1. Cell Biology
  2. Developmental Biology

Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

  1. Claudia G Vasquez
  2. Sarah M Heissler
  3. Neil Billington
  4. James R Sellers  Is a corresponding author
  5. Adam C Martin  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
  2. National Heart, Lung, and Blood Institute, National Institutes of Health, United States
https://doi.org/10.7554/eLife.20828
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Cite this article
  1. Claudia G Vasquez
  2. Sarah M Heissler
  3. Neil Billington
  4. James R Sellers
  5. Adam C Martin
(2016)
Drosophila non-muscle myosin II motor activity determines the rate of tissue folding
eLife 5:e20828.
https://doi.org/10.7554/eLife.20828
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4 figures and 4 tables

Figures

Figure 1 with 2 supplements
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Biochemical characterization of RLC-TS and RLC mutants.

(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 …

https://doi.org/10.7554/eLife.20828.002
Figure 1—figure supplement 1
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Purification and characterization of wild-type and mutant Drosophila myosin (A) PageBlue-stained 4–12% Bis-Tris gel showing recombinant HMM (160 kDa) and full-length (228 kDa) RLC-TS or RLC mutants.

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 …

https://doi.org/10.7554/eLife.20828.003
Figure 1—figure supplement 2
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Steady-state ATP-hydrolysis of RLC-TS (A) and RLC mutants (B–F).

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 …

https://doi.org/10.7554/eLife.20828.004
Figure 2
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Appearance and assembly of RLC-TS and RLC mutants into filaments.

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 …

https://doi.org/10.7554/eLife.20828.008
Figure 3 with 1 supplement
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Myosin mutants with reduced motor activity exhibit reduced tissue recoil following laser ablation.

(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 …

https://doi.org/10.7554/eLife.20828.009
Figure 3—figure supplement 1
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RLC-TS and RLC mutant do not exhibit clear differences in viscoelastic properties.

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 …

https://doi.org/10.7554/eLife.20828.010
Figure 4 with 1 supplement
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Apical constriction rate scales with measured in vitro myosin motor activity.

(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 …

https://doi.org/10.7554/eLife.20828.011
Figure 4—figure supplement 1
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Timing of tissue contraction scales with in vitro myosin motor activity.

(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 …

https://doi.org/10.7554/eLife.20828.012

Tables

Table 1

Comparison of the steady-state kinetic parameters between phosphorylated Drosophila and mammalian myosins.

https://doi.org/10.7554/eLife.20828.005

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

  1. Abbreviations used: Dm: Drosophila melanogaster; Hs: Homo sapiens; Mm: Mus musculus.

  2. *From this study.

  3. †From Kim et al. (2005).

  4. ‡From Kovács et al. (2004a).

Table 2

Steady-state ATP hydrolysis rates of RLC-TS and RLC mutants.

https://doi.org/10.7554/eLife.20828.006

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

  1. 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.

Table 3

Comparison of Drosophila and mammalian myosin filament dimensions.

https://doi.org/10.7554/eLife.20828.007

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.

  1. *From this study.

  2. †From Kiehart and Feghali (1986).

  3. ‡From Billington et al. (2013).

  4. §From Cuda et al. (1997).

Table 4

Fly stocks.

https://doi.org/10.7554/eLife.20828.013

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

  1. 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).

  2. Gap43 = sqh promoter with N-terminal 20 amino acids of rat Gap43 gene which contains a myristoylation sequence (Martin et al., 2010).

  3. *FlyBase IDs (RRID: SCR_006549)

  4. Sources: (1) From Vasquez et al. (2014); (2) Bloomington Drosophila Stock Center; (3) This study;

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