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Tropomyosin and myosin-II cellular levels promote actomyosin ring assembly in fission yeast.

Stark BC, Sladewski TE, Pollard LW, Lord M - Mol. Biol. Cell (2010)

Bottom Line: Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function.Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p.This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

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Doubling Myo2p expression levels influences contractile ring dynamics. (A) Kymographs comparing contractile ring lifetime and dynamics in representative 1x and 2xYFP-myo2 cells. Each kymograph is made up of a series of thin slices centered on the contractile ring using images captured every 2 min. Slice height, 4.4 μm. Kymographs start from the point at which rings have assembled, spanning dwell and constriction phases. Kymographs are aligned based on the point at which constriction begins. (B) Comparing the relative levels of Rlc1p at contractile rings in 1x and 2xYFP-myo2 cells. Colocalization of YFP-Myo2p and Rlc1p-CFP in representative cells was performed by epifluorescence microscopy. 1x and 2xYFP-myo2 strains were grown separately before mixing and imaging by light microscopy using DIC (left), YFP (center), and CFP (right) filters. The SPB marker Sad1p-GFP was incorporated into the genome of the 2xYFP-myo2 strain to differentiate it from the 1x strain. (C) Plot charting the average assembly times, dwell times, and constriction rates for contractile rings in 1x and 2xYFP-myo2 cells (n = 20) harboring excess Rlc1p (expressed from a multicopy plasmid). Horizontal lines to the left of the y-axis represent the average assembly time. On assembly ring diameters were measured through dwell and constriction phases and then collectively aligned at the point in time when rings initiate constriction and averaged. The dwell phases (horizontal line to right of y-axis) reflect the mean of the dataset (rounded up to the nearest 2-min time point).
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Figure 2: Doubling Myo2p expression levels influences contractile ring dynamics. (A) Kymographs comparing contractile ring lifetime and dynamics in representative 1x and 2xYFP-myo2 cells. Each kymograph is made up of a series of thin slices centered on the contractile ring using images captured every 2 min. Slice height, 4.4 μm. Kymographs start from the point at which rings have assembled, spanning dwell and constriction phases. Kymographs are aligned based on the point at which constriction begins. (B) Comparing the relative levels of Rlc1p at contractile rings in 1x and 2xYFP-myo2 cells. Colocalization of YFP-Myo2p and Rlc1p-CFP in representative cells was performed by epifluorescence microscopy. 1x and 2xYFP-myo2 strains were grown separately before mixing and imaging by light microscopy using DIC (left), YFP (center), and CFP (right) filters. The SPB marker Sad1p-GFP was incorporated into the genome of the 2xYFP-myo2 strain to differentiate it from the 1x strain. (C) Plot charting the average assembly times, dwell times, and constriction rates for contractile rings in 1x and 2xYFP-myo2 cells (n = 20) harboring excess Rlc1p (expressed from a multicopy plasmid). Horizontal lines to the left of the y-axis represent the average assembly time. On assembly ring diameters were measured through dwell and constriction phases and then collectively aligned at the point in time when rings initiate constriction and averaged. The dwell phases (horizontal line to right of y-axis) reflect the mean of the dataset (rounded up to the nearest 2-min time point).

Mentions: The impact of doubling Myo2p levels on cytokinesis was assessed by comparing the phenotypes of 1xYFP-myo2 and 2xYFP-myo2 cells. Both strains showed normal cell morphology and exhibited no obvious defects in cytokinesis (Figure 1, B and C). We next compared contractile ring performance. Four different properties of the ring were measured: assembly: time taken for Myo2p to compact into a ring after its appearance as a broad band of nodes; dwell: time from completion of ring assembly until initiation of constriction; constriction: change in ring circumference over time; and lifetime: time from completion of ring assembly to completion of constriction. Our analysis revealed that rings assembled about twofold quicker in the 2xYFP-myo2 strain (Table 2). These rings had a ∼1.5 longer lifetime than those in the 1xYFP-myo2 strain, which was reflected by a ∼1.5-fold longer dwell time and ∼1.5 slower rate of constriction (Figure 2A, Table 2).


