Distinct Functional Roles of beta-Tubulin Isotypes inMicrotubule Arrays of Tetrahymena thermophila, aModel Single-Celled Organism

Background The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2. Methodology/Principal Findings With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically. Conclusion/Significance We conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis

Sub-Antarctic and High Antarctic Notothenioid Fishes: Ecology and Adaptational Biology Revealed by the ICEFISH 2004 Cruise of RVIB Nathaniel B. Palmer

The goal of the ICEFISH 2004 cruise, which was conducted on board RVIB Nathaniel B. Palmer and traversed the transitional zones linking the South Atlantic to the Southern Ocean, was to compare the evolution, ecology, adaptational biology, community structure, and population dynamics of Antarctic notothenioid fishes relative to the cool/temperate notothenioids of the sub-Antarctic. To place this work in a comprehensive ecological context, cruise participants surveyed the benthos and geology of the biogeographic provinces and island shelves on either side of the Antarctic Polar Front (or Antarctic Convergence). Genome-enabled comparison of the responses of cold-living and temperate notothenioids to heat stress confirmed the sensitivity of the former to a warming Southern Ocean. Successful implementation of the international and interdisciplinary ICEFISH research cruise provides a model for future exploration of the sub-Antarctic sectors of the Indian and Pacific Oceans

A parasite outbreak in notothenioid fish in an Antarctic fjord

20 pages, 4 figures, supplemental information https://doi.org/10.1016/j.isci.2022.104588.-- Data and code availability: • All data have been deposited at NCBI GenBank: OL630144 and OL630145, NCBI SRA BioProject: PRJNA789574, at MorphoSource Project: 000405843, and at USAP-DC Project: p0010221, and are publicly available as of the date of publication. Biological materials have been deposited at the Zoological Museum of the University of Copenhagen. Additional accession numbers and DOIs are listed in the key resources. • This paper does not report original code. • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon requestClimate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystemsThis work was funded by the National Science Foundation grants OPP-1947040 (JHP and ArV), PLR-1444167 (HWD), and OPP-1543383 (JHP, TD, and HWD)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Genomics of cold adaptations in the Antarctic notothenioid fish radiation

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes

Cold Fusion: Massive Karyotype Evolution in the Antarctic Bullhead Notothen Notothenia coriiceps

Half of all vertebrate species share a series of chromosome fusions that preceded the teleost genome duplication (TGD), but we do not understand the causative evolutionary mechanisms. The Robertsonian-translocation hypothesis suggests a regular fusion of each ancestral acro- or telocentric chromosome to just one other by centromere fusions, thus halving the karyotype. An alternative genome-stirring hypothesis posits haphazard and repeated fusions, inversions, and reciprocal and nonreciprocal translocations. To study large-scale karyotype reduction, we investigated the decrease of chromosome numbers in Antarctic notothenioid fish. Most notothenioids have 24 haploid chromosomes, but bullhead notothen (Notothenia coriiceps) has 1. To understand mechanisms, we made a RAD-tag meiotic map with approximate to 10,000 polymorphic markers. Comparative genomics aligned about a thousand orthologs of platyfish and stickleback genes along bullhead chromosomes. Results revealed that 9 of 11 bullhead chromosomes arose by fusion of just two ancestral chromosomes and two others by fusion of three ancestral chromosomes. All markers from each ancestral chromosome remained contiguous, implying no inversions across fusion borders. Karyotype comparisons support a history of: (1) Robertsonian fusions of 22 ancestral chromosomes in pairs to yield 11 fused plus two small unfused chromosomes, like N. angustata; (2) fusion of one of the remaining two ancestral chromosomes to a preexisting fused pair, giving 12 chromosomes like N. rossii; and (3) fusion of the remaining ancestral chromosome to another fused pair, giving 11 chromosomes in N. coriiceps. These results raise the question of what selective forces promoted the systematic fusion of chromosomes in pairs and the suppression of pericentric inversions in this lineage, and provide a model for chromosome fusions in stem teleosts

Amino acid substitutions of BLT1 and BLT4 with respect to BTU2^a.

a<p>BTU2/residue/(BLT1)BLT4.</p

Distribution of BLT1 during conjugation. Upper panel:

