation. Mean lines from Figure 1I data on graph for comparison. DdMyo7 versus I426A uncoupler, p=0.07, not significant (see also Figure 7—source data 1 and 2). (D) Fraction of DdMyo7 cosedimenting with the cytoskeleton, symbols with the same shape are technical replicates, students t-test ****p<0.0001 (see also Figure 7—figure supplement 1, Figure 7—source data 3). (E) Quantification of cortical recruitment of DdMyo7 and mutants (see also Figure 7—source data 4). (F) Filopodia number per cell of wildtype and DdMyo7 mutants (see also Figure 7—source data 6). (E–F) Data for KKAA is taken from Figure 6, experimental means shown as circles. (C, E, and F) One-way ANOVA with multiple comparison correction, p*<0.05, p***<0.001, p****<0.0001, ns not significant (see also Figure 7—source data 5 and 7).

Figure 7—source data 1. Values of the cortical standard deviation measurements (cortical asymmetry) measured for each cell for Figure 7C, see also Figure 1—source data 1 and Figure 6—source data 5.
Figure 7—source data 2. Statistical test results for Figure 7C.
Figure 7—source data 3. Data and statistics test results for Figure 7D.
Figure 7—source data 4. Cortex: cell ratio values for each cell for lines analyzed in Figure 7E.
Figure 7—source data 5. Statistical test results for Figure 7E.
Figure 7—source data 6. Filopodia per cell values for each cell for lines analyzed in Figure 7F.
Figure 7—source data 7. Statistical test results for Figure 7F.

Figure 7.

Figure 7—figure supplement 1. Conservation of the DdMyo7 motor domain.

Figure 7—figure supplement 1.

(A) M-coffee sequence alignment (Wallace, 2006) of the relay helix region of different myosin motor domains, left column number is amino acid position. Switch two and L50 subdomain are shaded, circled columns indicate the highly conserved glutamic acid in switch 2 (non-hydrolyzer, DdMyo7 E386V) and hydrophobic residue in relay loop (uncoupler, DdMyo7 I426A). Symbols below indicate degree of conservation between sequences: ‘*’ identical, ‘:’ strongly similar, ‘.’ weakly similar. (B) Representative western blot analysis of two cytoskeleton prep supernatants (S) and pellets (P) from myo7 null cells expressing either wildtype or the E386V mutant. Band at 270 kDa is DdMyo7, band at 42 kDa is actin.