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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

Fluorescence recovery after photobleaching (FRAP) measurements to elucidate NMMII isoform assembly stability in SFs.(A) Typical FRAP experiment using MIIB-GFP and confocal optical sectioning. The image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). An image of the enlarged photobleached region is shown (bottom), with ROIs highlighted for two peripheral SFs (red), a central SF (blue), and cytoplasm (green). A reference region in a non-photobleached area was used for the correction of unintentional bleaching (purple). A negative control region (gray), also outside the photobleached area, was selected to confirm successful correction for unintentional photobleaching, following the correction steps using the reference region (see below). (B) Fluorescence recovery dynamics of MIIA-GFP (top) and MIIB-GFP (bottom) in central (blue curves) and peripheral (red curves) SF subpopulations as well as in cytoplasm (green curves). The negative controls (black curves) indicate fluorescence dynamics from non-photobleached areas in the corresponding FRAP experiments. (C) Quantification of immobile fraction (top) and half-life (bottom) of the fluorescence recovery dynamics. N ≥ 50 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*). NS: not significant. Scale bar: 15 μm.
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f5: Fluorescence recovery after photobleaching (FRAP) measurements to elucidate NMMII isoform assembly stability in SFs.(A) Typical FRAP experiment using MIIB-GFP and confocal optical sectioning. The image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). An image of the enlarged photobleached region is shown (bottom), with ROIs highlighted for two peripheral SFs (red), a central SF (blue), and cytoplasm (green). A reference region in a non-photobleached area was used for the correction of unintentional bleaching (purple). A negative control region (gray), also outside the photobleached area, was selected to confirm successful correction for unintentional photobleaching, following the correction steps using the reference region (see below). (B) Fluorescence recovery dynamics of MIIA-GFP (top) and MIIB-GFP (bottom) in central (blue curves) and peripheral (red curves) SF subpopulations as well as in cytoplasm (green curves). The negative controls (black curves) indicate fluorescence dynamics from non-photobleached areas in the corresponding FRAP experiments. (C) Quantification of immobile fraction (top) and half-life (bottom) of the fluorescence recovery dynamics. N ≥ 50 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*). NS: not significant. Scale bar: 15 μm.

Mentions: While these studies suggest differences in isoform localization, these differences are relatively subtle, which is perhaps not surprising given that MIIA and MIIB can co-assemble within single fibers37. This would be consistent with the notion that MIIA and MIIB associate with both populations of SFs, with the stoichiometry dependent upon location. Moreover, these modest localization differences are unlikely to solely explain the differential effects of each isoform on SF mechanics. However, we reasoned that while both isoforms localize with both SF pools, they might do so with different degrees of stability. To explore this possibility, we separately expressed GFP-tagged MIIA and MIIB and performed fluorescence recovery after photobleaching (FRAP) measurements on peripheral and central SFs (Fig. 5A,B). We then fit the recovery curves to a standard first-order model3839 to obtain the half-life of recovery and the immobile fraction (i.e., the fraction of fluorescent subunits incapable of exchanging with the bulk). Within each SF population, a larger population of MIIB was immobile, indicating that MIIB more stably associates with both SF populations than MIIA, consistent with previous studies (Fig. 5C, top plot)1736. Superimposed upon these differences, we found that a larger fraction of MIIB was immobile within peripheral SFs than within central SFs, consistent with a model in which MIIB associates more stably with peripheral SFs than with central SFs. Recovery half-life results (Fig. 5C, bottom plot) were consistent with this observation; MIIB within peripheral SFs required a longer half life of recovery than MIIA within peripheral SFs, indicating slower or less frequent exchange of MIIB molecules in peripheral SFs and those in the surrounding cytoplasm. This, in turn further implies more stable assembly for the mobile fraction of MIIB at periphery. Together, these data suggest that MIIB more stably associates with peripheral SFs.


