Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.

Polymer Modeling Reveals Interplay between Physical Properties of Chromosomal DNA and the Size and Distribution of Condensin-Based Chromatin Loops.

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead-spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain.

Adaptive partitioning of a gene locus to the nuclear envelope in Saccharomyces cerevisiae is driven by polymer-polymer phase separation.

Partitioning of active gene loci to the nuclear envelope (NE) is a mechanism by which organisms increase the speed of adaptation and metabolic robustness to fluctuating resources in the environment. In the yeast Saccharomyces cerevisiae, adaptation to nutrient depletion or other stresses, manifests as relocalization of active gene loci from nucleoplasm to the NE, resulting in more efficient transport and translation of mRNA. The mechanism by which this partitioning occurs remains a mystery. Here, we demonstrate that the yeast inositol depletion-responsive gene locus INO1 partitions to the nuclear envelope, driven by local histone acetylation-induced polymer-polymer phase separation from the nucleoplasmic phase. This demixing is consistent with recent evidence for chromatin phase separation by acetylation-mediated dissolution of multivalent histone association and fits a physical model where increased bending stiffness of acetylated chromatin polymer causes its phase separation from de-acetylated chromatin. Increased chromatin spring stiffness could explain nucleation of transcriptional machinery at active gene loci.

The power of weak, transient interactions across biology: A paradigm of emergent behavior.

A growing list of diverse biological systems and their equally diverse functionalities provides realizations of a paradigm of emergent behavior. In each of these biological systems, pervasive ensembles of weak, short-lived, spatially local interactions act autonomously to convey functionalities at larger spatial and temporal scales. In this article, a range of diverse systems and functionalities are presented in a cursory manner with literature citations for further details. Then two systems and their properties are discussed in more detail: yeast chromosome biology and human respiratory mucus.

Mechanisms of DNA Mobilization and Sequestration.

The entire genome becomes mobilized following DNA damage. Understanding the mechanisms that act at the genome level requires that we embrace experimental and computational strategies to capture the behavior of the long-chain DNA polymer, which is the building block for the chromosome. Long-chain polymers exhibit constrained, sub-diffusive motion in the nucleus. Cross-linking proteins, including cohesin and condensin, have a disproportionate effect on genome organization in their ability to stabilize transient interactions. Cross-linking proteins can segregate the genome into sub-domains through polymer-polymer phase separation (PPPS) and can drive the formation of gene clusters through small changes in their binding kinetics. Principles from polymer physics provide a means to unravel the mysteries hidden in the chains of life.

The rDNA is biomolecular condensate formed by polymer-polymer phase separation and is sequestered in the nucleolus by transcription and R-loops.

The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer-polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14's nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA-binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA-DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer-polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.

Characterization and immune regulation role of an immobilization antigen from Cryptocaryon irritans on groupers.

Immobilization antigens (i-antigens) are surface membrane proteins that are widely recognized to be the ideal candidates as vaccines antigens for immunization against Cryptocaryon irritans. In this study, we cloned a putative i-antigen gene from C. irritans, which was expressed in all three stages of the C. irritans life-cycle, and localized primarily to the cell surface. The recombinant GDCI3 i-antigen was expressed and purified using the free-living ciliate, Tetrahymena thermophila as an expression system. The purified recombinant protein was recognized by rabbit anti-C. irritans antiserum and was capable of eliciting immobilizing antibodies in rabbits and fish suggesting that the antigen itself was correctly folded. Following immunization and parasite challenge, groupers vaccinated with, recombinant GDCI3 i-antigen had a 25% cumulative percent survival rate compared to 8.3% for controls. Both non-specific and parasite-specific IgMs were generated in fish following immunization, with the levels of both increasing following challenge. Parasite-specific IgM in mucus could only be elicited after challenge of the GDCI3 i-antigen vaccinated groupers. To our knowledge, this is the first report using the Tetrahymena expression system to generate C. irritans i-antigens and investigate their use for fish vaccination.

