Michael Knop

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR‐primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C‐terminal tagged genes or for the exchange of the promoter and N‐terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C‐terminal epitope tags as well as nine different promoter substitutions in combination with N‐terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast‐optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches. Copyright

Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines

Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR‐based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope‐tagged proteins. Copyright

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called ‘der’ for ‘degradation in the ER’. DER1 was cloned by complementation of the der1–2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1‐deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

A screen for genes required for meiosis and spore formation based on whole-genome expression

BACKGROUND Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Spc98p and Spc97p of the yeast γ‐tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p

Previously, we have shown that the yeast γ‐tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N‐terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N‐terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.

Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3PP) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3PP activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3PP-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.

The spindle pole body component Spc97p interacts with the γ-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication

Previously, we have shown that the γ‐tubulin Tub4p and the spindle pole body component Spc98p are involved in microtubule organization by the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC97 encoding an essential SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules. Evidence is provided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature‐sensitive spc97(ts) mutants are suppressed by high gene dosage of SPC98 or TUB4. Second, Spc97p interacts with Spc98p and Tub4p in the two‐hybrid system. Finally, immunoprecipitation and fractionation studies revealed complexes containing Tub4p, Spc98p and Spc97p. Further support for a direct interaction of Tub4p, Spc98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co‐overexpression of TUB4 or SPC98. Analysis of temperature‐sensitive spc97(ts) alleles revealed multiple spindle defects. While spc97‐14 cells are either impaired in SPB separation or mitotic spindle formation, spc97‐20 cells show an additional defect in SPB duplication. We discuss a model in which the Tub4p–Spc98p–Spc97p complex is part of the microtubule attachment site at the SPB.

The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment.

Tub4p is a novel tubulin found in Saccharomyces cerevisiae. It most resembles gamma‐tubulin and, like it, is localized to the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC98 as a dosage‐dependent suppressor of the conditional lethal tub4–1 allele. SPC98 encodes an SPB component of 98 kDa which is identical to the previously described 90 kDa SPB protein. Strong overexpression of SPC98 is toxic, causing cells to arrest with a large bud, defective microtubule structures, undivided nucleus and replicated DNA. The toxicity of SPC98 overexpression was relieved by co‐overexpression of TUB4. Further evidence for an interaction between Tub4p and Spc98p came from the synthetic toxicity of tub4–1 and spc98–1 alleles, the dosage‐dependent suppression of spc98–4 by TUB4, the binding of Tub4p to Spc98p in the two‐hybrid system and the co‐immunoprecipitation of Tub4p and Spc98p. In addition, Spc98–1p is defective in its interaction with Tub4p in the two‐hybrid system. We suggest a model in which Tub4p and Spc98p form a complex involved in microtubule organization by the SPB.

Directional tissue migration through a self-generated chemokine gradient

The directed migration of cell collectives is a driving force of embryogenesis. The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients. One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them, a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion.

Receptors determine the cellular localization of a γ‐tubulin complex and thereby the site of microtubule formation

The yeast microtubule organizing centre (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules which are functionally and spatially distinct. Microtubule organization requires the yeast γ‐tubulin complex (Tub4p complex) which binds to the nuclear side of the SPB at the N‐terminal domain of Spc110p. Here, we describe the identification of the essential SPB component Spc72p whose N‐terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. We further report that this Tub4p complex‐binding domain of Spc72p is essential and that temperature‐sensitive alleles of SPC72 or overexpression of a binding domain‐deleted variant of SPC72 (ΔN‐SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulated in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules compared with wild‐type. Finally, exchange of the Tub4p complex‐binding domains of Spc110p and Spc72p established that the Spc110p domain, when attached to ΔN‐Spc72p, was functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p fused to ΔN‐Spc110p led to a dominant‐negative effect. These results suggest that different components of MTOCs act as receptors for γ‐tubulin complexes and that they are essential for the function of MTOCs.