ScienceWeek

CELL BIOLOGY: ON YEAST

The following points are made by D.M. Truckses et al (Science 2004 306:1509):

1) The most familiar fungal cell is baker's yeast (Saccharomyces cerevisiae), a budding yeast that has become a valuable model for examining the eukaryotic way of life at the molecular level. Although relatively benign, S. cerevisiae has shed light on fundamental mechanisms in fungal pathogenesis because it adopts two distinct morphologies: a spherical or ovoid (yeastlike) form, which proliferates by budding, and a filamentous form. For fungal pathogens, such as Candida albicans, the ability to undergo this dimorphic transition is strongly correlated with invasion of host tissue and virulence [1,2].

2) On rich medium, S. cerevisiae proliferates as separate spherical cells. However, in response to nitrogen limitation [3], the cells become thin and oblong, and after a daughter buds, it remains adherent and connected end-to-end with its mother, resembling a link in a sausage string. Elucidating the signaling pathways necessary to elicit such filamentous growth has led to insights about how cells respond to different stimuli and how they share components with other signaling pathways yet evoke unique responses that are physiologically appropriate.

3) The switch from budding to pseudohyphal growth in S. cerevisiae is controlled by at least three signaling modalities [5]. Each input involves the action of different classes of protein kinases. The core of one pathway is a three-tiered mitogen-activated protein kinase (MAPK) cascade. Each kinase catalyzes the transfer of phosphate from adenosine 5'-triphosphate (ATP) to serine, threonine, or tyrosine residues in the next kinase, which provides for switchlike amplification of signal propagation. The MAPK cascade operates in concert with a second input that acts by means of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), best studied in yeast for its role in glucose metabolism in vegetatively growing cells. When the supply of glucose is exhausted, a third input that acts through 5'-AMP-activated protein kinase (AMPK) ensures continued robust filamentous growth on fermentation end products and other nonglucose carbon sources. AMPK (Snf1) may act upstream of or in conjunction with the action of the atypical protein kinases, Tor1 and Tor2.

4) In summary: Fungi are nonmotile organisms that obtain carbon from compounds in their immediate surroundings. Confronted with nutrient limitation, the yeast Saccharomyces cerevisiae undergoes a dimorphic transition, switching from spherical cells to filaments of adherent, elongated cells that can invade the substratum. A complex web of sensing mechanisms and cooperation among signaling networks (including a mitogen-activated protein kinase cascade, cyclic adenosine monophosphate-dependent protein kinase, and 5'-adenosine monophosphate-activated protein kinase) elicits the appropriate changes in physiology, cell cycle progression, cell polarity, and gene expression to achieve this differentiation. Highly related signaling processes control filamentation and virulence of many human fungal pathogens.

References (abridged):

1. C. Sanchez-Martinez, J. Perez-Martin, Curr. Opin. Microbiol. 4, 214 (2001)

2. P. Sudbery, N. Gow, J. Berman, Trends Microbiol. 12, 317 (2004)

3. C. J. Gimeno, P. O. Ljungdahl, C. A. Styles, G. R. Fink, Cell 68, 1077 (1992)

4. D. M. Truckses, L. S. Garrenton, J. Thorner, Filamentous Growth Pathway in Yeast. Sci. STKE (Connections Map, as seen November 2004), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_14554.

5. M. Gagiano, F. F. Bauer, I. S. Pretorius, FEMS Yeast Res. 2, 433 (2002)

Science http://www.sciencemag.org

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CELL BIOLOGY: ON LASER MICROSURGERY IN YEAST CELLS

The following points are made by P. Carvalho and D. Pellman (Current Biology 2004 14:R748):

1) Many biologists prefer experiments involving manipulation of cells rather than observational approaches. For the most part, this began with developmental biologists who used transplantation experiments to identify regions of embryos that specified different cell fates, and then extended to classic experiments that continue to define some of the central issues in modern cell biology.

2) Microsurgery experiments by Ray Rappaport [1] revealed the role of overlapping microtubule arrays in determining the cell cleavage plane during cytokinesis. Nicklas and Koch [2] demonstrated the importance of tension on kinetochores to signal the completion of chromosome alignment on the mitotic spindle. Laser cutting experiments [3,4] uncovered the existence of pulling forces generated by astral microtubules on the anaphase spindle in certain eukaryotic cell types.