Diversity and Universality of Endosymbiotic Rickettsia in the Fish Parasite Ichthyophthirius multifiliis.

Although the presence of endosymbiotic rickettsial bacteria, specifically Candidatus Megaira, has been reported in diverse habitats and a wide range of eukaryotic hosts, it remains unclear how broadly Ca. Megaira are distributed in a single host species. In this study we seek to address whether Ca. Megaira are present in most, if not all isolates, of the parasitic ciliate Ichthyophthirius multifiliis. Conserved regions of bacterial 16S rRNA genes were either PCR amplified, or assembled from deep sequencing data, from 18 isolates/populations of I. multifiliis sampled worldwide (Brazil, Taiwan, and USA). We found that rickettsial rRNA sequences belonging to three out of four Ca. Megaira subclades could be consistently detected in all I. multifiliis samples. I. multifiliis collected from local fish farms tend to be inhabited by the same subclade of Ca. Megaira, whereas those derived from pet fish are often inhabited by more than one subclade of Ca. Megaira. Distributions of Ca. Megaira in I. multifiliis thus better reflect the travel history, but not the phylogeny, of I. multifiliis. In summary, our results suggest that I. multifiliis may be dependent on this endosymbiotic relationship, and the association between Ca. Megaira and I. multifiliis is more diverse than previously thought.

Function of the male-gamete-specific fusion protein HAP2 in a seven-sexed ciliate.

HAP2, a male-gamete-specific protein conserved across vast evolutionary distances, has garnered considerable attention as a potential membrane fusogen required for fertilization in taxa ranging from protozoa and green algae to flowering plants and invertebrate animals [1-6]. However, its presence in Tetrahymena thermophila, a ciliated protozoan with seven sexes or mating types that bypasses the production of male gametes, raises interesting questions regarding the evolutionary origins of gamete-specific functions in sexually dimorphic species. Here we show that HAP2 is expressed in all seven mating types of T. thermophila and that fertility is only blocked when the gene is deleted from both cells of a mating pair. HAP2 deletion strains of complementary mating types can recognize one another and form pairs; however, pair stability is compromised and membrane pore formation at the nuclear exchange junction is blocked. The absence of pore formation is consistent with previous studies suggesting a role for HAP2 in gamete fusion in other systems. We propose a model in which each of the several hundred membrane pores established at the conjugation junction of mating Tetrahymena represents the equivalent of a male/female interface, and that pore formation is driven on both sides of the junction by the presence of HAP2. Such a model supports the idea that many of the disparate functions of sperm and egg were shared by the "isogametes" of early eukaryotes and became partitioned to either male or female sex cells later in evolution.

Protein expression profiling of postmortem brain in schizophrenia.

Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) enables the sensitive, high-throughput protein profiling of complex biological mixtures. In combination with bioinformatics, this technology has the potential to identify combinations of spectral peaks that can differentiate individuals with a particular disease from normal controls. SELDI-TOF-MS was used to screen postmortem tissue derived from the dorsolateral prefrontal cortex of individuals with schizophrenia (n = 34) and matched controls (n = 35), obtained from the Stanley Foundation Neuropathology Consortium. Tissue samples were homogenized in urea buffer, applied to four different chip arrays which possess different chromatographic surfaces, and analyzed using the Ciphergen ProteinChip Biomarkers System (Model PBS II). Protein expression profiles of the schizophrenia and control groups were compared and analyzed using the Ciphergen Express (CE) and Biomarker Patterns Software (BPS) package. We detected several protein peaks whose intensities differed between the schizophrenia and control groups to a highly significant degree. A combination of these peaks was capable of distinguishing between schizophrenia and controls with a sensitivity and specificity of about 70%. The classification model that distinguished schizophrenia from controls was complex, suggesting that the biochemical abnormalities underlying schizophrenia are heterogeneous. Our results suggest that SELDI-TOF-MS has the potential for distinguishing individuals with schizophrenia from normal controls and may eventually lead to a better understanding of the classification, diagnosis and pathogenesis of this disorder.