Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre in which vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research

Lactate signalling regulates fungal β-glucan masking and immune evasion

AJPB: This work was supported by the European Research Council (STRIFE, ERC- 2009-AdG-249793), The UK Medical Research Council (MR/M026663/1), the UK Biotechnology and Biological Research Council (BB/K017365/1), the Wellcome Trust (080088; 097377). ERB: This work was supported by the UK Biotechnology and Biological Research Council (BB/M014525/1). GMA: Supported by the CNPq-Brazil (Science without Borders fellowship 202976/2014-9). GDB: Wellcome Trust (102705). CAM: This work was supported by the UK Medical Research Council (G0400284). DMM: This work was supported by UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/K000306/1). NARG/JW: Wellcome Trust (086827, 075470,101873) and Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377). ALL: This work was supported by the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPostprin

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD

Mice Null for Calsequestrin 1 Exhibit Deficits in Functional Performance and Sarcoplasmic Reticulum Calcium Handling

In skeletal muscle, the release of calcium (Ca2+) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca2+ release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca2+ buffering as well as its potential for modulating RyR1, the L-type Ca2+ channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca2+]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca2+ content and SR Ca2+ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca2+ release with single action potentials and a collapse of the Ca2+ release with repetitive trains. Under voltage clamp, SR Ca2+ release flux and total SR Ca2+ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca2+ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas the rapidly decaying component of SR Ca2+ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca2+] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca2+]free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca2+ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca2+ release

Depletion of cardiac cardiolipin synthase alters systolic and diastolic function

Cardiolipin (CL) is a major cardiac mitochondrial phospholipid maintaining regular mitochondrial morphology and function in cardiomyocytes. Cardiac CL production includes its biosynthesis and a CL remodeling process. Here we studied the impact of CL biosynthesis and the enzyme cardiolipin synthase (CLS) on cardiac function. CLS and cardiac CL species were significantly downregulated in cardiomyocytes following catecholamine-induced cardiac damage in mice, accompanied by increased oxygen consumption rates, signs of oxidative stress, and mitochondrial uncoupling. RNAi-mediated cardiomyocyte-specific knockdown of CLS in Drosophila melanogaster resulted in marked cardiac dilatation, severe impairment of systolic performance, and slower diastolic filling velocity assessed by fluorescence-based heart imaging. Finally, we showed that CL72:8 is significantly decreased in cardiac samples from patients with heart failure with reduced ejection fraction (HFrEF). In summary, we identified CLS as a regulator of cardiac function. Considering the cardiac depletion of CL species in HFrEF, pharmacological targeting of CLS may be a promising therapeutic approach

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response

Signaling of the human P2Y(1) receptor measured by a yeast growth assay with comparisons to assays of phospholipase C and calcium mobilization in 1321N1 human astrocytoma cells

The human P2Y(1) receptor was expressed in the yeast Saccharomyces cerevisiae strain MPY578q5, which is engineered to couple to mammalian G protein-coupled receptors (GPCRs) and requires agonist-induced activation for growth. A range of known P2Y(1) receptor agonists were examined with the yeast growth assay system, and the results were validated by comparing with potencies in the transfected 1321N1 astrocytoma cell line, in which calcium mobilization was measured with a FLIPR (fluorometric-imaging plate reader). The data were also compared with those from phospholipase C activation and radioligand binding with the use of a newly available radioligand [(3)H]MRS2279 (2-chloro-N(6)-methyl-(N)-methanocarba-2’-deoxyadenosine-3’,5’bisphosphate). In the yeast growth assay, the rank order of potency of 2-MeSADP (2-methylthioadenosine 5’-diphosphate), ADP (adenosine 5’-diphosphate), and ATP (adenosine 5’-triphosphate) is the same as those in other assay systems, i.e., 2-MeSADP>ADP>ATP. The P2Y(1)-selective antagonist MRS2179 (N(6)-methyl-2-deoxyadenosine-3’,5’-bisphosphate) was shown to act as an antagonist with similar potency in all systems. The results suggest that the yeast expression system is suitable for screening P2Y(1) receptor ligands, both agonists and antagonists. The yeast system should be useful for random mutagenesis of GPCRs to identify mutants with certain properties, such as selective potency enhancement for small synthetic molecules and constitutive activity