Molecular-level evidence of force maintenance by smooth muscle myosin during LC20 dephosphorylation

Figures

• 

  Figure S1.. Schematic diagrams of the assay chamber used for all experiments. (A–C) Exploded (A) and assembled (B) views of the flow-through chamber components are shown. A preinjection droplet and silicone grease (C) were used to minimize bulk flow during MLCP injections performed with a pipette tip (see Materials and methods for details). Dimensions are not to scale.

• 

  Figure S2.. The experimental setup used for the laser trap and in vitro motility assays. An assay chamber is secured to a metal frame (red arrow), placed on the heated microscope slide holder (yellow arrow), and then positioned such that the region below the microporous membrane is located directly above the objective lens. An acrylic plastic cover (blue arrow) is used to support the pipette tip (green arrow) during injections.

• 

  Figure S3.. Injection of MgATP during in vitro motility assays containing actin filaments bound to SkMM in rigor. Each color represents the fmot data collected from individual assays (n = 3) as movement is initiated from rigor to maximum motility. MgATP was injected at time = 0 s. The parameter settings of the video analysis software used to measure fmot were the same as those used for the in vitro motility mixture assay (Table S1).

• 

  Figure S4.. Representative in vitro motility assay data fitted with the sigmoid model. (A and B) Blue lines: fmot (A) or v of actin movement (B) for phasic SMM (n = 1); black lines: model fit (Eq. 1). MLCP was injected at time = 0 s.

• 

  Figure S5.. Images from the laser trap assay. (A and B) In the bright-field (A) and fluorescence (B) images shown, the myosin (not visible) on a pedestal (black arrow) is pulling on a fluorescently labeled actin filament (white arrow) attached to a trapped microsphere (blue arrows). The myosin, pedestal, and trapped microsphere are not fluorescently labeled; the outline of the trapped microsphere is visible in B because of small pieces of attached, fluorescently labeled actin. Scale bars: 5 μm.

• 

  Figure S6.. Laser trap kT calibration. (A) At 6.0% laser power (LP), Δx was measured as it was being moved at constant velocity (vc). kT was calculated at increasing vc using the Stokes force (Ff) exerted on the microsphere (Eqs. 3 and 4) and then averaged. (B) kT was calculated at increasing LP (6.0–9.0%) across multiple weeks. Linear fits were determined for each trial (n = 5), represented by different colors, and their slopes were averaged to obtain an interpolation constant (kc) that was used to calculate kT for each individual laser trap assay experiment (Eq. 5). kT for the experiments reported in this study varied between 58 and 71 pN/μm.

• 

  Figure 1.. Injection of MLCP in the laser trap assay. (A and B) The injection was performed at time = 0 s during plateau force generation by phasic SMM (n = 5; A) or tonic SMM (n = 5; B). Each color represents the data collected from an individual assay. TInj: time of the injection following the start of force generation (exemplified using the blue phasic SMM trace); THold: time of force maintenance after the injection (exemplified using the blue phasic SMM trace); (TInj)avg: average TInj; (THold)med: median THold.

• 

  Figure 2.. Injection of control buffer in the laser trap assay. (A and B) The injection was performed at time = 0 s during plateau force generation by phasic SMM (n = 5; A) or tonic SMM (n = 5; B). Each color represents the data collected from an individual assay. (TInj)avg: average time of the injection following the start of force generation; (THold)med: median time of force maintenance after the injection. Inset: A portion of the gray phasic SMM trace wherein the sawtooth pattern can be observed.

• 

  Figure 3.. Injection of MLCP in the in vitro motility assay. (A–D) Normalized fmot and v of actin movement for phasic SMM (n = 11; A and C) or tonic SMM (n = 14; B and D). Gray lines: data collected from individual assays; solid black lines: averaged data; t0: average time at the sigmoid model fit midpoint (Eq. 1). MLCP was injected at time = 0 s. Individual curves were normalized with respect to the sum of the sigmoid model parameters (A and C; Eq. 1).

• 

  Figure 4.. Injection of MLCP in the in vitro motility mixture assay. (A–D) Normalized v of actin movement and fmot for 75% phasic SMM + 25% SkMM (n = 4; A and C) or 75% tonic SMM + 25% SkMM (n = 5; B and D). Each color represents the data collected from an individual assay. MLCP was injected at time = 0 s. v was normalized with respect to the sigmoid model parameter C (Eq. 1). fmot was normalized with respect to the value at the time of MLCP injection.

• 

  Figure S7.. Injection of MLCP during in vitro motility assays performed with SkMM. (A and B) Gray lines: fmot (A) or v of actin movement (B) data collected from individual assays (SkMM = 125 μg/ml; n = 8); solid black lines: averaged data. MLCP was injected at time = 0 s. The parameter settings of the video analysis software used to measure fmot were the same as those used for the in vitro motility mixture assay (Table S1).

• 

  Figure S8.. Western blot of SMM. Total SMM heavy chain (SMMHC), the (+)insert (SMB) isoform and the (−)insert (SMA) isoform content in purified phasic SMM (chicken gizzard) and tonic SMM (pig stomach fundus). Black arrows: 200-kD molecular weight. Source data are available for this figure: SourceData FS8.