Tropomyosin and myosin-II cellular levels promote actomyosin ring assembly in fission yeast.

Stark BC, Sladewski TE, Pollard LW, Lord M - Mol. Biol. Cell (2010)

Doubling Myo2p expression levels influences contractile ring dynamics. (A) Kymographs comparing contractile ring lifetime and dynamics in representative 1x and 2xYFP-myo2 cells. Each kymograph is made up of a series of thin slices centered on the contractile ring using images captured every 2 min. Slice height, 4.4 μm. Kymographs start from the point at which rings have assembled, spanning dwell and constriction phases. Kymographs are aligned based on the point at which constriction begins. (B) Comparing the relative levels of Rlc1p at contractile rings in 1x and 2xYFP-myo2 cells. Colocalization of YFP-Myo2p and Rlc1p-CFP in representative cells was performed by epifluorescence microscopy. 1x and 2xYFP-myo2 strains were grown separately before mixing and imaging by light microscopy using DIC (left), YFP (center), and CFP (right) filters. The SPB marker Sad1p-GFP was incorporated into the genome of the 2xYFP-myo2 strain to differentiate it from the 1x strain. (C) Plot charting the average assembly times, dwell times, and constriction rates for contractile rings in 1x and 2xYFP-myo2 cells (n = 20) harboring excess Rlc1p (expressed from a multicopy plasmid). Horizontal lines to the left of the y-axis represent the average assembly time. On assembly ring diameters were measured through dwell and constriction phases and then collectively aligned at the point in time when rings initiate constriction and averaged. The dwell phases (horizontal line to right of y-axis) reflect the mean of the dataset (rounded up to the nearest 2-min time point).
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Related In: Results  -  Collection

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Figure 2: Doubling Myo2p expression levels influences contractile ring dynamics. (A) Kymographs comparing contractile ring lifetime and dynamics in representative 1x and 2xYFP-myo2 cells. Each kymograph is made up of a series of thin slices centered on the contractile ring using images captured every 2 min. Slice height, 4.4 μm. Kymographs start from the point at which rings have assembled, spanning dwell and constriction phases. Kymographs are aligned based on the point at which constriction begins. (B) Comparing the relative levels of Rlc1p at contractile rings in 1x and 2xYFP-myo2 cells. Colocalization of YFP-Myo2p and Rlc1p-CFP in representative cells was performed by epifluorescence microscopy. 1x and 2xYFP-myo2 strains were grown separately before mixing and imaging by light microscopy using DIC (left), YFP (center), and CFP (right) filters. The SPB marker Sad1p-GFP was incorporated into the genome of the 2xYFP-myo2 strain to differentiate it from the 1x strain. (C) Plot charting the average assembly times, dwell times, and constriction rates for contractile rings in 1x and 2xYFP-myo2 cells (n = 20) harboring excess Rlc1p (expressed from a multicopy plasmid). Horizontal lines to the left of the y-axis represent the average assembly time. On assembly ring diameters were measured through dwell and constriction phases and then collectively aligned at the point in time when rings initiate constriction and averaged. The dwell phases (horizontal line to right of y-axis) reflect the mean of the dataset (rounded up to the nearest 2-min time point).
Mentions: The impact of doubling Myo2p levels on cytokinesis was assessed by comparing the phenotypes of 1xYFP-myo2 and 2xYFP-myo2 cells. Both strains showed normal cell morphology and exhibited no obvious defects in cytokinesis (Figure 1, B and C). We next compared contractile ring performance. Four different properties of the ring were measured: assembly: time taken for Myo2p to compact into a ring after its appearance as a broad band of nodes; dwell: time from completion of ring assembly until initiation of constriction; constriction: change in ring circumference over time; and lifetime: time from completion of ring assembly to completion of constriction. Our analysis revealed that rings assembled about twofold quicker in the 2xYFP-myo2 strain (Table 2). These rings had a ∼1.5 longer lifetime than those in the 1xYFP-myo2 strain, which was reflected by a ∼1.5-fold longer dwell time and ∼1.5 slower rate of constriction (Figure 2A, Table 2).

Bottom Line: Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function.Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p.This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

Show MeSH
Related in: MedlinePlus