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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

Immunofluorescence confocal optical section images illustrating colocalization of MLCK and MIIB, and of ROCK1 and MIIA.(Top row) Co-staining of ROCK1 and MLCK. Yellow arrows indicate ROCK1-associated central SFs, and light blue arrows indicate MLCK-associated peripheral SFs. (Middle row) Co-staining of MIIB and MLCK. (Bottom row) Co-staining of MIIA and ROCK1. In the top and bottom rows, the image brightness was adjusted to more clearly depict stress fibers in the lamellar regions of the cell, thus necessitating saturation at the cell center. Scale bar: 30 μm.
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f2: Immunofluorescence confocal optical section images illustrating colocalization of MLCK and MIIB, and of ROCK1 and MIIA.(Top row) Co-staining of ROCK1 and MLCK. Yellow arrows indicate ROCK1-associated central SFs, and light blue arrows indicate MLCK-associated peripheral SFs. (Middle row) Co-staining of MIIB and MLCK. (Bottom row) Co-staining of MIIA and ROCK1. In the top and bottom rows, the image brightness was adjusted to more clearly depict stress fibers in the lamellar regions of the cell, thus necessitating saturation at the cell center. Scale bar: 30 μm.

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)

Immunofluorescence confocal optical section images illustrating colocalization of MLCK and MIIB, and of ROCK1 and MIIA.(Top row) Co-staining of ROCK1 and MLCK. Yellow arrows indicate ROCK1-associated central SFs, and light blue arrows indicate MLCK-associated peripheral SFs. (Middle row) Co-staining of MIIB and MLCK. (Bottom row) Co-staining of MIIA and ROCK1. In the top and bottom rows, the image brightness was adjusted to more clearly depict stress fibers in the lamellar regions of the cell, thus necessitating saturation at the cell center. Scale bar: 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Immunofluorescence confocal optical section images illustrating colocalization of MLCK and MIIB, and of ROCK1 and MIIA.(Top row) Co-staining of ROCK1 and MLCK. Yellow arrows indicate ROCK1-associated central SFs, and light blue arrows indicate MLCK-associated peripheral SFs. (Middle row) Co-staining of MIIB and MLCK. (Bottom row) Co-staining of MIIA and ROCK1. In the top and bottom rows, the image brightness was adjusted to more clearly depict stress fibers in the lamellar regions of the cell, thus necessitating saturation at the cell center. Scale bar: 30 μm.
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