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Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans.

Severson AF, Bowerman B - J. Cell Biol. (2003)

Bottom Line: Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces.Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex.This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA.

ABSTRACT
In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

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Intact MFs are required for cortical PAR localization, centrosome flattening, and mitotic spindle orientation. (a and b) Disrupting MF assembly with LatA results in a loss of cortical PAR-3 and PAR-2. (e and f) Cortical PAR protein levels also are reduced in pfn-1(RNAi) embryos. (c and g) Cortical MF assembly is disrupted in LatA-treated and pfn-1 mutant embryos (red). Both the anterior and posterior centrosomes remain rounded (green), and the first mitotic spindle does not become posteriorly displaced. (d and h) Centrosomes remain rounded in pfn-1(RNAi); par-3(it71) double mutants and in par-3(it71) mutants treated with LatA, suggesting that PAR-3 does not inhibit centrosome flattening in embryos with disrupted F-actin. Instead, we suggest that intact MFs are required for centrosome flattening itself. (i–k) Mitotic spindle orientation at the two-cell stage. Asterisks indicate the position of spindle poles. (i) In wild-type embryos, the posterior spindle lies along the a-p axis, perpendicular to the anterior spindle. (j) Both spindles are transverse in LatA-treated embryos. (k) Both spindles are parallel to the a-p axis in par-3(it71) mutant embryos. (l) Both spindles are transverse in par-3(it71) embryos treated with LatA.
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fig2: Intact MFs are required for cortical PAR localization, centrosome flattening, and mitotic spindle orientation. (a and b) Disrupting MF assembly with LatA results in a loss of cortical PAR-3 and PAR-2. (e and f) Cortical PAR protein levels also are reduced in pfn-1(RNAi) embryos. (c and g) Cortical MF assembly is disrupted in LatA-treated and pfn-1 mutant embryos (red). Both the anterior and posterior centrosomes remain rounded (green), and the first mitotic spindle does not become posteriorly displaced. (d and h) Centrosomes remain rounded in pfn-1(RNAi); par-3(it71) double mutants and in par-3(it71) mutants treated with LatA, suggesting that PAR-3 does not inhibit centrosome flattening in embryos with disrupted F-actin. Instead, we suggest that intact MFs are required for centrosome flattening itself. (i–k) Mitotic spindle orientation at the two-cell stage. Asterisks indicate the position of spindle poles. (i) In wild-type embryos, the posterior spindle lies along the a-p axis, perpendicular to the anterior spindle. (j) Both spindles are transverse in LatA-treated embryos. (k) Both spindles are parallel to the a-p axis in par-3(it71) mutant embryos. (l) Both spindles are transverse in par-3(it71) embryos treated with LatA.

Mentions: Disruption of MF assembly results in a-p polarity defects similar to those caused by mutations in par-2. In wild-type embryos treated with cytochalasin D (Hill and Strome, 1988) or latrunculin A (LatA; Fig. 2 c; eight out of nine embryos), neither centrosome flattened. The failure of either pole to flatten could result from mislocalized PAR-3 inhibiting flattening at both poles as in par-2 mutants (Cheng et al., 1995). Moreover, MFs might be required for cortical localization of the PAR proteins, with such localization being important for their function. Therefore, we examined the localization of PAR-2 and PAR-3 in embryos exposed to LatA. We found that PAR-2 and PAR-3 both require intact MFs to localize to the cortex. Both were undetectable at the cortex, or present at severely reduced levels, in the presence of LatA (Fig. 2, a and b; n ≥ 5 for each; see Materials and methods). PAR-2 accumulated around the centrosomes of LatA-treated embryos as was observed recently in pod mutants with defects in a-p polarity (Rappleye et al., 2002). We also examined centrosome flattening and PAR localization in embryos with reduced levels of the profilin PFN-1, which we have recently shown is required for the assembly of cortical MFs (Severson et al., 2002) (Fig. 2 g). Consistent with our findings in LatA-treated embryos, the posterior centrosome failed to flatten in embryos depleted of PFN-1 using dsRNA-mediated gene silencing, or RNAi (Fig. 2 g), and PAR-2 was undetectable at the cortex but instead localized around centrosomes (Fig. 2 f; 10 out of 12 embryos). Although PAR-3 was always detected at the cortex in PFN-1–depleted embryos, it was present at much reduced levels compared with wild-type embryos fixed on the same slides (Fig. 2 e; six out of six embryos). The remaining cortical PAR-3 may simply reflect residual MF assembly because low levels of cortical F-actin still assemble in embryos with reduced levels of profilin (Severson et al., 2002). We conclude that centrosome flattening and the cortical localization of PAR-2 and PAR-3 all require an intact MF cytoskeleton.


Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans.

