Analysis of the gut microbiome in sled dogs reveals glucosamine- and activity-related effects on gut microbial composition.

The composition of the microbiome influences many aspects of physiology and health, and can be altered by environmental factors, including diet and activity. Glucosamine is a dietary supplement often administered to address arthritic symptoms in humans, dogs, and other mammals. To investigate how gut microbial composition varies with glucosamine supplementation, we performed 16S rRNA sequence analysis of fecal samples from 24 Alaskan and Inuit huskies and used mixed effects models to investigate associations with activity, age, and additional factors. Glucosamine ingestion, age, activity, sex, and diet were correlated with differences in alpha-diversity, with diversity decreasing in dogs consuming glucosamine. Beta-diversity analysis revealed clustering of dogs based on glucosamine supplementation status. Glucosamine supplementation and exercise-related activity were associated with greater inter-individual pairwise distances. At the family level, Lactobacillaceae and Anaerovoracaceae relative abundances were lower in supplemented dogs when activity was accounted for. At the genus level, Eubacterium [brachy], Sellimonus, Parvibacter, and an unclassified genus belonging to the same family as Parvibacter (Eggerthellaceae) all were lower in supplemented dogs, but only significantly so post-activity. Our findings suggest that glucosamine supplementation alters microbiome composition in sled dogs, particularly in the context of exercise-related activity.

Machine learning and network analysis of the gut microbiome from patients with schizophrenia and non-psychiatric subject controls reveal behavioral risk factors and bacterial interactions.

Recent findings have supported an association between deviations in gut microbiome composition and schizophrenia. However, the extent to which the gut microbiota contributes to schizophrenia remains unclear. Moreover, studies have yet to explore variations in ecological associations among bacterial types in subjects with schizophrenia, which can reveal differences in community interactions and gut stability. We examined the dataset collected by Nguyen et al. (2021) to investigate the similarities and differences in gut microbial constituents between 48 subjects with schizophrenia and 48 matched non-psychiatric comparison cases. We re-analyzed alpha- and beta-diversity differences and completed modified differential abundance analyses and confirmed the findings of Nguyen et al. (2021) that there was little variation in alpha-diversity but significant differences in beta-diversity between individuals with schizophrenia and non-psychiatric subjects. We also conducted mediation analysis, developed a machine learning (ML) model to predict schizophrenia, and completed network analysis to examine community-level interactions among bacterial taxa. Our study offers new insights, suggesting that the gut microbiome mediates the effects between schizophrenia and smoking status, BMI, anxiety score, and depression score. Our differential abundance and network analysis findings suggest that the differential abundance of Lachnospiraceae and Ruminococcaceae taxa fosters a decrease in stabilizing competitive interactions in the gut microbiome of subjects with schizophrenia. Loss of this competition may promote ecological instability and dysbiosis, altering gut-brain axis interactions in these subjects.

Exportin Crm1 is important for Swi6 nuclear shuttling and MBF transcription activation in Saccharomyces cerevisiae.

BACKGROUND: Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities. RESULTS: Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5. CONCLUSIONS: These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression.

Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae.

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

Microarray Analysis of Gene Expression in Saccharomyces cerevisiae kap108Δ Mutants upon Addition of Oxidative Stress.

Protein transport between the nucleus and cytoplasm of eukaryotic cells is tightly regulated, providing a mechanism for controlling intracellular localization of proteins, and regulating gene expression. In this study, we have investigated the importance of nucleocytoplasmic transport mediated by the karyopherin Kap108 in regulating cellular responses to oxidative stress in Saccharomyces cerevisiae We carried out microarray analyses on wild-type and kap108 mutant cells grown under normal conditions, shortly after introduction of oxidative stress, after 1 hr of oxidative stress, and 1 hr after oxidative stress was removed. We observe more than 500 genes that undergo a 40% or greater change in differential expression between wild-type and kap108Δ cells under at least one of these conditions. Genes undergoing changes in expression can be categorized in two general groups: 1) those that are differentially expressed between wild-type and kap108Δ cells, no matter the oxidative stress conditions; and 2) those that have patterns of response dependent upon both the absence of Kap108, and introduction or removal of oxidative stress. Gene ontology analysis reveals that, among the genes whose expression is reduced in the absence of Kap108 are those involved in stress response and intracellular transport, while those overexpressed are largely involved in mating and pheromone response. We also identified 25 clusters of genes that undergo similar patterns of change in gene expression when oxidative stresses are added and subsequently removed, including genes involved in stress response, oxidation-reduction processing, iron homeostasis, ascospore wall assembly, transmembrane transport, and cell fusion during mating. These data suggest that Kap108 is important for regulating expression of genes involved in a variety of specific cell functions.

