ScienceWeek

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)

Science http://www.sciencemag.org

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GENOMIC BIOLOGY: ON THE YEAST PROTEOME

The following points are made by J.A. Wohlschlegel and J.R. Yates (Nature 2003 425:671):

1) Saccharomyces cerevisiae was the first eukaryote -- the type of organism characterized by a nucleus and membrane-bound organelles, which also includes humans -- to have its genome sequenced(3). Work with this organism has since led the way in functional genomics. Experiments pioneered in yeast have set the standard for the global analysis of cellular processes and paved the way for similar approaches in other organisms. They have also generated genome-wide collections of reagents that have been tremendously valuable.

2) Open reading frames (ORFs) are commonly the center of attention in genome biology. These are stretches of DNA that have the characteristics of protein-coding capacity; that is, they may be genes. Collections of yeast strains now exist in which the expected ORFs have been either deleted or fused to various protein tags(4,5). Arrays have been created by using yeast strains expressing proteins that carry so-called affinity tags, allowing large numbers of proteins to be rapidly purified, then immobilized on a solid support. Large-scale studies involving various techniques -- protein arrays, and yeast two-hybrid or co-immunoprecipitation assays -- have revealed the identities of proteins that interact with individual proteins, large macromolecular complexes, or even specific small molecules.

3) All in all, yeast biologists have led the charge in developing approaches to understanding eukaryotic genomes. Huh et al(1) and Ghaemmaghami et al(2) continue that tradition. Their goal was to tag and study the gene products of all recognized ORFs in the yeast genome. A key component of these studies was the tagging method used: artificially altering a protein's expression level can lead to results, such as mislocalization, that do not reflect its characteristics when it is expressed normally.

4) In technical terms, Huh et al and Ghaemmaghami et al used homologous recombination to integrate a DNA sequence, encoding either a tandem affinity purification tag (TAP) or green fluorescent protein (GFP), in-frame with the 3'-end of the coding sequence of each gene in its original chromosomal location. Because a gene's promoter and upstream regulatory sequences are not affected in this approach, it is likely that the behavior of these fusion genes is nearly identical to that of their normal counterparts.

5) These studies have achieved three major results: First, we now have data on protein abundance and localization for 75% of the predicted yeast ORFs. Second, we have a value for the number of proteins present in a yeast cell during normal growth. Previously, a fun game to play with yeast biologists was to ask how many proteins they thought should be present under a given set of conditions. Numbers ranged between 2500 and 5000. It appears that the higher number was correct. Last -- and most important -- reagents have been developed for tracking a large majority of yeast genes while keeping them under native regulatory control. The reagents will be tools for further studies.

References (abridged):

1. Huh, W-K et al. Nature 425, 686-691 (2003)

2. Ghaemmaghami, S. et al. Nature 425, 737-741 (2003)

3. Goffeau, A. et al. Science 274, 546, 563-567 (1996)

4. Winzeler, E. A. et al. Science 285, 901-906 (1999)

5. Martzen, M. R. et al. Science 286, 1153-1155 (1999)

Nature http://www.nature.com/nature

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