Biology subjects

Slabodnick, M. M.

Publications and source records attributed to Slabodnick, M. M..

5 recordsLinked to original sources

bioRxiv2025

Microinjected dsRNA triggers a robust RNAi response in Stentor coeruleus

Stentor coeruleus is an emerging model system for the study of morphogenesis and regeneration as well as behaviors like habituation and photosensation. While Stentors genome has been sequenced, genetic tools are currently limited to RNA interference by feeding. Here we show that microinjection of long double-stranded RNA is an effective method for triggering a robust RNAi response. Additionally, microinjection of mob1 dsRNA resulted in cell phenotypes appearing 24 hours earlier than by feeding bacterially expressed dsRNA. This work expands the genetic toolkit in Stentor when RNAi by feeding is not ideal for an experiment.

cell biology↗

bioRxiv2024

The cell biology and genome of Stentor pyriformis, a giant cell that embeds symbiotic algae in a microtubule meshwork

Endosymbiotic events in which an endosymbiont is retained within a cell that remains capable of phagocytosis, a situation known as mixotrophy, provide potentially important clues about the evolutionary origins of eukaryotes, particularly regarding the relative evolutionary sequence of phagocytosis and endosymbiosis. Mixotrophy in ciliates is commonplace, but has been investigated at a cellular and molecular level almost entirely in one organism, Paramecium bursaria. Reliance on just one model system makes it difficult to know which cell biological aspects of this system represent general features of ciliate mixotrophy versus accidental features of the specific organism. Here we describe the cell biology and genome of the giant heterotrichous ciliate Stentor pyriformis. We show that this giant unicellular organism contains Chlorella variabilis as its endosymbiont, that the Chlorella can live freely outside the host, that within the host the Chlorella cells are docked near the cell surface, surrounded by microtubule "baskets", and that the photosynthetic efficiency of the Chlorella is reduced inside the Stentor cell compared to when it is free-living outside the host, with photon energy instead being shunted to non-photochemical quenching. Compared to the non-mixotrophic Stentor coeruleus, S. pyriformis has several distinct cellular features that may be related to endosymbiosis: the presence of microtubule baskets, which are absent in S. coeruleus; positive rather than negative phototaxis, which is likely an adaptation to allow the photosynthetic symbionts access to sufficient light; and a lack of pigment in the host cell, which may be an adaptation to tolerate high light levels. Compared to P. bursaria, S. pyriformis has several similar cellular features: in both organisms, the symbiont is a strain of Chlorella variabilis; the Chlorella endosymbiont retains the ability to live freely when separated from the host; and the algal symbionts contained in perialgal vesicles are docked at the cell surface. One potentially informative difference between P. bursaria and S. pyriformis is that S. pyriformis employs a standard genetic code, similar to other Stentor species but different from most other ciliates, including P. bursaria, which use a non-standard code in which one or more stop-codons are respecified to encode amino acids. This difference in genetic code could serve as a barrier to impede gene transfer from symbiont to host in other ciliates, but this would not be a factor in S. pyriformis. A second cell biological difference is that whereas P. bursaria performs phototaxis by a kinetic accumulation mechanism, in which swimming is non-directional but cells slow down in regions of higher light intensity, S. pyriformis performs directed swimming towards the direction of high light intensity. However, as in P. bursaria, phototaxis in S. pyriformis requires the presence of the Chlorella, implying a potential flow of information from the symbiont to direct the orientation and swimming of the host cell. We propose that S. pyriformis will serve as a useful model system for studying the evolution of mixotrophy and endosymbiosis, with unique advantages in terms of size and regenerative ability as well as distinct cellular and genomic features compared with other mixotrophic ciliate models.

cell biology↗

bioRxiv2022

SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-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 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 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 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 is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1 is constitutively open. A good candidate is AFD-1/Afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1 is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/Afadin increases cleft closure defects in srgp-1 and hmp-1R551/554A backgrounds. We propose that SRGP-1 promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1 opens, allowing maturing junctions to transition from recruitment of SRGP-1 to AFD-1. Our work identifies new roles for -catenin interactors during a process crucial to metazoan development.

developmental biology↗

bioRxiv2022

Afadin and zyxin contribute 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 -catenin and {beta}-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 to describe rates of myosin and membrane movement, which we then used to discover 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.

genetics↗

bioRxiv2019

Demonstration of RNA interference by bacterial feeding in Stentor polymorphus

Stentor 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 -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.

cell biology↗