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Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

Newman-Smith E, Kourakis MJ, Reeves W, Veeman M, Smith WC - Elife (2015)

Bottom Line: Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization.On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization.Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, United States.

ABSTRACT
The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

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Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.Arrows in A indicate contacts of cells with the basement membrane, and arrowheads in C indicate anteriorly localized protein. Densitometry readings across the center of four cells demonstrate the transition in MRLC9/12 localization from stage 21 to stage 22 (charts in B and C). (D) At stage 24, the anterior localization of both proteins persists. Anterior is to the right in all panels. Scale bars are 10 μm.DOI:http://dx.doi.org/10.7554/eLife.05361.004
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fig2: Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.Arrows in A indicate contacts of cells with the basement membrane, and arrowheads in C indicate anteriorly localized protein. Densitometry readings across the center of four cells demonstrate the transition in MRLC9/12 localization from stage 21 to stage 22 (charts in B and C). (D) At stage 24, the anterior localization of both proteins persists. Anterior is to the right in all panels. Scale bars are 10 μm.DOI:http://dx.doi.org/10.7554/eLife.05361.004

Mentions: To visualize myosin assemblies in developing notochord cells, we constructed fusion proteins of MRLC9/12 and Myosin10/11/14/9 with Venus fluorescent protein and myc antigen, respectively. The resulting cDNAs were fused to the Brachyury cis-regulatory region to drive notochord expression (Corbo et al., 1997), and then introduced to embryos by electroporation. Low doses of plasmid were used to encourage highly mosaic expression. This allows the visualization of the exogenous proteins in cells isolated from the expression of neighboring cells, which would otherwise make the determination of anterior vs posterior protein localization difficult (Jiang et al., 2005; Kourakis et al., 2014). At the earliest developmental stage analyzed, stage 19—when the notochord cells are mid-intercalation, both Myosin10/11/14/9-myc and MRLC9/12-Venus were observed throughout the cells, with the highest localization on the perinotochordal surfaces where cells contact the basement membrane that surrounds the developing notochord (white arrows, stage 19; Figure 2A; see also Veeman et al., 2008). At the immediate completion of intercalation (stage 21, Figure 2B), the localization of both fusion proteins was still the strongest at the basal/lateral edges. However, less than an hour later, at stage 22, both proteins were found localized to the anterior pole of each notochord cell (arrowhead, Figure 2C), as well as continuing to be found at the basal/lateral edges. Quantification of MRLC9/12-Venus fluorescence signal along the anterior/posterior axis of notochord cells confirms a distinct transition in MRLC9/12-Venus localization between stages 21 and 22 (insets, Figure 2B,C). During notochord cell elongation, and at its completion (stage 24), the anterior localization of both proteins persists (Figure 2D). The onset of anterior/posterior localization of myosin assemblies closely matches the localization of Pk-myc, with A/P localization of Pk-myc starting at stage 22 (Kourakis et al., 2014). However, the Pk-myc and MRLC9/12 proteins do not appear to completely colocalize spatially. At stage 22, Pk-myc, Myosin10/11/14/9-myc, and MRLC9/12-Venus are each found to be enriched on the anterior side of the cells. However, as elongation progresses, Pk-myc localization becomes restricted to a small region within the center of this domain (Jiang et al., 2005; Kourakis et al., 2014), while Myosin10/11/14/9-myc and MRLC9/12-Venus remain more dispersed (Figure 2).10.7554/eLife.05361.004Figure 2.Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.


Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

Newman-Smith E, Kourakis MJ, Reeves W, Veeman M, Smith WC - Elife (2015)

Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.Arrows in A indicate contacts of cells with the basement membrane, and arrowheads in C indicate anteriorly localized protein. Densitometry readings across the center of four cells demonstrate the transition in MRLC9/12 localization from stage 21 to stage 22 (charts in B and C). (D) At stage 24, the anterior localization of both proteins persists. Anterior is to the right in all panels. Scale bars are 10 μm.DOI:http://dx.doi.org/10.7554/eLife.05361.004
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fig2: Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.Arrows in A indicate contacts of cells with the basement membrane, and arrowheads in C indicate anteriorly localized protein. Densitometry readings across the center of four cells demonstrate the transition in MRLC9/12 localization from stage 21 to stage 22 (charts in B and C). (D) At stage 24, the anterior localization of both proteins persists. Anterior is to the right in all panels. Scale bars are 10 μm.DOI:http://dx.doi.org/10.7554/eLife.05361.004
Mentions: To visualize myosin assemblies in developing notochord cells, we constructed fusion proteins of MRLC9/12 and Myosin10/11/14/9 with Venus fluorescent protein and myc antigen, respectively. The resulting cDNAs were fused to the Brachyury cis-regulatory region to drive notochord expression (Corbo et al., 1997), and then introduced to embryos by electroporation. Low doses of plasmid were used to encourage highly mosaic expression. This allows the visualization of the exogenous proteins in cells isolated from the expression of neighboring cells, which would otherwise make the determination of anterior vs posterior protein localization difficult (Jiang et al., 2005; Kourakis et al., 2014). At the earliest developmental stage analyzed, stage 19—when the notochord cells are mid-intercalation, both Myosin10/11/14/9-myc and MRLC9/12-Venus were observed throughout the cells, with the highest localization on the perinotochordal surfaces where cells contact the basement membrane that surrounds the developing notochord (white arrows, stage 19; Figure 2A; see also Veeman et al., 2008). At the immediate completion of intercalation (stage 21, Figure 2B), the localization of both fusion proteins was still the strongest at the basal/lateral edges. However, less than an hour later, at stage 22, both proteins were found localized to the anterior pole of each notochord cell (arrowhead, Figure 2C), as well as continuing to be found at the basal/lateral edges. Quantification of MRLC9/12-Venus fluorescence signal along the anterior/posterior axis of notochord cells confirms a distinct transition in MRLC9/12-Venus localization between stages 21 and 22 (insets, Figure 2B,C). During notochord cell elongation, and at its completion (stage 24), the anterior localization of both proteins persists (Figure 2D). The onset of anterior/posterior localization of myosin assemblies closely matches the localization of Pk-myc, with A/P localization of Pk-myc starting at stage 22 (Kourakis et al., 2014). However, the Pk-myc and MRLC9/12 proteins do not appear to completely colocalize spatially. At stage 22, Pk-myc, Myosin10/11/14/9-myc, and MRLC9/12-Venus are each found to be enriched on the anterior side of the cells. However, as elongation progresses, Pk-myc localization becomes restricted to a small region within the center of this domain (Jiang et al., 2005; Kourakis et al., 2014), while Myosin10/11/14/9-myc and MRLC9/12-Venus remain more dispersed (Figure 2).10.7554/eLife.05361.004Figure 2.Time course for the subcellular localization of myc-tagged myosin10/11/14/9-tagged myosin regulatory light chain (MRLC) and Venus-tagged myosin regulatory light chain (MRLC9/12-Venus) at the stages indicated.

Bottom Line: Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization.On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization.Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, United States.

ABSTRACT
The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

Show MeSH
Related in: MedlinePlus