SummaryPrevious studies have suggested that breast cancer stem cells (BCSCs) mediate metastasis, are resistant to radiation and chemotherapy, and contribute to relapse. Although several BCSC markers have been described, it is unclear whether these markers identify the same or independent BCSCs. Here, we show that BCSCs exist in distinct mesenchymal-like (epithelial-mesenchymal transition [EMT]) and epithelial-like (mesenchymal-epithelial transition [MET]) states. Mesenchymal-like BCSCs characterized as CD24−CD44+ are primarily quiescent and localized at the tumor invasive front, whereas epithelial-like BCSCs express aldehyde dehydrogenase (ALDH), are proliferative, and are located more centrally. The gene-expression profiles of mesenchymal-like and epithelial-like BCSCs are remarkably similar across different molecular subtypes of breast cancer, and resemble those of distinct basal and luminal stem cells found in the normal breast. We propose that the plasticity of BCSCs that allows them to transition between EMT- and MET-like states endows these cells with the capacity for tissue invasion, dissemination, and growth at metastatic sites.

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochromelabeled phallotoxins after fixing and permeabilizing the cells by several methods . The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers . Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope.The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud .Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds.These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle .The cell-division cycle of the yeast Saccharomyces cerevisiae includes the following sequential morphogenetic events (see references 16, 71, and references cited therein): (a) selection of a nonrandom site at which budding will occur ; (b) formation of a ring ofchitin (the "bud scar") in the largely nonchitinous cell wall at that site; (c) localization of new cell-wall growth to the region bounded by the chitin ring, resulting in the appearance and selective growth ofa bud; (d) localization of new cell-wall growth to the tip of the growing bud; (e) cytokinesis and the formation of septal cell wall. In addition, it seems that periods of uniform growth of the bud cell wall precede and follow the period of tip growth; presumably, the relative amounts of tip growth and of uniform growth are adjusted to yield the normal ellipsoidal shape ofthe daughter cell (21). In addition, when the bud is largely grown, the nucleus migrates from a position within the mother cell into the neck connecting mother and bud, and...

The distribution of actin and tubulin during the cell cycle of the budding yeast Saccharomyces was mapped by immunofluorescence using fixed cells from which the walls had been removed by digestion . The intranuclear mitotic spindle was shown clearly by staining with a monoclonal antitubulin ; the presence of extensive bundles of cytoplasmic microtubules is reported. In cells containing short spindles still entirely within the mother cells, one of the bundles of cytoplasmic microtubules nearly always extended to (or into) the bud . Two independent reagents (anti-yeast actin and fluorescent phalloidin) revealed an unusual distribution of actin : it was present as a set of cortical dots or patches and also as distinct fibers that were presumably bundles of actin filaments . Double labeling showed that at no stage in the cell cycle do the distributions of actin and tubulin coincide for any significant length, and, in particular, that the mitotic spindle did not stain detestably for actin . However, both microtubule and actin staining patterns change in a characteristic way during the cell cycle . In particular, the actin dots clustered in rings about the bases of very small buds and at the sites on unbudded cells at which bud emergence was apparently imminent . Later in the budding cycle, the actin dots were present largely in the buds and, in many strains, primarily at the tips of these buds. At about the time of cytokinesis the actin dots clustered in the neck region between the separating cells. These aspects of actin distribution suggest that it may have a role in the localized deposition of new cell wall material.Actin and tubulin are major structural proteins in all eucaryote cells. They form distinct filamentous structures in the interphase cytoskeleton and during mitosis, also they may be involved in many motile events in eucaryotes. However, in nonmuscle cells only a few of their functions such as the role of actin in cytokinesis (52) and tubulin in flagellar movement (19) are reasonably well established . Their role in other cellular motility events such as intracellular movement of organelles or chromosome movement is as yet unclear. A genetic analysis of these problems might illuminate the precise functions of actin, of tubulin and, in particular, of proteins associated with them. For this purpose, yeast, as a genetically tractable eucaryote microorganism possessing tubulin and actin closely similar to those of higher organisms (21,30,36,60), may be of great value. The genes for yeast actin (18,44,53) and tubulin (43) are already under intensive study and most cloned yeast genes can now be mutagenized in vitro and substituted for the wild-type gene in the correct chromosomal position (51). Moreover studies of"pseudorevertants" of such 922

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Abstract. Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindies), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.A important class of questions about the cell division cycle concerns the dependency relationships or other coordinating mechanisms that ensure that cell-cycle events occur in an appropriate sequence. Such questions have been investigated in the yeast Saccharomyces cerevisiae by using mutations and inhibitors that block specific cellcycle events (Hartwell et al., 1974;Pringle, 1978;Pringle and Hartwell, 1981; Moir and Botstein, 1982;Wood and Hartwell, 1982;Jacobs et al., 1988; Hartwell and Weinert, 1989). In this context, temperature-sensitive (Ts-) ~ mutants defective in gene CDC24 have been of great interest. The observation that such mutants can continue DNA synthesis and nuclear division while bud emergence is blocked (Hartwell et al., 1973(Hartwell et al., , 1974 suggests that the nuclear cycle is not dependent on the cytoplasmic processes involved in budding. Conversely, experiments with a variety of other mutations and inhibitors suggest that bud emergence is not dependent on the nuclear cycle (Hartwell et al., 1974;Pringle and Hartwell, 1981). Thus, it appears that many of the events of the yeast cell cycle are organized into two parallel and indepen-A. E. M. Adams' present address is