Severson AF, Bowerman B - J. Cell Biol. (2003)

Intact MFs are required for cortical PAR localization, centrosome flattening, and mitotic spindle orientation. (a and b) Disrupting MF assembly with LatA results in a loss of cortical PAR-3 and PAR-2. (e and f) Cortical PAR protein levels also are reduced in pfn-1(RNAi) embryos. (c and g) Cortical MF assembly is disrupted in LatA-treated and pfn-1 mutant embryos (red). Both the anterior and posterior centrosomes remain rounded (green), and the first mitotic spindle does not become posteriorly displaced. (d and h) Centrosomes remain rounded in pfn-1(RNAi); par-3(it71) double mutants and in par-3(it71) mutants treated with LatA, suggesting that PAR-3 does not inhibit centrosome flattening in embryos with disrupted F-actin. Instead, we suggest that intact MFs are required for centrosome flattening itself. (i–k) Mitotic spindle orientation at the two-cell stage. Asterisks indicate the position of spindle poles. (i) In wild-type embryos, the posterior spindle lies along the a-p axis, perpendicular to the anterior spindle. (j) Both spindles are transverse in LatA-treated embryos. (k) Both spindles are parallel to the a-p axis in par-3(it71) mutant embryos. (l) Both spindles are transverse in par-3(it71) embryos treated with LatA.
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Related In: Results  -  Collection

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fig2: Intact MFs are required for cortical PAR localization, centrosome flattening, and mitotic spindle orientation. (a and b) Disrupting MF assembly with LatA results in a loss of cortical PAR-3 and PAR-2. (e and f) Cortical PAR protein levels also are reduced in pfn-1(RNAi) embryos. (c and g) Cortical MF assembly is disrupted in LatA-treated and pfn-1 mutant embryos (red). Both the anterior and posterior centrosomes remain rounded (green), and the first mitotic spindle does not become posteriorly displaced. (d and h) Centrosomes remain rounded in pfn-1(RNAi); par-3(it71) double mutants and in par-3(it71) mutants treated with LatA, suggesting that PAR-3 does not inhibit centrosome flattening in embryos with disrupted F-actin. Instead, we suggest that intact MFs are required for centrosome flattening itself. (i–k) Mitotic spindle orientation at the two-cell stage. Asterisks indicate the position of spindle poles. (i) In wild-type embryos, the posterior spindle lies along the a-p axis, perpendicular to the anterior spindle. (j) Both spindles are transverse in LatA-treated embryos. (k) Both spindles are parallel to the a-p axis in par-3(it71) mutant embryos. (l) Both spindles are transverse in par-3(it71) embryos treated with LatA.
Mentions: Disruption of MF assembly results in a-p polarity defects similar to those caused by mutations in par-2. In wild-type embryos treated with cytochalasin D (Hill and Strome, 1988) or latrunculin A (LatA; Fig. 2 c; eight out of nine embryos), neither centrosome flattened. The failure of either pole to flatten could result from mislocalized PAR-3 inhibiting flattening at both poles as in par-2 mutants (Cheng et al., 1995). Moreover, MFs might be required for cortical localization of the PAR proteins, with such localization being important for their function. Therefore, we examined the localization of PAR-2 and PAR-3 in embryos exposed to LatA. We found that PAR-2 and PAR-3 both require intact MFs to localize to the cortex. Both were undetectable at the cortex, or present at severely reduced levels, in the presence of LatA (Fig. 2, a and b; n ≥ 5 for each; see Materials and methods). PAR-2 accumulated around the centrosomes of LatA-treated embryos as was observed recently in pod mutants with defects in a-p polarity (Rappleye et al., 2002). We also examined centrosome flattening and PAR localization in embryos with reduced levels of the profilin PFN-1, which we have recently shown is required for the assembly of cortical MFs (Severson et al., 2002) (Fig. 2 g). Consistent with our findings in LatA-treated embryos, the posterior centrosome failed to flatten in embryos depleted of PFN-1 using dsRNA-mediated gene silencing, or RNAi (Fig. 2 g), and PAR-2 was undetectable at the cortex but instead localized around centrosomes (Fig. 2 f; 10 out of 12 embryos). Although PAR-3 was always detected at the cortex in PFN-1–depleted embryos, it was present at much reduced levels compared with wild-type embryos fixed on the same slides (Fig. 2 e; six out of six embryos). The remaining cortical PAR-3 may simply reflect residual MF assembly because low levels of cortical F-actin still assemble in embryos with reduced levels of profilin (Severson et al., 2002). We conclude that centrosome flattening and the cortical localization of PAR-2 and PAR-3 all require an intact MF cytoskeleton.

Bottom Line: Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces.Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex.This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA.

ABSTRACT
In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

Show MeSH