The Macronuclear Genome of \u3cem\u3eStentor coeruleus\u3c/em\u3e Reveals Tiny Introns in a Giant Cell

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities—if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor

Zyxin contributes to coupling between cell junctions and contractile actomyosin networks during apical constriction

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and β-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions’ centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved

SRGP-1/srGAP and AFD-1/afadin stabilize HMP-1/⍺-catenin at rosettes to seal internalization sites following gastrulation in C. elegans.

A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1/srGAP C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1/srGAP C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/⍺-catenin in surface cells during cleft closure. A mutant form of HMP-1/⍺-catenin with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1/⍺-catenin is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1/⍺-catenin is constitutively open. A good candidate is AFD-1/afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1/afadin is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/afadin increases cleft closure defects in srgp-1/srGAP and hmp-1R551/554A/⍺-catenin backgrounds. We propose that SRGP-1/srGAP promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1/⍺-catenin opens, allowing maturing junctions to transition from recruitment of SRGP-1/srGAP to AFD-1/afadin. Our work identifies new roles for ⍺-catenin interactors during a process crucial to metazoan development

The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system

<i>β-tubulin(RNAi)</i> results in aberrant cell morphologies.

<p>(A) qRT-PCR results showing relative expression of β-tubulin normalized to GAPDH expression in both <i>control(RNAi)</i> and <i>β-tubulin(RNAi)</i> cells. (B, C) Brightfield images of control and <i>β-tubulin(RNAi)</i> cells showing dramatic alteration in cell shape. (D, E) Immunofluorescence images of stained control and <i>β-tubulin(RNAi)</i> cells highlighting cortical microtubule bundles (green, anti-α-tubulin). In contrast to the highly organized parallel microtubule rows seen in control cells, <i>β-tubulin(RNAi)</i> cells have improperly oriented (white arrow), broken (purple arrow), and discontinuous (yellow arrow) rows, indicating a disruption of normal cellular patterning.</p

The anatomy of <i>Stentor coeruleus</i>.

<p>(A) Cartoon of <i>Stentor coeruleus</i> highlighting key cellular structures, all of which have reproducible positions within the cell. (B) Cartoon representing <i>Stentor</i> regeneration after surgical bisection as initially reported by Thomas Hunt Morgan in 1901. (C) A neighbor-joining phylogenetic analysis of protein sequences of <i>Stentor</i> argonaute homologs along with sequences from <i>Paramecium</i> (<i>Pt</i>), <i>Tetrahymena</i> (<i>Tt</i>), human (Hs), mouse (Mm), <i>C. elegans</i> (<i>Ce</i>), <i>Drosophila</i> (<i>Dm</i>), and <i>Arabidopsis</i> (<i>At</i>). The three major classes of Argonaute proteins—PIWI, WAGO, and AGO—are indicated (Sciwi proteins listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001861#pbio.1001861.s014" target="_blank">Table S1</a>, gene IDs in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001861#pbio.1001861.s015" target="_blank">Table S2</a>).</p

Presence of residual Mob1 protein in <i>Mob1(RNAi)</i> cells.

<p>(A) Immunofluorescence images of stained <i>control(RNAi)</i> cells showing Mob1 localization (green, anti-Mob1), macronucleus (blue, DAPI), and cortical rows (red, stentorin-autofluorescence). (B, C) Immunofluorescence images of stained elongated (B) and medusoid (C) <i>Mob1(RNAi)</i> cells; Mob1 (green, anti-Mob1), macronucleus (blue, DAPI), and cortical rows (red, stentorin autofluorescence) (compare to A). Cortical aberrations are seen in the cortical rows (red arrows). Both the elongated and medusoid cells were too large to fit in a single frame, and two separate images were taken with identical settings, then manually stitched together using the cortical rows for alignment (join-lines between images are indicated by a dashed yellow line).</p