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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 co-localization of MIIA and MIIB in SFs.(A–E) Images of MIIB (green) and MIIA (red) in immunostained cells. (F) Quantification of the MIIA/MIIB fluorescence intensity ratio in peripheral SF and central SF sub-populations. N = 22 cells. Error bars represent SEM. The average ratios are statistically significantly different (p < 0.05; two-tailed Student’s t-test), indicated by a star sign (*). In all images, the image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). Scale bar: 30 μm. See Methods for the approach to image compartmentalization for different SF sub-populations.
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f4: Immunofluorescence confocal optical section images illustrating co-localization of MIIA and MIIB in SFs.(A–E) Images of MIIB (green) and MIIA (red) in immunostained cells. (F) Quantification of the MIIA/MIIB fluorescence intensity ratio in peripheral SF and central SF sub-populations. N = 22 cells. Error bars represent SEM. The average ratios are statistically significantly different (p < 0.05; two-tailed Student’s t-test), indicated by a star sign (*). In all images, the image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). Scale bar: 30 μm. See Methods for the approach to image compartmentalization for different SF sub-populations.

Mentions: If MIIA and MIIB indeed preferentially contribute to central and peripheral SF contractility, respectively, then one might expect this to be reflected in the regional localization of each isoform. We therefore performed dual-color localization studies in which we immunostained for MIIA and MIIB using isoform-specific antibodies. We readily identified SFs at the cell periphery in which both isoforms localized but were modestly enriched in MIIB (Fig. 4A–E). Where lamellipodia were present, MIIA localized to the lamellipodial edge, consistent with past reports14181936. In our image quantification, we excluded the edges of lamellar areas (where there were no SFs) and defined SFs within 20 pixels (~8.2 μm) from cell edge to be peripheral SFs. The enrichment of MIIB at cell periphery was then verified by quantitative ratiometric analysis (Fig. 4F).


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 co-localization of MIIA and MIIB in SFs.(A–E) Images of MIIB (green) and MIIA (red) in immunostained cells. (F) Quantification of the MIIA/MIIB fluorescence intensity ratio in peripheral SF and central SF sub-populations. N = 22 cells. Error bars represent SEM. The average ratios are statistically significantly different (p < 0.05; two-tailed Student’s t-test), indicated by a star sign (*). In all images, the image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). Scale bar: 30 μm. See Methods for the approach to image compartmentalization for different SF sub-populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Immunofluorescence confocal optical section images illustrating co-localization of MIIA and MIIB in SFs.(A–E) Images of MIIB (green) and MIIA (red) in immunostained cells. (F) Quantification of the MIIA/MIIB fluorescence intensity ratio in peripheral SF and central SF sub-populations. N = 22 cells. Error bars represent SEM. The average ratios are statistically significantly different (p < 0.05; two-tailed Student’s t-test), indicated by a star sign (*). In all images, the image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). Scale bar: 30 μm. See Methods for the approach to image compartmentalization for different SF sub-populations.
Mentions: If MIIA and MIIB indeed preferentially contribute to central and peripheral SF contractility, respectively, then one might expect this to be reflected in the regional localization of each isoform. We therefore performed dual-color localization studies in which we immunostained for MIIA and MIIB using isoform-specific antibodies. We readily identified SFs at the cell periphery in which both isoforms localized but were modestly enriched in MIIB (Fig. 4A–E). Where lamellipodia were present, MIIA localized to the lamellipodial edge, consistent with past reports14181936. In our image quantification, we excluded the edges of lamellar areas (where there were no SFs) and defined SFs within 20 pixels (~8.2 μm) from cell edge to be peripheral SFs. The enrichment of MIIB at cell periphery was then verified by quantitative ratiometric analysis (Fig. 4F).

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