A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export.

The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae.

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.

Nuclear localisation is crucial for the proapoptotic activity of the HtrA-like serine protease Nma111p.

Programmed cell death is induced by the activation of a subset of intracellular proteins in response to specific extra- and intracellular signals. In the yeast Saccharomyces cerevisiae, Nma111p functions as a nuclear serine protease that is necessary for apoptosis under cellular stress conditions, such as elevated temperature or treatment of cells with hydrogen peroxide to induce cell death. We have examined the role of nuclear protein import in the function of Nma111p in apoptosis. Nma111p contains two small clusters of basic residues towards its N-terminus, both of which are necessary for efficient translocation into the nucleus. Nma111p does not shuttle between the nucleus and cytoplasm during either normal growth conditions or under environmental stresses that induce apoptosis. The N-terminal half of Nma111p is sufficient to provide the apoptosis-inducing activity of the protein, and the nuclear-localisation signal (NLS) sequences and catalytic serine 235 are both necessary for this function. We provide compelling evidence that intranuclear Nma111p activity is necessary for apoptosis in yeast.

Using affinity chromatography to investigate novel protein-protein interactions in an undergraduate cell and molecular biology lab course.

Inquiry-driven lab exercises require students to think carefully about a question, carry out an investigation of that question, and critically analyze the results of their investigation. Here, we describe the implementation and assessment of an inquiry-based laboratory exercise in which students obtain and analyze novel data that contribute to our understanding of macromolecular trafficking between the nucleus and cytoplasm in eukaryotic cells. Although many of the proteins involved in nucleocytoplasmic transport are known, the physical interactions between some of these polypeptides remain uncharacterized. In this cell and molecular biology lab exercise, students investigate novel protein-protein interactions between factors involved in nuclear RNA export. Using recombinant protein expression, protein extraction, affinity chromatography, SDS-polyacrylamide gel electrophoresis, and Western blotting, undergraduates in a sophomore-level lab course identified a previously unreported association between the soluble mRNA transport factor Mex67 and the C-terminal region of the yeast nuclear pore complex protein Nup1. This exercise immersed students in the process of investigative science, from proposing and performing experiments through analyzing data and reporting outcomes. On completion of this investigative lab sequence, students reported enhanced understanding of the scientific process, increased proficiency with cellular and molecular methods and content, greater understanding of data analysis and the importance of appropriate controls, an enhanced ability to communicate science effectively, and an increased enthusiasm for scientific research and for the lab component of the course. The modular nature of this exercise and its focus on asking novel questions about protein-protein interactions make it easily transferable to undergraduate lab courses performed in a wide variety of contexts.

