Project description:Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles in this process by providing linkage between chromosomes and spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Eeuglenozoans forms a highly diversespeciose group of unicellular flagellates -including kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have a canonical onekinetochore system. It remains unclear what kind of kinetochores are present in diplonemids, a group of extremely diverse and abundant marine flagellatesplankton. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid nor canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like the CLK kinase and the meiotic synaptonemal complex proteins SYCP2 that function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLK, PpSYCP2L1 and INCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results raise a possibility that diplonemids evolved a hitherto unknown type of kinetochore system.
Project description:Microarray profiling of GFP-marked epithelial cells isolated by FACS from 1st and 3rd instar wild type and mutant Drosophila larvae. Multiple independent replicates collected for each population/timepoint combination. RNA isolated and amplified from FACS-sorted populations. Hybridized as two-color experiment against a common pooled reference.
Project description:Microarray profiling of GFP-marked neuronal and non-neuronal cellular populations isolated by FACS at 24h intervals across development of Drosophila larvae. Multiple independent replicates collected for each population/timepoint combination. RNA isolated and amplified from FACS-sorted populations. Hybridized as two-color experiment against a common pooled reference.
Project description:A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a non-functional Tuft1 (tuftelin 1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene. Recently, we identified a recessive mutation, an abnormal coat appearance, in BXH6 strain, a member of the HXB/BXH set of recombinant inbred strains derived from the SHR (spontaneously hypertensive rat) and BN-Lx (Brown Norway rat) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the Tuft1 (tuftelin 1) gene on chromosome 2, which resulted in a premature stop codon. Compared to wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin, and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum MCP-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and beta oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for an important role of Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.
Project description:Recently, red beetroot has attracted attention as a health promoting functional food. Studies showed that beetroot administration can reduce blood pressure and ameliorate parameters of glucose and lipid metabolism, however, mechanisms underlying these beneficial effects of beetroot are not fully understood. In the current study, we analyzed effects of beetroot on parameters of glucose and lipid metabolism in two models of metabolic syndrome, (i) transgenic spontaneously hypertensive rats expressing human C-reactive protein (SHR-CRP rats), and (ii) hereditary hypertriglyceridemic (HHTg) rats. Treatment with beetroot juice for 4 weeks was in both models associated with amelioration of oxidative stress, reduced circulating lipids, smaller visceral fat depots, and lower ectopic fat accumulation in the liver, compared to their respective untreated controls. On the other hand, beetroot treatment had no significant effects on sensitivity of muscle and adipose tissue to insulin action in both models. Analyses of hepatic proteome revealed significantly deregulated proteins involved in glycerophospholipid metabolism, mTOR signalling, inflammation, and cytoskeleton rearrangement.
Project description:Microarray profiling of whole blood of immunodeficient mouse strains at 24h after infection with Plasmodium chabaudi. Multiple independent replicates collected for each knockout. RNA isolated and amplified. Hybridized as two-color experiment against a common pooled reference.
Project description:Microarray profiling of adoptively transferred GFP-marked NK cells during MCMV infection in C57BL/6 mice Multiple independent replicates were collected per time point. NK cells were isolated from spleens of mice by flow cytometry. RNA isolated, amplified, and hybridized as two-color experiment against a common pooled reference.
Project description:CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe a new mechanism of CK1 regulation conserved from yeast to human – autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site – that inhibits kinase activity. Consequently, yeast and human CK1 enzymes with phosphoablating mutations at this site are hyperactive in vitro. We used quantitative phosphoproteomics to show that disruption of this regulatory mechanism rewires CK1 signaling in Schizosaccharomyces pombe. In accord, we found that a known CK1 pathway, a mitotic checkpoint, is downregulated in these mutants, while new pathways that confer heat shock resistance and suppress meiotic transcripts are upregulated. Molecular dynamics simulations demonstrated that phosphorylation on the L-EF loop alters the conformation of the substrate docking site, and we propose that this affects which CK1 substrates can be phosphorylated. Due to the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.