Yeast becomes a cell biologist
http://www.100md.com
《细胞学杂志》
For her graduate work, Adams wanted to study what actin was doing in yeast, specifically using immunofluorescence (IF) for localization. But her committee members (and many others in the field) were skeptical, because the impermeable yeast cell wall would block antibody penetration. Pringle says he distinctly remembers "having pessimistic conversations" about small, round yeast cells making bad candidates for IF compared with the large, flattened cells that were in vogue for the technique.
Actin (top) and tubulin (bottom) can be tracked during the budding yeast cell cycle.
ADAMS
The two went to Woods Hole to see if Bob and Anne Goldman's antibodies to mammalian cytoskeletal proteins would recognize yeast proteins. By chance, Kilmartin was there with his new monoclonal antitubulin antibody, which he had already managed to get into spheroplasts. The spheroplasts showed good IF, but had lost the original cell's shape and organization. "We decided to try to fix the cells before removing the cell wall," Adams recalls. It worked. "It was exciting to see cytoplasmic microtubules in yeast that are hard to see by EM, but by IF they really stood out."
IF tools now in hand, Adams and Pringle returned to the University of Michigan (Ann Arbor, MI), and Kilmartin to the MRC Laboratory of Molecular Biology (Cambridge, UK) to delve further into the roles of actin and microtubules. Kilmartin examined actin by IF while Adams stained it with the newly available fluorescent phalloidin.
In two papers, they described the distribution of actin in cortical patches and cytoplasmic cables that ran along the long axis of mother–bud pairs (Adams and Pringle, 1984; Kilmartin and Adams, 1984), a phenomenon particularly clear in mutants with elongated buds. The studies also revealed that the IF patterns of actin and tubulin never significantly overlapped during the cell cycle. And actin was seen around the base of small, forming buds and clustered in the neck region during cytokinesis.
This last observation suggested that maybe the neck-localized actin was driving the new cell wall growth of bud formation—a distinct switch from the prevailing view that microtubules served this function. A few years later, this idea was solidified when Peter Novick and David Botstein showed that temperature-sensitive actin mutants were defective for polarized secretion to the bud (Novick and Botstein, 1985). The Botstein and Pringle labs also showed bud growth occurring normally in the absence of microtubules (Huffaker et al., 1988; Jacobs et al., 1988). As for the actin patches, they are now thought to act as endosome coats (Huckaba et al., 2004).
The studies' biggest contribution—IF of internal yeast structures—got only a brief mention. "Effective IF procedures for yeasts," the authors noted, "should greatly facilitate use of these genetically tractable organisms for study of various problems in cell biology." Adams says she did not appreciate the full potential of the technique at the time. "We didn't even have a fluorescence scope in the lab," she says. "I had to hike 20 minutes over to the medical school and call back to John to describe what I was seeing."
When the findings were presented at the 1983 yeast meeting, however, there was a palpable buzz from interested colleagues. David Drubin, whose current studies of yeast actin draw heavily on real-time fluorescence microscopy, says he remembers the impact the breakthrough made on his choice of post-doctoral positions. "People tended to think of yeast as a big bacterium—you couldn't use it for questions of spatial organization," he says. "Now, you could have really powerful genetics and see how the structures in a cell changed. It just opened up for yeast." KP
Adams, A.E.M., and J.R. Pringle. 1984. J. Cell Biol. 98:934–945.
Huckaba, T.M., et al. 2004. J. Cell Biol. 167:519–530.
Huffaker, T.C., et al. 1988. J. Cell Biol. 106:1997–2010.
Jacobs, C.W., et al. 1988. J. Cell Biol. 107:1409–1426.
Kilmartin, J.V., and A.E.M. Adams. 1984. J. Cell Biol. 98:922–933.
Novick, P., and D. Botstein. 1985. Cell. 40:405–416.(Summer research collaborations at the Wo)
Actin (top) and tubulin (bottom) can be tracked during the budding yeast cell cycle.
ADAMS
The two went to Woods Hole to see if Bob and Anne Goldman's antibodies to mammalian cytoskeletal proteins would recognize yeast proteins. By chance, Kilmartin was there with his new monoclonal antitubulin antibody, which he had already managed to get into spheroplasts. The spheroplasts showed good IF, but had lost the original cell's shape and organization. "We decided to try to fix the cells before removing the cell wall," Adams recalls. It worked. "It was exciting to see cytoplasmic microtubules in yeast that are hard to see by EM, but by IF they really stood out."
IF tools now in hand, Adams and Pringle returned to the University of Michigan (Ann Arbor, MI), and Kilmartin to the MRC Laboratory of Molecular Biology (Cambridge, UK) to delve further into the roles of actin and microtubules. Kilmartin examined actin by IF while Adams stained it with the newly available fluorescent phalloidin.
In two papers, they described the distribution of actin in cortical patches and cytoplasmic cables that ran along the long axis of mother–bud pairs (Adams and Pringle, 1984; Kilmartin and Adams, 1984), a phenomenon particularly clear in mutants with elongated buds. The studies also revealed that the IF patterns of actin and tubulin never significantly overlapped during the cell cycle. And actin was seen around the base of small, forming buds and clustered in the neck region during cytokinesis.
This last observation suggested that maybe the neck-localized actin was driving the new cell wall growth of bud formation—a distinct switch from the prevailing view that microtubules served this function. A few years later, this idea was solidified when Peter Novick and David Botstein showed that temperature-sensitive actin mutants were defective for polarized secretion to the bud (Novick and Botstein, 1985). The Botstein and Pringle labs also showed bud growth occurring normally in the absence of microtubules (Huffaker et al., 1988; Jacobs et al., 1988). As for the actin patches, they are now thought to act as endosome coats (Huckaba et al., 2004).
The studies' biggest contribution—IF of internal yeast structures—got only a brief mention. "Effective IF procedures for yeasts," the authors noted, "should greatly facilitate use of these genetically tractable organisms for study of various problems in cell biology." Adams says she did not appreciate the full potential of the technique at the time. "We didn't even have a fluorescence scope in the lab," she says. "I had to hike 20 minutes over to the medical school and call back to John to describe what I was seeing."
When the findings were presented at the 1983 yeast meeting, however, there was a palpable buzz from interested colleagues. David Drubin, whose current studies of yeast actin draw heavily on real-time fluorescence microscopy, says he remembers the impact the breakthrough made on his choice of post-doctoral positions. "People tended to think of yeast as a big bacterium—you couldn't use it for questions of spatial organization," he says. "Now, you could have really powerful genetics and see how the structures in a cell changed. It just opened up for yeast." KP
Adams, A.E.M., and J.R. Pringle. 1984. J. Cell Biol. 98:934–945.
Huckaba, T.M., et al. 2004. J. Cell Biol. 167:519–530.
Huffaker, T.C., et al. 1988. J. Cell Biol. 106:1997–2010.
Jacobs, C.W., et al. 1988. J. Cell Biol. 107:1409–1426.
Kilmartin, J.V., and A.E.M. Adams. 1984. J. Cell Biol. 98:922–933.
Novick, P., and D. Botstein. 1985. Cell. 40:405–416.(Summer research collaborations at the Wo)