Limits...
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

Contributions of NMMII isoforms to SF retraction dynamics.(A,B) Mean retraction traces of multiple central SFs (A) and peripheral SFs (B) following transduction with a non-targeting shRNA, or an shRNA directed against MIIA or MIIB. Error bars represent the standard error of the mean (SEM). Insets show representative curves and their fits. Laser ablation was performed immediately after time = 0 and therefore the origin was not included in the fitting model. Instead, a non-zero y-intercept was assigned as one of the fitting parameters. See Methods for the fitting model and detailed description. (C,D) Viscoelastic time constant (τ) and retraction plateau distance (L0) obtained from the Kelvin-Voigt fits. N ≥ 13 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*) versus the corresponding n. t. (non-targeting) shRNA control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4559901&req=5

f1: Contributions of NMMII isoforms to SF retraction dynamics.(A,B) Mean retraction traces of multiple central SFs (A) and peripheral SFs (B) following transduction with a non-targeting shRNA, or an shRNA directed against MIIA or MIIB. Error bars represent the standard error of the mean (SEM). Insets show representative curves and their fits. Laser ablation was performed immediately after time = 0 and therefore the origin was not included in the fitting model. Instead, a non-zero y-intercept was assigned as one of the fitting parameters. See Methods for the fitting model and detailed description. (C,D) Viscoelastic time constant (τ) and retraction plateau distance (L0) obtained from the Kelvin-Voigt fits. N ≥ 13 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*) versus the corresponding n. t. (non-targeting) shRNA control.

Mentions: To investigate the contributions of MIIA and MIIB to SF viscoelastic properties, we performed loss-of-function studies in which we suppressed MIIA and MIIB in U373 MG cells with lentiviral shRNA. Successful isoform-specific suppression was confirmed by immunoblot (Figure S2), with >99% MIIA knockdown (KD), 63% MIIB KD, and no cross-isoform suppression. To confirm that each isoform could still assemble into SFs when the other one was knocked down, we conducted immunofluorescence imaging of MIIA KD or MIIB KD cells along with naïve cells (for direct comparison), which indicated that the other isoform could indeed incorporate into SFs (Figure S3). We then applied femtosecond laser ablation to photodisrupt single SFs in MIIA KD, MIIB KD, or control (non-targeting shRNA-transduced) cells and tracked SF retraction following fiber scission (Figure S4). Following our previous studies, we compartmentalized SFs according to whether they were located at the cell periphery (peripheral SFs) or more centrally (central SFs), based on our finding that these two populations possess different viscoelastic properties and are controlled by different myosin activators3031. Retraction curves (retraction distance vs. time; Fig. 1A,B, with insets showing representative curves and fits) reveal that MIIA KD and MIIB KD produce distinct shifts in retraction mechanics and that the nature of these shifts depends on the location of the SF severed. As in our previous studies, we fit each individual retraction curve to a Kelvin-Voigt viscoelastic cable model described by a characteristic time constant (τ), which reflects the fiber’s effective viscosity/elasticity ratio, and a plateau retraction distance (Lo), which equals the strained SF length before ablation, and performed statistical analysis on the fitted parameters for many SFs (Fig. 1C,D). This analysis revealed that MIIA KD preferentially reduces the retraction time constant for central SFs, whereas MIIB KD reduces the value of that parameter for peripheral SFs (Fig. 1C). MIIA KD did not significantly affect the retraction plateau distance (L0) of either SF population, while MIIB KD significantly increased that of central SFs (Fig. 1A,B,D).


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

Chang CW, Kumar S - Sci Rep (2015)

Contributions of NMMII isoforms to SF retraction dynamics.(A,B) Mean retraction traces of multiple central SFs (A) and peripheral SFs (B) following transduction with a non-targeting shRNA, or an shRNA directed against MIIA or MIIB. Error bars represent the standard error of the mean (SEM). Insets show representative curves and their fits. Laser ablation was performed immediately after time = 0 and therefore the origin was not included in the fitting model. Instead, a non-zero y-intercept was assigned as one of the fitting parameters. See Methods for the fitting model and detailed description. (C,D) Viscoelastic time constant (τ) and retraction plateau distance (L0) obtained from the Kelvin-Voigt fits. N ≥ 13 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*) versus the corresponding n. t. (non-targeting) shRNA control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Contributions of NMMII isoforms to SF retraction dynamics.(A,B) Mean retraction traces of multiple central SFs (A) and peripheral SFs (B) following transduction with a non-targeting shRNA, or an shRNA directed against MIIA or MIIB. Error bars represent the standard error of the mean (SEM). Insets show representative curves and their fits. Laser ablation was performed immediately after time = 0 and therefore the origin was not included in the fitting model. Instead, a non-zero y-intercept was assigned as one of the fitting parameters. See Methods for the fitting model and detailed description. (C,D) Viscoelastic time constant (τ) and retraction plateau distance (L0) obtained from the Kelvin-Voigt fits. N ≥ 13 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*) versus the corresponding n. t. (non-targeting) shRNA control.
Mentions: To investigate the contributions of MIIA and MIIB to SF viscoelastic properties, we performed loss-of-function studies in which we suppressed MIIA and MIIB in U373 MG cells with lentiviral shRNA. Successful isoform-specific suppression was confirmed by immunoblot (Figure S2), with >99% MIIA knockdown (KD), 63% MIIB KD, and no cross-isoform suppression. To confirm that each isoform could still assemble into SFs when the other one was knocked down, we conducted immunofluorescence imaging of MIIA KD or MIIB KD cells along with naïve cells (for direct comparison), which indicated that the other isoform could indeed incorporate into SFs (Figure S3). We then applied femtosecond laser ablation to photodisrupt single SFs in MIIA KD, MIIB KD, or control (non-targeting shRNA-transduced) cells and tracked SF retraction following fiber scission (Figure S4). Following our previous studies, we compartmentalized SFs according to whether they were located at the cell periphery (peripheral SFs) or more centrally (central SFs), based on our finding that these two populations possess different viscoelastic properties and are controlled by different myosin activators3031. Retraction curves (retraction distance vs. time; Fig. 1A,B, with insets showing representative curves and fits) reveal that MIIA KD and MIIB KD produce distinct shifts in retraction mechanics and that the nature of these shifts depends on the location of the SF severed. As in our previous studies, we fit each individual retraction curve to a Kelvin-Voigt viscoelastic cable model described by a characteristic time constant (τ), which reflects the fiber’s effective viscosity/elasticity ratio, and a plateau retraction distance (Lo), which equals the strained SF length before ablation, and performed statistical analysis on the fitted parameters for many SFs (Fig. 1C,D). This analysis revealed that MIIA KD preferentially reduces the retraction time constant for central SFs, whereas MIIB KD reduces the value of that parameter for peripheral SFs (Fig. 1C). MIIA KD did not significantly affect the retraction plateau distance (L0) of either SF population, while MIIB KD significantly increased that of central SFs (Fig. 1A,B,D).

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