Mutations affecting spindle pole body and mitotic exit network function are synthetically lethal with a deletion of the nucleoporin NUP1 in S. cerevisiae.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope of eukaryotic cells and function to regulate passage of macromolecules in and out of the nucleus. Nup1 is one of 30 nucleoporins comprising the NPC of the yeast Saccharomyces cerevisiae and is located on the nucleoplasmic face of the NPC where it plays a role in mRNA export and protein transport. In order to further characterize the function of Nup1 we used a genetic approach to identify mutations that are synthetically lethal in combination with a deletion of NUP1 (nup1Delta). We have identified one such nup1 lethal mutant (nle6) as a temperature sensitive allele of nud1. NUD1 encodes a component of the yeast spindle pole body (SPB) and acts as scaffolding for the mitotic exit network (MEN). We observe that nle6/nud1 mutant cells have a normal distribution of NPCs within the nuclear envelope and exhibit normal rates of nuclear protein import at both the permissive and restrictive temperatures. nup1Delta also exhibits synthetic lethality with bub2Delta and bfa1Delta, both of which encode proteins that colocalize with Nud1 at spindle pole bodies and function in the mitotic exit network. However, we do not observe genetic interactions among nle6/nud1, bub2Delta, or bfa1Delta and mutations in the nucleoporin encoding genes NUP60 or NUP170, nor is nup1Delta synthetically lethal with the absence of components downstream in the mitotic exit network, including Lte1, Swi5, and Dbf2. Our results suggest a novel functional connection between Nup1 and proteins comprising both the spindle pole body and early mitotic exit network.

Nuclear pore complex function in Saccharomyces cerevisiae is influenced by glycosylation of the transmembrane nucleoporin Pom152p.

The regulated transport of proteins across the nuclear envelope occurs through nuclear pore complexes (NPCs), which are composed of >30 different protein subunits termed nucleoporins. While some nucleoporins are glycosylated, little about the role of glycosylation in NPC activity is understood. We have identified loss-of-function alleles of ALG12, encoding a mannosyltransferase, as suppressors of a temperature-sensitive mutation in the gene encoding the FXFG-nucleoporin NUP1. We observe that nup1Delta cells import nucleophilic proteins more efficiently when ALG12 is absent, suggesting that glycosylation may influence nuclear transport. Conditional nup1 and nup82 mutations are partially suppressed by the glycosylation inhibitor tunicamycin, while nic96 and nup116 alleles are hypersensitive to tunicamycin treatment, further implicating glycosylation in NPC function. Because Pom152p is a glycosylated, transmembrane nucleoporin, we examined genetic interactions between pom152 mutants and nup1Delta. A nup1 deletion is lethal in combination with pom152Delta, as well as with truncations of the N-terminal and transmembrane regions of Pom152p. However, truncations of the N-glycosylated, lumenal domain of Pom152p and pom152 mutants lacking N-linked glycosylation sites are viable in combination with nup1Delta, suppress nup1Delta temperature sensitivity, and partially suppress the nuclear protein import defects associated with the deletion of NUP1. These data provide compelling evidence for a role for glycosylation in influencing NPC function.

Crm1-mediated nuclear export of Cdc14 is required for the completion of cytokinesis in budding yeast.

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.

The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and performs a function distinct from translocation in RPA protein import.

Protein transport between the nucleus and cytoplasm requires interactions between nuclear pore complex proteins (nucleoporins) and soluble nuclear transport factors (karyopherins, importins, and exportins). Exactly how these interactions contribute to the nucleocytoplasmic transport of substrates remains unclear. Using a synthetic lethal screen with the nucleoporin NUP1, we have identified a conditional allele of NUP82, encoding an essential nuclear pore complex protein in Saccharomyces cerevisiae. This nup82-3 allele also exhibits synthetic genetic interactions with mutants of the karyopherin MSN5. nup82-3 mutants accumulate the Msn5 export substrate Pho4 within the nucleus at non-permissive temperatures. The nuclear import of the RPA complex subunit Rfa2 is impaired in nup82-3 and in mutants of the karyopherin KAP95, but is not affected by the loss of MSN5. Interestingly, deletion of MSN5 results in retention of Rfa2-GFP within the nucleus under conditions in which it normally diffuses out. These data provide evidence that Nup82 is important for Msn5-mediated nuclear protein export and Kap95-mediated protein import. In addition, Msn5 may play a role independent of import in the localization of Rfa2.

A signal peptide secretion screen in Fucus distichus embryos reveals expression of glucanase, EGF domain-containing, and LRR receptor kinase-like polypeptides during asymmetric cell growth.

Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis. In order to identify F. distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion. We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity. The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides. BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal beta-1, 3-exoglucanase. However, most of the isolated Fucus polypeptides lack similarity to known proteins. The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos.