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

Demonstration of RNA interference by bacterial feeding in <i>Stentor polymorphus</i>

AbstractStentor is a genus of large trumpet-shaped unicellular organisms in the ciliate phylum. Classically they have been used as models of cellular morphogenesis due to their large size and ability to regenerate, but some Stentor species have features that make them useful models for other types of studies as well. Stentor polymorphus is a widely distributed species that harbors green algal endosymbionts from the Chlorella genus. While interesting phenomenology in this species has been described, molecular tools have never been developed in this system. As technology has advanced, the use of emerging models like S. polymorphus has become more prevalent, and recently a set of transcriptomes for S. polymorphus was published. However, there are still technical hurdles to using S. polymorphus as an effective experimental system in the lab. Here I describe the identification and culture of a S. polymorphus population from North Carolina as well as the identification and cloning of homologs of α-tubulin and the morphogenesis gene mob-1. Additionally, I demonstrate that RNA interference (RNAi) by feeding is effective against both of these homologs in S. polymorphus. The phenotypes observed in S. polymorphus were similar to phenotypes previously validated in S. coeruleus, a related Stentor species. A direct comparison of feeding RNAi between the two species revealed that RNAi appeared to be less effective in S. polymorphus. The ability to perform RNAi in S. polymorphus strengthens its use as an emerging model for exploring mechanisms of unicellular morphogenesis and regeneration or host-symbiont interactions and suggests that RNAi by bacterial feeding might be more broadly effective across the Stentor genus.</jats:p

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.</jats:p

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

Genetic perturbation of <i>srgp-1</i> leads to cleft closure defects.

(A) Summary timeline of events relevant to this study at 20°C. The beginning of gastrulation in C. elegans is marked by the internalization of the endodermal precursors Ea/p. Ventral cleft formation and closure begin approximately 100 min. later. Internalizing cells are indicated by pink pseudo-coloring. (B) A map depicting the domains of SRGP-1 including mutants used in this study (srgp-1W122Stop, srgp-1R563A, srgp-1ΔF-BAR, and srgp-1ΔC). (C) DIC images of embryos over 230 minutes. White dotted lines depict the ventral cleft. The first row depicts a typical wild-type embryo proceeding through cleft closure and into early elongation. srgp-1W122Stop, srgp-1ΔF-BAR, and srgp-1ΔC mutants all display cleft closure failure, resulting in extruded endoderm (yellow dotted lines). Scale bar is 10 μm. (D) A graph depicting percentage cleft closure defects in wild-type and srgp-1 mutants. ****, p < 0.0001; *, p < 0.05.</p

AFD-1 accumulates at the vertex of the anterior rosette.

(A) A typical embryo expressing HMP-1::GFP and mKate2::AFD-1 before, during, and after anterior rosette formation. White dotted line outlines cells that form the rosette. Blue dotted lines mark the anterior cells that internalize prior to formation of the rosette. White arrowheads indicate the vertex of the rosette. Scale bar is 10 μm. (B) Images of HMP-1::GFP and mKate2::AFD-1 localization at the anterior rosette immediately following internalization in various genetic backgrounds. White dotted lines outline cells forming the rosette; yellow dotted lines indicate ventral cleft that remains open. Scale bar is 5 μm. (C) A graph depicting the total accumulation of HMP-1::GFP at the anterior rosette. (D) Graph depicting the area of HMP-1::GFP aggregation at the vertex of the anterior rosette. ****, p < 0.0001; ***, p<0.001; **, p<0.01; *, p < 0.05.</p