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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

Quantification of immunofluorescence confocal optical section images indicating spatial colocalization between MLCK and MIIB (left halves of each panel), and between ROCK and MIIA (right halves of each panel).(A) Percentage of cells exhibiting expression of a given protein in its stress fibers. The third (MIIB + MLCK) and sixth (MIIA + ROCK) columns report the percentage of cells with stress fibers containing both listed components. For example, MIIB + MLCK refers to the percentage of cells containing stress fibers that stain positively for both MIIB and MLCK. (B) Percentage of myosin-isoform-positive and kinase-positive stress fibers that also stained positively for the indicated molecules. All bars in both panels represent data from N > 130 cells taken from multiple biological replicates.
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f3: Quantification of immunofluorescence confocal optical section images indicating spatial colocalization between MLCK and MIIB (left halves of each panel), and between ROCK and MIIA (right halves of each panel).(A) Percentage of cells exhibiting expression of a given protein in its stress fibers. The third (MIIB + MLCK) and sixth (MIIA + ROCK) columns report the percentage of cells with stress fibers containing both listed components. For example, MIIB + MLCK refers to the percentage of cells containing stress fibers that stain positively for both MIIB and MLCK. (B) Percentage of myosin-isoform-positive and kinase-positive stress fibers that also stained positively for the indicated molecules. All bars in both panels represent data from N > 130 cells taken from multiple biological replicates.

Mentions: To further substantiate these correlations, we then performed dual immunofluorescence imaging of ROCK1/MLCK, MIIB/MLCK, and MIIA/ROCK1. As reported previously, ROCK1 and MLCK were observed to localize both to stress fibers and to the cytoplasmic background32333435. For cells in which both kinases unambiguously localized to SFs, MLCK preferentially localized to peripheral SFs while ROCK1 localized to central SFs (Fig. 2, top row), consistent with past observations that ROCK and MLCK selectively regulate these two pools of SFs3033. More importantly, co-staining of MLCK and MIIB indicated that for cells in which MLCK clearly localized to SFs, the MLCK-positive SFs nearly always (>95%) also stained positively for MIIB (Fig. 2, middle row; Fig. 3, left halves of each panel). The same relationship held for ROCK1 and MIIA (>90% colocalization; Fig. 2, bottom row; Fig. 3, right halves of each panel). These observations further support the notion that MIIB and MLCK are associated with common pools of SFs, as are MIIA and ROCK.


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

Chang CW, Kumar S - Sci Rep (2015)

Quantification of immunofluorescence confocal optical section images indicating spatial colocalization between MLCK and MIIB (left halves of each panel), and between ROCK and MIIA (right halves of each panel).(A) Percentage of cells exhibiting expression of a given protein in its stress fibers. The third (MIIB + MLCK) and sixth (MIIA + ROCK) columns report the percentage of cells with stress fibers containing both listed components. For example, MIIB + MLCK refers to the percentage of cells containing stress fibers that stain positively for both MIIB and MLCK. (B) Percentage of myosin-isoform-positive and kinase-positive stress fibers that also stained positively for the indicated molecules. All bars in both panels represent data from N > 130 cells taken from multiple biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Quantification of immunofluorescence confocal optical section images indicating spatial colocalization between MLCK and MIIB (left halves of each panel), and between ROCK and MIIA (right halves of each panel).(A) Percentage of cells exhibiting expression of a given protein in its stress fibers. The third (MIIB + MLCK) and sixth (MIIA + ROCK) columns report the percentage of cells with stress fibers containing both listed components. For example, MIIB + MLCK refers to the percentage of cells containing stress fibers that stain positively for both MIIB and MLCK. (B) Percentage of myosin-isoform-positive and kinase-positive stress fibers that also stained positively for the indicated molecules. All bars in both panels represent data from N > 130 cells taken from multiple biological replicates.
Mentions: To further substantiate these correlations, we then performed dual immunofluorescence imaging of ROCK1/MLCK, MIIB/MLCK, and MIIA/ROCK1. As reported previously, ROCK1 and MLCK were observed to localize both to stress fibers and to the cytoplasmic background32333435. For cells in which both kinases unambiguously localized to SFs, MLCK preferentially localized to peripheral SFs while ROCK1 localized to central SFs (Fig. 2, top row), consistent with past observations that ROCK and MLCK selectively regulate these two pools of SFs3033. More importantly, co-staining of MLCK and MIIB indicated that for cells in which MLCK clearly localized to SFs, the MLCK-positive SFs nearly always (>95%) also stained positively for MIIB (Fig. 2, middle row; Fig. 3, left halves of each panel). The same relationship held for ROCK1 and MIIA (>90% colocalization; Fig. 2, bottom row; Fig. 3, right halves of each panel). These observations further support the notion that MIIB and MLCK are associated with common pools of SFs, as are MIIA and ROCK.

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