<p>Fluorescence microscopic images of <i>Tetrahymena</i> cells expressing GFP-BTL1 <i>in vivo</i>. To assist interpretation, each image contains an inset that schematically represents the microtubule structures present during the main stages of conjugation as described by Gaertig and Fleury <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039694#pone.0039694-Gaertig5" target="_blank">[73]</a>. <b>Bottom panel</b>: Fluorescence microscopic images of <i>Tetrahymena</i> cells expressing GFP-BTL1 after fixation and nuclear staining with propidium iodide; the micronuclei (asterisks) and macronuclei (arrowheads) of the conjugants stained red. (<b>A</b>) <i>Tetrahymena</i> cells shortly after formation of the conjugating pair (stage 1 of prophase). GFP-BTL1 was visible in the micronuclei (arrows), whereas the macronuclei were negative for GFP-BLT1 fluorescence. (<b>B</b> and <b>a</b>) During prophase stage 4 of the first meiotic division, GFP-BLT1 forms curved microtubule bundles within the elongating micronucleus (arrows). (<b>C</b>) At metaphase of meiosis I, GFP-BLT1 was observed in normal spindles (arrows). At anaphase of the first (<b>D</b> and <b>b</b>) and second (<b>E</b>) meiotic divisions, metaphase spindles depolymerized and their tubulin dimers were used to form elongating separation spindles (arrows). (<b>F</b> and <b>c)</b> Four haploid nuclei were formed, one of which underwent a mitotic division (prezygotic mitosis) as shown by the GFP-BLT1 fluorescent signal and indicated by the arrows; the three remaining haploid meiotic products degenerated (not shown). (<b>G</b> and <b>d</b>) Subsequently, GFP-BLT1 is found in the micronuclei that exchanged between mating partners, and in the mitotic spindles of the first and second postzygotic divisions (<b>H, </b><b>I</b> and <b>e</b>). (<b>H</b> and <b>I)</b> The conjugating cells are slightly asynchronous in the first and second postzygotic divisions such that elongated mitotic spindles are visible in only one member of each pair. Micrographs were recorded from different cells at the conjugation stages specified. Bars, 10 μm.</p

Analysis of the subcellular localization of the BLT1, BLT4, and BTU2 isotypes by biochemical fractionation and Western blotting.

<p>(<b>A</b>) Nuclei were purified from non-synchronous cells (ns, lanes 1, 3, and 5) and from synchronously dividing cells (s, lanes 2, 4, and 6) after induction of GFP-BTU2, GFP-BLT1, or GFP-BLT4 synthesis (see Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039694#s4" target="_blank">Methods</a>). After samples were subjected to SDS-PAGE and electrotransfer to nitrocellulose, blots were developed with anti-β-tubulin or anti-GFP primary antibodies and appropriate secondary antibodies (Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039694#s4" target="_blank">Methods</a>). The “anti-β-tubulin” panel contains lanes derived from two different blots, whereas the “anti-GFP” panel derived from the same blot. (<b>B</b>) Cilia were separated from <i>Tetrahymena</i> cell bodies after induction of GFP-BTU2 or GFP-BLT4. Blots were developed as for (A). <b>Results</b>: GFP-BLT1 and GFP-BLT4 were predominantly found in samples containing nuclei purified from synchronously dividing cells (panel A, lanes 4 and 6) or in deciliated cell bodies (panel B, lanes 4 and 6). In contrast, GFP-BTU2 was present mainly in purified cilia (panel B, lanes 1).</p

Cortical distribution of BTU2 and BLT4 in living <i>T. thermophila</i> cells during interphase.

<p>(<b>A, B</b>) Fluorescence microscopic images of the cortices of <i>T. thermophila</i> cells transformed with vectors encoding GFP-BLT4 or GFP-BTU2, respectively. (<b>B</b>') Labeling of the oral apparatus by GFP-BTU2. (<b>C</b>, <b>D</b>) Black-and-white enlargements of subregions from (A) and (B), respectively. Structural components of the microtubule cytoskeleton that incorporated GFP-BLT4 (A) or GFP-BTU2 (B) tubulins are keyed to the schematic representation of the subcortical cytoskeleton of a <i>Tetrahymena</i> cell (slightly modified from Allen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039694#pone.0039694-Allen1" target="_blank">[72]</a>). GFP-BLT4 (and GFP-BLT1, not shown) was found in the longitudinal and transverse microtubule bundles (A, C), whereas GFP-BTU2 was present in somatic cilia, basal microtubules, and postciliary microtubules (B, D). Bar, 10 μm.</p

Distribution of BLT4 and BTU2 during conjugation.

<p>Fluorescence microscopic images of <i>Tetrahymena</i> conjugant cell pairs expressing GFP-BTU2 (<b>A</b> and <b>B</b>) or GFP-BTL4 (<b>C</b> and <b>D</b>). (A) and (C) are micrographs of living cells, whereas (B) and (D) are images of cells after fixation and staining with propidium iodide. In (A) and (C), the nuclei are negative for GFP-tubulin fluorescence. (B) Propidium iodide stained the newly formed micronuclei (arrowheads) and the parental macronuclei (arrows) in conjugants at the first post-zygotic division. (D) Propidium iodide stained the macronuclei (arrows) and the elongating micronuclei (arrowheads) in conjugants during anaphase of meiosis I. Although GFP-BTU2 and GFP-BLT4 did not label nuclear microtubules during meiosis, GFP-BTU2 was visible in cilia of the conjugant cells (A and B). Micrographs were recorded from different cells at the conjugation stages specified. Bar, 10 μm.</p