The oxoglutarate dehydrogenase complex is involved in myofibril growth and Z-disc assembly in Drosophila.

Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc.

SAGA Complex Subunits in Candida albicans Differentially Regulate Filamentation, Invasiveness, and Biofilm Formation.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a highly conserved, multiprotein co-activator complex that consists of five distinct modules. It has two enzymatic functions, a histone acetyltransferase (HAT) and a deubiquitinase (DUB) and plays a central role in processes such as transcription initiation, elongation, protein stability, and telomere maintenance. We analyzed conditional and null mutants of the SAGA complex module components in the fungal pathogen Candida albicans; Ngg1, (the HAT module); Ubp8, (the DUB module); Tra1, (the recruitment module), Spt7, (the architecture module) and Spt8, (the TBP interaction unit), and assessed their roles in a variety of cellular processes. We observed that spt7Δ/Δ and spt8Δ/Δ strains have a filamentous phenotype, and both are highly invasive in yeast growing conditions as compared to the wild type, while ngg1Δ/Δ and ubp8Δ/Δ are in yeast-locked state and non-invasive in both YPD media and filamentous induced conditions compared to wild type. RNA-sequencing-based transcriptional profiling of SAGA mutants reveals upregulation of hyphal specific genes in spt7Δ/Δ and spt8Δ/Δ strains and downregulation of ergosterol metabolism pathway. As well, spt7Δ/Δ and spt8Δ/Δ confer susceptibility to antifungal drugs, to acidic and alkaline pH, to high temperature, and to osmotic, oxidative, cell wall, and DNA damage stresses, indicating that these proteins are important for genotoxic and cellular stress responses. Despite having similar morphological phenotypes (constitutively filamentous and invasive) spt7 and spt8 mutants displayed variation in nuclear distribution where spt7Δ/Δ cells were frequently binucleate and spt8Δ/Δ cells were consistently mononucleate. We also observed that spt7Δ/Δ and spt8Δ/Δ mutants were quickly engulfed by macrophages compared to ngg1Δ/Δ and ubp8Δ/Δ strains. All these findings suggest that the SAGA complex modules can have contrasting functions where loss of Spt7 or Spt8 enhances filamentation and invasiveness while loss of Ngg1 or Ubp8 blocks these processes.

Screening of Candida albicans GRACE library revealed a unique pattern of biofilm formation under repression of the essential gene ILS1.

Candida albicans biofilm formation is governed by a regulatory circuit comprising nine transcription factors which control a network of target genes. However, there are still unknown genes contributing to biofilm features. Thus, the GRACE library was screened to identify genes involved in mature biofilm development. Twenty-nine conditional mutants were selected for a second screening revealing three groups of genes: twenty- two conditional mutants were defective for normal growth and unable to form biofilms; six strains, conditionally defective in genes ARC40, ARC35, ORF19.2438, SKP1, ERG6, and ADE5,7 that are likely essential or involved in general cell processes, grew normally as free-floating cells but produced less biofilm; finally, the conditional strain for a putative essential isoleucyl- tRNA synthetase gene, ILS1, was unable to grow as yeast-phase cells but was capable of producing a tridimensional biofilm structure in spite of reduced metabolic activity. This unique biofilm still relied on the classical biofilm genes, while it differentially induced groups of genes involved in adhesion, protein synthesis, cell wall organization, and protein folding. Although the conditional mutant repressed genes annotated for morphology and homeostasis processes affecting morphology and metabolism, the dynamic cell growth enabled the formation of a complex biofilm community independent of ILS1.

Negative regulation of filamentous growth in Candida albicans by Dig1p.

Transcriptional regulation involves both positive and negative regulatory elements. The Dig1 negative regulators are part of a fungal-specific module that includes a transcription factor (a Ste12 family member) and a Dig1 family member. In Saccharomyces cerevisiae, the post-genome-duplication Dig1/Dig2 proteins regulate MAP kinase controlled signalling pathways involved in mating and filamentous growth. We have identified the single Dig1 orthologue in the fungal pathogen Candida albicans. Genetic studies and transcriptional profiling experiments show that this single protein is implicated in the regulation of MAP kinase-controlled processes involved in mating, filamentous growth and biofilm formation, and also influences cAMP-regulated processes. This suggests that the multiple cellular roles of the Dig1 protein are ancestral and predate the sub-functionalization apparent in S. cerevisiae after the genome duplication. Intriguingly, even though loss of Dig1 function in C. albicans enhances filamentous growth and biofilm formation, colonization of the murine gastrointestinal tract is reduced in the mutant. The complexity of the processes influenced by Dig1 in C. albicans, and the observation that Dig1 is one of the few regulatory proteins that were retained in the duplicated state after the whole genome duplication event in yeast, emphasizes the important role of these negative regulators in fungal transcriptional control.