Three-dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle.

The actin cytoskeleton of Saccharomyces cerevisiae is composed of a single, conventional actin isoform that is 86% identical to mammalian actins in addition to a battery of similarly conserved associated proteins. This conservation of components and their functions has led yeast geneticists and cell biologists to use the experimental power of yeast toward the study of the regulation of actin cytoskeleton assembly and the basic, housekeeping functions of the actin cytoskeleton (for a comprehensive review, see Botstein et al., 1997 ). Important to these studies has been the visualization of the actin cytoskeleton through the cell cycle and how mutations in regulators and components of the yeast cytoskeleton affect this organization. The first images of the yeast actin cytoskeleton showed that it consists of two filament-based structures: the actin cortical patch and the actin cables (Adams and Pringle, 1984 ; Kilmartin and Adams, 1984 ). The actin cortical patches show a polarized distribution that changes during the cell cycle: first they appear at the incipient bud site, suggesting a role in bud emergence; soon thereafter they are also found within the growing bud, indicating a role in bud growth; and late in the cell cycle they reorganize into two rings in the neck, where they are believed to be involved in septation and cytokinesis. By electron microscopy, the actin cortical patches have been shown to be invaginations of the plasma membrane around which actin filaments and actin-associated proteins are organized (Mulholland et al., 1994 ). Recently it has been shown that subsets of actin cortical patches can move at speeds of up to 1 μm/s (Doyle and Botstein, 1996 ; Waddle et al., 1996 ). The actin cables, which consist of bundled actin filaments, were observed to generally run along the long axis of budding cells. This organization fits well with the understanding that actin is involved in polarized cell growth, dynamic reorganization of the cell cortex, membrane trafficking at the cell cortex, and organelle segregation at cell division. Because certain aspects of the organization of the yeast actin cytoskeleton cannot be well addressed or documented by conventional two-dimensional microscopy, I have investigated the use of a DeltaVision deconvolution microscope for visualization of the yeast actin cytoskeleton in three dimensions. This instrument digitally captures focal sections in the Z plane and mathematically removes out-of-focus light by examining neighboring focal sections, from each section in a process called iterative deconvolution (Agard et al., 1989 ; Scalettar et al., 1996 ). The clarified focal sections can then be assembled to produce high-resolution three-dimensional images. I have found that this technology is extremely powerful for the study of yeast cell anatomy. With respect to the organization of the actin cytoskeleton, I have confirmed that the actin cables (like the patches) are cortical in their arrangement and can be observed to attach both at their ends and laterally to the cortical patches. In some cases, multiple cables can be seen to attach to single cortical patches. In addition, I have observed the earliest stages of bud emergence and have found that buds first emerge through a ring of cortical patches, and only after this stage do the cortical patches migrate into the bud. In my strain background (S288C), I do not observe a polarized distribution of cortical patches in the bud at any time during bud growth.