Eric Grote

Cell–cell fusion

Cell–cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. Fusion may also play a role in the development of cancer and in tissue repair by stem cells. While virus–cell fusion and the fusion of intracellular membranes have been the subject of intense investigation during the past decade, cell–cell fusion remains poorly understood. Given the importance of this cell‐biological phenomenon, a number of investigators have begun analyses of the molecular mechanisms that mediate the specialized fusion events of a variety of cell types and species. We discuss recent genetic and biochemical studies that are beginning to yield exciting insights into the fusion mechanisms of Saccharomyces cerevisiae mating pairs, Caenorhabditis elegans epithelial cells and gametes, Drosophila melanogaster and mammalian myoblasts, and mammalian macrophages.

Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast

Ergosterol depletion independently inhibits two aspects of yeast mating: pheromone signaling and plasma membrane fusion. In signaling, ergosterol participates in the recruitment of Ste5 to a polarized site on the plasma membrane. Ergosterol is thought to form microdomains within the membrane by interacting with the long acyl chains of sphingolipids. We find that although sphingolipid-free ergosterol is concentrated at sites of cell–cell contact, transmission of the pheromone signal at contact sites depends on a balanced ratio of ergosterol to sphingolipids. If a mating pair forms between ergosterol-depleted cells despite the attenuated pheromone response, the subsequent process of membrane fusion is retarded. Prm1 also participates in membrane fusion. However, ergosterol and Prm1 have independent functions and only prm1 mutant mating pairs are susceptible to contact-dependent lysis. In contrast to signaling, plasma membrane fusion is relatively insensitive to sphingolipid depletion. Thus, the sphingolipid-free pool of ergosterol promotes plasma membrane fusion.

Prm1 prevents contact-dependent lysis of yeast mating pairs.

ABSTRACT Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 μm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.

Constrained selected reaction monitoring: quantification of selected post-translational modifications and protein isoforms.

Selected reaction monitoring (SRM) is a mass spectrometry method that can target signature peptides to provide for the detection and quantitation of specific proteins in complex biological samples. When quantifying a protein, multiple peptides are generated using a specific protease such as trypsin, thereby allowing a choice of signature peptides with robust signals. In contrast, signature peptide selection can be constrained when the goal is to monitor a specific post-translational modification (PTM) or protein isoform, as the signature peptide must include the amino acid residue(s) of PTM attachment or sequence variation. This can force the selection of a signature peptide with a weak SRM response or one that is confounded by high background. In this article, we discuss steps that can be optimized to maximize peptide selection and assay performance of constrained SRM assays, including tuning instrument parameters, fragmenting product ions, using a different protease, and enriching the sample. Examples are provided for phosphorylated or citrullinated peptides and protein isoforms.

Cell Fusion Assays for Yeast Mating Pairs

Yeast mating provides an accessible genetic system for the discovery of fundamental mechanisms in eukaryotic cell fusion. Although aspects of yeast mating related to pheromone signaling and polarized growth have been intensively investigated, fusion itself is poorly understood. This chapter describes methods for measuring the overall efficiency of yeast cell fusion and for monitoring various stages of the fusion process including cell wall remodeling, plasma membrane fusion, and nuclear fusion.

An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine

BACKGROUND Many avenues have been proposed for a seamless transition between biomarker discovery data and selected reaction monitoring (SRM) assays for biomarker validation. Unfortunately, studies with the abundant urinary protein uromodulin have shown that these methods do not converge on a consistent set of surrogate peptides for targeted mass spectrometry. As an alternative, we present an empirical peptide selection work flow for robust protein quantification. METHODS We compared the relative SRM signal intensity of 12 uromodulin-derived peptides between tryptic digests of 9 urine samples. Pairwise CVs between the 12 peptides were 0.19-0.99. We used a correlation matrix to identify peptides that reproducibly tracked the amount of uromodulin protein and selected 4 peptides with robust and highly correlated SRM signals. Absolute quantification was performed with stable isotope-labeled versions of these peptides as internal standards and a standard curve prepared from a tryptic digest of purified uromodulin. RESULTS Absolute quantification of uromodulin in 40 clinical urine samples yielded interpeptide correlations of ≥0.984 and correlations of ≥0.912 with ELISA data. The SRM assays were linear over >3 orders of magnitude and had typical interdigest CVs of <10%, interinjection CVs of <7%, and intertransition CVs of <7%. CONCLUSIONS Comparing the apparent abundance of a plurality of peptides derived from the same target protein makes it possible to select signature peptides that are unaffected by the unpredictable confounding factors inevitably present in biological samples.

Using pure protein to build a multiple reaction monitoring mass spectrometry assay for targeted detection and quantitation.

Multiple reaction monitoring (MRM) is an increasingly popular mass spectrometry-based method to simultaneously detect and quantify multiple proteins. MRM is particularly useful for validating biomarkers discovered with a mass spectrometer and any analite discovered by MS can be monitored by MR because an MRM assay can be developed without the need to generate specific antibodies. In this chapter, we present a robust and systematic procedure to rapidly build a high-sensitivity MRM assay using purified protein as the starting material. Theoretical digestion of the protein with trypsin is used to identify mass spectrometry--compatible peptides and to generate preliminary MRM transitions to detect these peptides. Peptides generated by trypsin cleavage of the actual protein are then run on a liquid chromatography column coupled to a triple quadrupole mass spectrometer, which is programmed with the preliminary transitions. Whenever a transition is detected, it triggers dissociation of the corresponding peptide and collection of a full mass range scan of the resulting fragment ions. From this scan, fragment ions yielding the strongest and most reproducible signals are utilized to design empirical MRM transitions. The assay is further refined by optimizing the collision energy and creating a standard curve to measure sensitivity. Once MRM transitions have been established for a particular protein, they can be combined with transitions for other target proteins to create multiplex assays and used to quantify proteins in samples arising from serum, urine, subcellular fractions, or any other specemen of interest.

Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

ABSTRACT Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.