(A) Domain organization of the myosin heavy chain and myosin fragments used to study the biochemical properties of myosin. The top panel shows the myosin hexamer composed of two myosin heavy chains …
RLC and mutant RLC (19 kDa) and the ELC (16 kDa) bind in a 1:1 stoichiometry to the myosin heavy chain, indicating that mutant RLCs bind with a similar affinity as RLC-TS. Data shown for RLC-AS HMM …
The dependence of steady-state ATP hydrolysis rate is plotted as a function of actin in the concentration range up to 100 µM. Data sets corresponding to the ATP hydrolysis rates in the absence of …
RLC-TS and RLC mutants form predominantly filaments in the absence of ATP (−ATP) and disassemble into the 10S conformation in the presence of ATP (+ATP). Phosphorylation of Threonine-20 or …
(A–B) Myosin intensity in pair of embryos expressing RLC-TS and RLC-TA (A) or RLC-TS and RLC-AE (B) are comparable. The images represent embryos mounted side-by-side and imaged at the same time to …
Exponents (α) of power laws fit (A) and time decay constants (τ) of Kelvin-Voigt fits (B) to displacements of wound edges after tissue ablation are not significantly different between RLC-TS, RLC-TA …
(A) RLC-AS, RLC-TA, and RLC-AE cells generate weaker constrictions than RLC-TS cells. Central line in box plot is the median, the box edges are the 25th and 75th percentiles, and the whiskers …
(A) Aligned mean apical area over time for representative embryos. (B) Time it takes for tissue contraction to 50% the starting mean apical area (A0). RLC-AE tissue constricts slowest compared to …
Comparison of the steady-state kinetic parameters between phosphorylated Drosophila and mammalian myosins.
Parameter | Dm myosin II* | Hs myosin IIA‡ | Hs myosin IIB ‡† | Mm myosin IIC ‡† |
---|---|---|---|---|
kbasal (s−1) | 0.01 ± 0.001 | 0.021 ± 0.003 | <0.004 | <0.004 |
kcat (s−1) | 1.23 ± 0.07 | 0.45 ± 0.03 | 0.17 ± 0.04 | 0.18 ± 0.04 |
Kapp (µM) | 38.9 ± 7.32 | 9 ± 2 | 3.4 ± 1.8 | 4.2 ± 1.1 |
kcat/Kapp (µM−1·s−1) | ~0.03 | ~0.05 | ~0.05 | ~0.04 |
Abbreviations used: Dm: Drosophila melanogaster; Hs: Homo sapiens; Mm: Mus musculus.
*From this study.
†From Kim et al. (2005).
‡From Kovács et al. (2004a).
Steady-state ATP hydrolysis rates of RLC-TS and RLC mutants.
RLC | kbasal (s−1) | s.d. | k100 (s−1) | s.d. |
---|---|---|---|---|
RLC-TS | 0.013 | 0.007 | 0.12 | 0.03 |
RLC-TS | 0.010 | 0.001 | 0.82 | 0.01 |
RLC-AS | 0.024 | 0.005 | 0.15 | 0.02 |
RLC-AS | 0.024 | 0.008 | 0.81 | 0.04 |
RLC-TA | 0.009 | 0.002 | 0.12 | 0.01 |
RLC-TA | 0.008 | 0.002 | 0.37 | 0.01 |
RLC-AA | 0.014 | 0.006 | 0.16 | 0.03 |
RLC-AA | 0.012 | 0.002 | 0.23 | 0.02 |
RLC-AE | 0.010 | 0.002 | 0.21 | 0.03 |
RLC-AE | 0.009 | 0.002 | 0.23 | 0.01 |
RLC-EE | 0.011 | 0.007 | 0.19 | 0.01 |
RLC-EE | 0.010 | 0.002 | 0.22 | 0.01 |
The actin-activated ATPase activity was measured at 25°C, as described under ‘Materials and methods’. Treatment of RLC with MLCK is indicated by bold print. For comparison, kbasal the steady-state ATPase activity in the absence of actin filaments, and k100, the steady-state ATPase activity at 100 µM actin filaments, are listed.
Comparison of Drosophila and mammalian myosin filament dimensions.
Parameter | Dm myosin-II*,† | Hs myosin IIA‡ | Hs myosin IIB‡ | Hs myosin IIC‡ |
---|---|---|---|---|
Mean number of myosins per filament | 12.8 ± 4.5* 14.9 ± 3† | 29 | 30 | 14 |
Mean bare zone length (nm) | 194 ± 21* 204 ± 19† | 167 ± 19 | 166 ± 16 | 219 ± 13 |
Filament length (nm) | 312 ± 25* | 301 ± 24 | 323 ± 24 | 293 ± 33 |
Sliding velocity (nm·s−1) | 247 ± 37 | 82§ | n.d. | n.d. |
*From this study.
†From Kiehart and Feghali (1986).
‡From Billington et al. (2013).
§From Cuda et al. (1997).
Fly stocks.
Genotype | Source | RRID for associated stocks or alleles* | |
---|---|---|---|
Oregon-R-C (wild type) | 2 | RRID: FBst0000005 | |
ovoD1FRT101/Y; hsFLP-38/hsFLP-38 | 2 | ovoD1 | RRID: FBst0001813 |
sqh1FRT101/FM7; P{w+sqh-TS::GFP}attP1/CyO | 1 | sqh1 sqh-TS | RRID: FBal0016066 RRID: FBal0298052 |
sqh1FRT101/FM7; P{w+sqh-AS::GFP}attP1/CyO | 1 | sqh1 sqh-AS | RRID: FBal0016066 RRID: FBal0298055 |
sqh1FRT101/FM7; P{w+sqh-TA::GFP}attP1/CyO | 1 | sqh1 sqh-TA | RRID: FBal0016066 RRID: FBal0298054 |
sqh1FRT101/FM7; P{w+sqh-AE::GFP}attP1/CyO | 1 | sqh1 sqh-AE | RRID: FBal0016066 RRID: FBal0298057 |
sqh1FRT101/FM7; P{w+sqh-TS::GFP}attP1 P{w+Gap43::mCherry}attP40/CyO | 1 | sqh1 sqh-TS Gap43 | RRID: FBal0016066 RRID: FBal0298052 RRID: FBtp0087760 |
sqh1FRT101/FM7; P{w+sqh-AS::GFP}attP1 P{w+Gap43::mCherry}attP40/CyO | 3 | sqh1 sqh-AS Gap43 | RRID: FBal0016066 RRID: FBal0298055 RRID: FBtp0087760 |
sqh1FRT101/FM7; P{w+sqh-TA::GFP}attP1 P{w+Gap43::mCherry}attP40/CyO | 1 | sqh1 sqh-TA Gap43 | RRID: FBal0016066 RRID: FBal0298054 RRID: FBtp0087760 |
sqh1FRT101/FM7; P{w+sqh-AE::GFP}attP1 P{w+Gap43::mCherry}attP40/CyO | 1 | sqh1 sqh-AE Gap43 | RRID: FBal0016066 RRID: FBal0298057 RRID: FBtp0087760 |
sqh-XX = sqh promoter and ORF with site-directed mutagenesis at Threonine-20 and Serine-21 as noted in Figure 1B (spaghetti squash, sqh is the Drosophila RLC gene name).
Gap43 = sqh promoter with N-terminal 20 amino acids of rat Gap43 gene which contains a myristoylation sequence (Martin et al., 2010).
*FlyBase IDs (RRID: SCR_006549)
Sources: (1) From Vasquez et al. (2014); (2) Bloomington Drosophila Stock Center; (3) This study;