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Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

Chang CW, Kumar S - Sci Rep (2015)

Bottom Line: Here we combine biophotonic and genetic approaches to address these open questions.Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively.Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720.

ABSTRACT
While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.

No MeSH data available.


Related in: MedlinePlus

Model describing NMMII isoform localization and stability in SF sub-populations as well as the contribution of NMMII head domain to SF viscoelasticity.(Left) The two-headed arrows represent exchanges of isoforms in SF assembly with those in cytoplasm, with faster exchange reflected by thicker arrows. In this model, we propose that MIIB preferentially contributes to the viscoelastic properties of peripheral SFs due to its greater localization and higher assembly stability in that subset of SFs. Preferential contribution of MIIA to the viscoelastic properties of central SFs can be explained in a similar way. (Right) The contribution of NMMII head domain to SF viscoelasticity is further illustrated in detail. The retraction of a model SF is depicted prior to (t0), during (t1), and long after (t∞) laser incision. Without the NMMII head domain (ΔHead), the severed ends of the SF retract more rapidly than a corresponding SF with the wild-type (WT) NMMII, reflected by the retraction distance at a given time point (t1). However, deletion of the head domain does not appear to significantly influence the final retraction distance (L0 at t∞).
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f8: Model describing NMMII isoform localization and stability in SF sub-populations as well as the contribution of NMMII head domain to SF viscoelasticity.(Left) The two-headed arrows represent exchanges of isoforms in SF assembly with those in cytoplasm, with faster exchange reflected by thicker arrows. In this model, we propose that MIIB preferentially contributes to the viscoelastic properties of peripheral SFs due to its greater localization and higher assembly stability in that subset of SFs. Preferential contribution of MIIA to the viscoelastic properties of central SFs can be explained in a similar way. (Right) The contribution of NMMII head domain to SF viscoelasticity is further illustrated in detail. The retraction of a model SF is depicted prior to (t0), during (t1), and long after (t∞) laser incision. Without the NMMII head domain (ΔHead), the severed ends of the SF retract more rapidly than a corresponding SF with the wild-type (WT) NMMII, reflected by the retraction distance at a given time point (t1). However, deletion of the head domain does not appear to significantly influence the final retraction distance (L0 at t∞).

Mentions: Thus, we transduced U373 MG cells with shRNA to reduce endogenous MIIA background and then rescued with the RNAi-resistant versions of these constructs. To verify that these mutations functionally affected cell-scale force generation, we performed traction force microscopy, in which cellular traction stresses and strain energy per unit area are measured based on the motions of fiduciary particles embedded in a compliant hydrogel substrate (Figure S6)29314041. None of the constructs gave rise to traction force and strain energy/area levels that were statistically distinguishable from the MIIA KD, with the exception of full length MIIA, which rescued traction forces and strain energy/area to roughly 80% and 40% of the values for naïve cells, respectively. We then performed SF photo-disruption experiments to study the contributions of these constructs to SF mechanics (Fig. 6). While the SF retraction plateau (L0) with the constructs did not appear to differ from the WT rescue (Fig. 6B), we were surprised to observe that deletion of the head domain reduced the time constant of SF retraction relative to rescue with full length MIIA (Fig. 6A,C,D; Fig. 7). This is consistent with a scenario in which the effective viscosity or internal friction of the stress fiber decreases in the absence of the head domain (∆Head) (Fig. 8). In this case, the retraction is presumably driven by MIIB and is associated with compromised whole-cell traction force.


Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

Chang CW, Kumar S - Sci Rep (2015)

Model describing NMMII isoform localization and stability in SF sub-populations as well as the contribution of NMMII head domain to SF viscoelasticity.(Left) The two-headed arrows represent exchanges of isoforms in SF assembly with those in cytoplasm, with faster exchange reflected by thicker arrows. In this model, we propose that MIIB preferentially contributes to the viscoelastic properties of peripheral SFs due to its greater localization and higher assembly stability in that subset of SFs. Preferential contribution of MIIA to the viscoelastic properties of central SFs can be explained in a similar way. (Right) The contribution of NMMII head domain to SF viscoelasticity is further illustrated in detail. The retraction of a model SF is depicted prior to (t0), during (t1), and long after (t∞) laser incision. Without the NMMII head domain (ΔHead), the severed ends of the SF retract more rapidly than a corresponding SF with the wild-type (WT) NMMII, reflected by the retraction distance at a given time point (t1). However, deletion of the head domain does not appear to significantly influence the final retraction distance (L0 at t∞).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559901&req=5

f8: Model describing NMMII isoform localization and stability in SF sub-populations as well as the contribution of NMMII head domain to SF viscoelasticity.(Left) The two-headed arrows represent exchanges of isoforms in SF assembly with those in cytoplasm, with faster exchange reflected by thicker arrows. In this model, we propose that MIIB preferentially contributes to the viscoelastic properties of peripheral SFs due to its greater localization and higher assembly stability in that subset of SFs. Preferential contribution of MIIA to the viscoelastic properties of central SFs can be explained in a similar way. (Right) The contribution of NMMII head domain to SF viscoelasticity is further illustrated in detail. The retraction of a model SF is depicted prior to (t0), during (t1), and long after (t∞) laser incision. Without the NMMII head domain (ΔHead), the severed ends of the SF retract more rapidly than a corresponding SF with the wild-type (WT) NMMII, reflected by the retraction distance at a given time point (t1). However, deletion of the head domain does not appear to significantly influence the final retraction distance (L0 at t∞).
Mentions: Thus, we transduced U373 MG cells with shRNA to reduce endogenous MIIA background and then rescued with the RNAi-resistant versions of these constructs. To verify that these mutations functionally affected cell-scale force generation, we performed traction force microscopy, in which cellular traction stresses and strain energy per unit area are measured based on the motions of fiduciary particles embedded in a compliant hydrogel substrate (Figure S6)29314041. None of the constructs gave rise to traction force and strain energy/area levels that were statistically distinguishable from the MIIA KD, with the exception of full length MIIA, which rescued traction forces and strain energy/area to roughly 80% and 40% of the values for naïve cells, respectively. We then performed SF photo-disruption experiments to study the contributions of these constructs to SF mechanics (Fig. 6). While the SF retraction plateau (L0) with the constructs did not appear to differ from the WT rescue (Fig. 6B), we were surprised to observe that deletion of the head domain reduced the time constant of SF retraction relative to rescue with full length MIIA (Fig. 6A,C,D; Fig. 7). This is consistent with a scenario in which the effective viscosity or internal friction of the stress fiber decreases in the absence of the head domain (∆Head) (Fig. 8). In this case, the retraction is presumably driven by MIIB and is associated with compromised whole-cell traction force.

Bottom Line: Here we combine biophotonic and genetic approaches to address these open questions.Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively.Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720.

ABSTRACT
While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.

No MeSH data available.


Related in: MedlinePlus