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

Chang CW, Kumar S - Sci Rep (2015)

Fluorescence recovery after photobleaching (FRAP) measurements to elucidate NMMII isoform assembly stability in SFs.(A) Typical FRAP experiment using MIIB-GFP and confocal optical sectioning. The image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). An image of the enlarged photobleached region is shown (bottom), with ROIs highlighted for two peripheral SFs (red), a central SF (blue), and cytoplasm (green). A reference region in a non-photobleached area was used for the correction of unintentional bleaching (purple). A negative control region (gray), also outside the photobleached area, was selected to confirm successful correction for unintentional photobleaching, following the correction steps using the reference region (see below). (B) Fluorescence recovery dynamics of MIIA-GFP (top) and MIIB-GFP (bottom) in central (blue curves) and peripheral (red curves) SF subpopulations as well as in cytoplasm (green curves). The negative controls (black curves) indicate fluorescence dynamics from non-photobleached areas in the corresponding FRAP experiments. (C) Quantification of immobile fraction (top) and half-life (bottom) of the fluorescence recovery dynamics. N ≥ 50 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*). NS: not significant. Scale bar: 15 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Fluorescence recovery after photobleaching (FRAP) measurements to elucidate NMMII isoform assembly stability in SFs.(A) Typical FRAP experiment using MIIB-GFP and confocal optical sectioning. The image brightness was adjusted to more clearly depict stress fibers (see Fig. 2 legend). An image of the enlarged photobleached region is shown (bottom), with ROIs highlighted for two peripheral SFs (red), a central SF (blue), and cytoplasm (green). A reference region in a non-photobleached area was used for the correction of unintentional bleaching (purple). A negative control region (gray), also outside the photobleached area, was selected to confirm successful correction for unintentional photobleaching, following the correction steps using the reference region (see below). (B) Fluorescence recovery dynamics of MIIA-GFP (top) and MIIB-GFP (bottom) in central (blue curves) and peripheral (red curves) SF subpopulations as well as in cytoplasm (green curves). The negative controls (black curves) indicate fluorescence dynamics from non-photobleached areas in the corresponding FRAP experiments. (C) Quantification of immobile fraction (top) and half-life (bottom) of the fluorescence recovery dynamics. N ≥ 50 per condition. Error bars represent SEM. With two-tailed Student’s t-tests, statistically significant differences (p < 0.05) are denoted by star signs (*). NS: not significant. Scale bar: 15 μm.
Mentions: While these studies suggest differences in isoform localization, these differences are relatively subtle, which is perhaps not surprising given that MIIA and MIIB can co-assemble within single fibers37. This would be consistent with the notion that MIIA and MIIB associate with both populations of SFs, with the stoichiometry dependent upon location. Moreover, these modest localization differences are unlikely to solely explain the differential effects of each isoform on SF mechanics. However, we reasoned that while both isoforms localize with both SF pools, they might do so with different degrees of stability. To explore this possibility, we separately expressed GFP-tagged MIIA and MIIB and performed fluorescence recovery after photobleaching (FRAP) measurements on peripheral and central SFs (Fig. 5A,B). We then fit the recovery curves to a standard first-order model3839 to obtain the half-life of recovery and the immobile fraction (i.e., the fraction of fluorescent subunits incapable of exchanging with the bulk). Within each SF population, a larger population of MIIB was immobile, indicating that MIIB more stably associates with both SF populations than MIIA, consistent with previous studies (Fig. 5C, top plot)1736. Superimposed upon these differences, we found that a larger fraction of MIIB was immobile within peripheral SFs than within central SFs, consistent with a model in which MIIB associates more stably with peripheral SFs than with central SFs. Recovery half-life results (Fig. 5C, bottom plot) were consistent with this observation; MIIB within peripheral SFs required a longer half life of recovery than MIIA within peripheral SFs, indicating slower or less frequent exchange of MIIB molecules in peripheral SFs and those in the surrounding cytoplasm. This, in turn further implies more stable assembly for the mobile fraction of MIIB at periphery. Together, these data suggest that MIIB more stably associates with peripheral SFs.

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