3) Because most cell biologists now study relatively small tissue culture cells, rather than the larger embryonic cells used in the pioneering studies, laser microsurgery has become a weapon of choice for the manipulation of cells [5]. More recently, the use of GFP-tagged proteins has enabled lasers to be targeted more specifically to even smaller structures, such as the centrosome. The manipulators continue to push the envelop of smallness, and recent reports have demonstrated the utility of laser microsurgery in yeast, the smallest and most genetically tractable eukaryotic system for studying cell division.

4) Although we like to think of genetic mutations as "smart bombs" to study gene function, disrupting a multifunctional gene product not uncommonly has more of a "scud missile" effect, producing hard to interpret collateral damage. This problem can sometimes be addressed by studying specific alleles, but often such informative alleles are not available. Furthermore, mutations tend to inactivate gene functions globally, while cell biologists often really want information about local activity. Cell biologists also need functional information with rapid time resolution, and functional inactivation through conditional mutations can be slow.

5) All of this makes an appealing case to try laser microsurgery on yeast. This appeal is only enhanced when one considers the fact that the engines driving intracellular movements are often "over-built", consisting of several overlapping mechanisms. Thus, genetics can be used to strip down over-built processes, and laser microsurgery can then be used to characterize individual mechanisms. The power of this combined genetic/microsurgery approach has been elegantly demonstrated by experiments in the nematode Caenorhabditis elegans[4]

References (abridged):

1. Rappaport, R. (1961). Experiments concerning the cleavage stimulus in sand dollar eggs. J. Exp. Zool. 148, 81-89

2. Nicklas, R.B. and Koch, C.A. (1969). Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes. J. Cell Biol. 43, 40-50

3. Aist, J.R. and Berns, M.W. (1981). Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments. J. Cell Biol. 91, 446-458

4. Grill, S.W., Gonczy, P., Stelzer, E.H. and Hyman, A.A. (2001). Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630-633

5. Berns, M.W., Wright, W.H. and Wiegand Steubing, R. (1991). Laser microbeam as a tool in cell biology. Int. Rev. Cytol. 129, 1-44

Current Biology http://www.current-biology.com

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MOLECULAR BIOLOGY: ON PROTEIN COMPLEXES IN YEAST

The following points are made by P. Aloy et al (Science 2004 303:2026):

1) Cell and structural biology share the common goal of understanding large, complex biological entities at the highest possible detail. Although different in outlook, the distinction diminishes as techniques improve. Cell biologists can now see structures like the nuclear pore (1), or even whole cells (2), at resolutions approaching 3 nm. Structural biologists, once restricted for technical reasons to small macromolecules, are now solving atomic resolution structures for large molecular machines like the ribosome (3) or RNA polymerases (4).

2) In spite of this blurring distinction, technical problems will delay atomic resolution structures of large cellular entities for several years. Microscopy has difficulties reaching this resolution, and expression and crystallization problems still slow x-ray crystallography for large complexes. The result is an information gap between low-resolution images of large cellular entities and atomic structures for the macromolecules that they contain.

3) Recent developments in functional genomics provide possibilities for bridging this gap. Genome-scale interaction discovery approaches, like the two-hybrid system (5) or affinity purifications, have uncovered details of the cell network, although without critical molecular details: what interacts with what, but not how. These details can sometimes come from similarities to interacting proteins of known three-dimensional (3D) structure.

4) The authors report the investigation of a large set of yeast interactions using structures to give the most complete view currently possible of complexes and their interrelationships. The authors screen complexes using electron microscopy (EM) and use low-resolution images to help assemble and validate models. The authors also predict links between complexes and provide a higher order, structure-based network of connected molecular machines within the cell.

5) In summary: Images of entire cells are preceding atomic structures of the separate molecular machines that they contain. The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy. The authors use interactions of known three-dimensional structure to model a large set of yeast complexes, which they also screen by electron microscopy. For 54 of 102 complexes, they obtain at least partial models of interacting subunits. For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures. The authors also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.

References (abridged):

1. Q. Yang, M. P. Rout, C. W. Akey, Mol. Cell 1, 223 (1998)

2. O. Medalia et al., Science 298, 1209 (2002)

3. N. Ban, P. Nissen, J. Hansen, P. B. Moore, T. A. Steitz, Science 289, 905 (2000)

4. C. L. Poglitsch et al., Cell 98, 791 (1999)

5. P. Uetz et al., Nature 403, 623 (2000)

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