Myo2p Dynamics in Saccharomyces cerevisiae

These movies relate to experments described in "Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae" by Tatiana S. Karpova, Samara L. Reck-Peterson, N. Barry Elkind, Mark S. Mooseker, Peter J. Novick, and John A. Cooper. Mol. Biol. Cell (2000) 11: 1727-1737.PubMed Citation

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AbstractWe examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae using several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion, but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell.
Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1D(las17D), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2D, had a severe defect in polarized growth.
The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expressing its tail domain completely abolished growth.
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Myo2p-GFP dynamics in living cells.

Control Cells
  • Control Movie 1. A small-budded cell with a Myo2p-GFP cap at the tip of the bud.

  • Control Movie 2. One cell with a small bud and one with a medium bud. Both have Myo2p-GFP caps at the tip of the bud.
In the movies above, note that caps of Myo2p-GFP consist of multiple moving particles of variable size. In the small bud, even though the particles move constantly, their movement is confined to a small region. Later in the cell cycle particles move larger distances and assume more dispersed distribution. Eventually these particles disappear. In the medium-sized bud in Movie 2, the particles are in process of disassembly. These movies were collected at 2 frames per second and displayed at 6 frames per second.
  • Time-lapse Myo2 Movie. Time-lapse projection movie of Myo2 through the cell cycle. At each time point, images at multiple focal planes were collected, then projected onto a single 2-D image.

  • Myo2 and Tub1 Movie. Simultaneous GFP labelling of Myo2 and microtubules to show the relationship of Myo2 structures to the mitotic spindle.
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Latrunculin Treatment
  • Latrunculin Movie. This movie shows two cells with small to medium-sized buds and two unbudded cells treated with 500 µM latrunculin A. Myo2p-GFP disappears from bud tip caps and appears throughout the cytoplasm of the bud and the mother as numerous dots. A Myo2p-GFP ring at the mother-bud neck, seen in the top cell, persists in latrunculin. Collected at 2 frames per second and displayed at 6 frames per second.