Biology subjects

Cooley, L.

Publications and source records attributed to Cooley, L..

9 recordsLinked to original sources

bioRxiv2025

Examination of germline and somatic intercellular bridges in Hydra vulgaris reveals insights into the evolutionarily conserved mechanism of intercellular bridge formation

Incomplete cytokinesis results in the formation of stable intercellular bridges that have been extensively studied in bilaterians, where they play essential roles in cell-cell communication and coordination of differentiation. However, little is known about their structure and molecular composition in non-bilaterian animals. This study characterizes germline and somatic intercellular bridges in the cnidarian Hydra vulgaris, providing insights into their evolutionary origins and functional significance. We identified key conserved components, including KIF23, F-actin, and phosphotyrosine. Notably, we observed microtubule localization within Hydra ring canals, suggesting previously unrecognized functions for this cytoskeletal component in intercellular bridge formation. Bioinformatic analyses confirmed the conserved expression of Kif23 and suggested its role as a molecular marker for identifying ring canal-associated components. EdU incorporation during DNA replication demonstrated that cells connected by ring canals exhibit synchronized cell cycles, which may be critical for the coordination of division and differentiation. Our findings reveal that the molecular and structural features of intercellular bridges in Hydra are conserved across evolutionary lineages, highlighting their ancient origins and functional significance in cellular connectivity. The presence of synchronized cell cycles in ring canal-connected cells underscores their role in promoting coordinated cellular behaviors, processes fundamental to multicellular organization. This study provides new perspectives on the evolution of incomplete cytokinesis and establishes a framework for comparative investigations into the diversity and conservation of intercellular bridge mechanisms across metazoans.

cell biology↗

bioRxiv2022

Evolutionarily conserved midbody reorganization precedes ring canal formation during gametogenesis

How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges called ring canals is poorly under-stood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs via reorganization of the germline mid-body, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germline midbodies reorganize from a focus into a ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function in stark contrast to its role in abscission during somatic cell cytokinesis. Our results challenge existing models of ring canal formation, and provide important insights into broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.

developmental biology↗

bioRxiv2020

3D Adaptive Optical Nanoscopy for Thick Specimen Imaging at sub-50 nm Resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here, we have developed a nanoscope that, by utilizing an advanced adaptive optics strategy, achieves sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue.

biophysics↗

bioRxiv2020

Tissue-specific dynamic codon redefinition in Drosophila

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

HtsRC-mediated accumulation of F-actin regulates ring canal size during Drosophila melanogaster oogenesis

Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC, a component specific to female germline ring canals, is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ring-canal-like ectopic actin structures in somatic follicle cells. Finally, we present findings which indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility, a result which is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

cell biology↗

bioRxiv2020

A multi-layered systems approach for renal cell carcinoma

Renal cell carcinoma (RCC) still lacks prognostic and predictive biomarkers to monitor the disease and the response to therapy. The usual strategy in translational research is to start from human samples, to identify molecular markers and gene networks and then to functionally validate them in vitro and in animal models. We devised herein a completely opposite strategy from "mouse to man" by performing an aggressiveness screen and used functional genomics, imaging, clinical data and computational approaches in order to discover molecular pathways and players in renal cancer development and metastasis. Multiple cell lines for primary tumor growth, survival in the blood circulation and lung metastasis or metastatic spread from the primary tumor were generated and analyzed using a multi-layered approach which includes large-scale transcriptome, genome and methylome analyses. Transcriptome and methylome analyses demonstrated distinct clustering in three different groups. Remarkably, DNA sequencing did not show significant genomic variations in the different groups which indicates absence of clonal selection during the in vivo amplification process. Transcriptome analysis revealed distinct signatures of tumor aggressiveness which were validated in patient cohorts. Methylome analysis of full-length DNA allowed clustering of the same groups and revealed clinically relevant signatures. Furthermore, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. We also uncovered IL34 as another soluble prognostic biomarker and key regulator of renal cell carcinoma (RCC) progression. This was also functionally validated in vivo, and a mathematical model of IL34-dependent primary tumor growth and metastasis development was provided. These results indicate that such multilayered analysis in a RCC animal model leads to meaningful results that are of translational significance. One Sentence SummaryAn aggressiveness screen with multilayer systems analysis to identify signatures and biomarkers for renal cell carcinoma aggressiveness.

cancer biology↗

bioRxiv2019

Ring canals permit extensive cytoplasm sharing among germline cells independent of fusomes in Drosophila testes

Intercellular bridges, also called ring canals (RCs), connect germline cells during gametogenesis in males and females. They form as a consequence of incomplete cytokinesis during cell division leaving groups of daughter cells connected in syncytia. In Drosophila females, RCs are required for oocyte growth but little is known about the role of RCs during spermatogenesis. Using live imaging, we document extensive intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids. Loss of the fusome, a large cytoplasmic structure extending through RCs that is known to be important during oogenesis, has minimal impact on RC development or intercellular protein movement during spermatogenesis. Our results reveal that male germline RCs remain persistently open and mediate extensive sharing of cytoplasmic information, supporting multiple roles for RCs throughout sperm development.

developmental biology↗

bioRxiv2019

Proximity labeling reveals novel interactomes in live Drosophila tissue

Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10m. While multiple proteins have been identified as components of ring canals (RCs), we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. We optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.

cell biology↗

bioRxiv2018

The ubiquitin ligase CRL3Kelch targets HtsRC to organize the Drosophila ring canal cytoskeleton

During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

developmental biology↗