Uniparental inheritance of mitochondria dominates among sexual eukaryotes. However, little is known about the mechanisms and genetic determinants. We have investigated the role of the plant pathogen Ustilago maydis genes lga2 and rga2 in uniparental mitochondrial DNA (mtDNA) inheritance during sexual development. The lga2 and rga2 genes are specific to the a2 mating-type locus and encode small mitochondrial proteins. On the basis of identified sequence polymorphisms due to variable intron numbers in mitochondrial genotypes, we could demonstrate that lga2 and rga2 decisively influence mtDNA inheritance in matings between a1 and a2 strains. Deletion of lga2 favored biparental inheritance and generation of recombinant mtDNA molecules in combinations in which inheritance of mtDNA of the a2 partner dominated. Conversely, deletion of rga2 resulted in predominant loss of a2-specific mtDNA and favored inheritance of the a1 mtDNA. Furthermore, expression of rga2 in the a1 partner protected the associated mtDNA from elimination. Our results indicate that Lga2 in conjunction with Rga2 directs uniparental mtDNA inheritance by mediating loss of the a1-associated mtDNA. This study shows for the first time an interplay of mitochondrial proteins in regulating uniparental mtDNA inheritance.
The a2 mating-type-locus gene lga2 of the basidiomycete Ustilago maydis encodes a mitochondrial protein that interferes with mitochondrial morphology and integrity, and that plays a role in uniparental inheritance of mitochondrial DNA. To address the mode of action of Lga2, we investigated its Dnm1 (a dynamin-related protein)-dependent effects. Here, we demonstrate that Dnm1 functions as a mitochondrial fission component in U. maydis and mediates Lga2-induced mitochondrial fragmentation. Mitochondrial fusion occurred very inefficiently in matings of U. maydis wild-type strains, but was strongly stimulated in the absence of dnm1 and highest in either wild-type or Δdnm1 combinations when the a2 partner was deleted in lga2. This indicates that Dnm1 plays a central role in opposing mitochondrial fusion in response to endogenous lga2 expression and that Lga2 additionally inhibits fusion in a dnm1-independent manner. Our results further show that Lga2 does not stimulate increased turnover of the putative fusion protein Fzo1 and causes mitochondrial branching, loss of mitochondrial DNA and fitness reduction independently of dnm1. We conclude that Lga2 acts upstream of Dnm1, but controls mitochondrial integrity independently of Dnm1-mediated fission. In addition, we demonstrate a role of dnm1 in fungal virulence.
Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free‐flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix‐free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state‐of‐the‐art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin‐positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.
The characterization of extracellular vesicles (EVs) has evolved rapidly in recent years due to advances in straightforward technologies. Based on these more sensitive methods, it is now possible to describe EV populations in their entirety more precisely. However, these applications require an equivalently delicate experiment design and optimization steps to draw valid conclusions in the end. One of these methods is represented by the highly sensitive nanoflow cytometry (nFCM), by which particles can be analyzed not only on their size (< 40 nm) and concentration but also concerning surface markers. In this work, we addressed some of the potential caveats of this method, especially when characterizing particles with fluorescently labelled antibodies. In particular, we show, when using low particle concentrations, which are inevitably encountered when working with EVs, the characterization of surface markers is prone to significantly varying. We hypothesized that these technical limitations could respond to the stickiness of EVs and should be properly counteracted. As a reference, we strongly recommend performing particle number-based comparisons with at least 109 particles as staining input in nFCM analyses. Moreover, we provided representative particle-number based immunoblotting results, underlying the significance of this parameter as a normalizer in future EV research.
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