Biology subjects

Gibney, T. V.

Publications and source records attributed to Gibney, T. V..

7 recordsLinked to original sources

bioRxiv2025

Engineering the C. elegans genome with a nested, self-excising selection cassette

C. elegans is a powerful model for dissecting biological processes in vivo. In particular, the ease of generating targeted knock-in alleles makes it possible to visualize and functionally modify endogenous proteins to gain fundamental insights into biological mechanisms. Methods for C. elegans genome engineering typically utilize selectable markers, visual screening for fluorescence, or PCR genotyping to identify successfully edited animals. A common genetic tool known as the Self-Excising Cassette (SEC) combines drug and phenotypic selection, which makes it possible to screen large numbers of progeny rapidly and with minimal hands-on effort. However, N-terminal and internal knock-ins using the SEC cause loss of function until the selectable marker cassette is excised, which makes it impossible to isolate homozygous lines for essential genes prior to SEC excision. To simplify generating knock-ins for essential genes, we developed a Nested, Self-Excising selection Cassette (NSEC) that is located entirely within a synthetic intron and does not interfere with the expression of endogenous, N-terminally-tagged NSEC-fusion proteins. This innovation makes it possible to isolate homozygous lines for N-terminally tagged genes prior to selectable marker excision and allows for a standardized workflow to generate N-terminal and internal tags in any background and without the need for genetic balancers. We designed versions of NSEC that include an optional auxin-inducible degron tag and mTurquoise2, GFP, mStayGold, mNeonGreen, or mScarlet-I fluorescent proteins for experimental flexibility. The NSEC expands our molecular toolbox and enhances the scalability, efficiency, and versatility of C. elegans genome engineering.

cell biology↗

bioRxiv2025

FGF-dependent, polarized SOS activity orchestrates directed migration of C. elegans muscle progenitors independently of canonical effectors in vivo

Directed cell migration is essential for animal development, tissue maintenance, regeneration, and disease states. Cells often migrate towards, or away from, sources of secreted signaling proteins that impart spatial information. How migrating cells interpret extracellular signals to orient and navigate within living animals is a fundamental question in biology. Receptor Tyrosine Kinase (RTK) signaling plays critical roles in cell migration, and aberrant RTK pathway activity is a key driver of multiple types of cancers. Yet, how RTKs control cell migration in living animals remains unclear, in part due to essential, pleiotropic roles of key proteins in development. To elucidate how RTK signaling controls cell migration in vivo, we dissected spatial and temporal requirements for key signal transduction and cytoskeletal regulatory proteins using C. elegans muscle progenitor migration as a tractable model. Cell type-specific depletion of endogenously tagged proteins revealed that homologs of FGFR, GRB2, SOS, and Ras control cell migration independently of their canonical ERK, PI3K, Akt, mTOR, and PLC{psi} effectors. Instead, we found that FGF-dependent, polarized SOS-1 orients migrating cells towards an FGF source, and mislocalizing SOS activity within migrating cells severely disrupts migration independent of ERK. Cell type-specific, gain-of-function experiments demonstrated that activated Ras is largely permissive for anterior migration in this context, and an intragenic revertant identified in a screen for suppressors of activated Ras/let-60 revealed that signal transduction in migrating muscle progenitors can be genetically uncoupled from Ras-ERK-dependent developmental processes. We found that conserved regulators of branched actin assembly control SM protrusive dynamics but are not essential for accurate, FGF-directed migration. Our findings provide a novel mechanism for RTK-directed cell migration in vivo and highlight the importance of cell type-specific approaches to elucidate signal transduction mechanisms in physiologically relevant contexts. Our work also outlines a comprehensive framework for investigating RTK-dependent processes in a multicellular organism and introduces a versatile genetic toolkit for dissecting spatial and temporal signaling dynamics fundamental to development, homeostasis, and disease.

cell biology↗

bioRxiv2025

FGF diffusion is required for directed migration of postembryonic muscle progenitors in C. elegans

Extracellular signaling molecules mediate crucial aspects of cell-cell communication and play essential roles in development and homeostasis. Fibroblast Growth Factors (FGFs) are a conserved family of secreted signaling proteins that can disperse long distances between cells and are often thought to form concentration gradients that encode spatial information. However, we know relatively little about the spatial distribution of FGFs in vivo, and endogenously tagged FGFs use different mechanisms to move between cells and form gradients in zebrafish and flies. We used FGF-dependent migration of C. elegans muscle progenitors called sex myoblasts (SMs) as a tractable system to elucidate FGF dispersal mechanisms and dissect how FGF guides migrating cells. Live imaging of cell dynamics and endogenously tagged FGF combined with membrane tethering and extracellular trapping approaches revealed that endogenous FGF is diffusible in vivo and extracellular dispersal is required for SM migration. Misexpression demonstrated FGF is a bona fide chemoattractant that orients SMs during a critical window, while an unidentified, short-range signal acts in concert to precisely position SMs. Our finding that an invertebrate FGF is endogenously diffusible suggests that this may be the ancestral mode for FGF dispersal. Summary StatementFibroblast Growth Factors (FGFs) are signaling proteins with crucial developmental roles. We demonstrated that a C. elegans FGF homolog is diffusible, and diffusion is required in vivo for cell migration guidance.

developmental biology↗

bioRxiv2024

A split-GAL4 driver line resource for Drosophila CNS cell types

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

neuroscience↗

bioRxiv2023

Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans

Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell in vitro essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and KLP-19 in C. elegans, the homologs of PRC1 and KIF4a, suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length and thus microtubule-based interactions in the spindle midzone, which affects spindle dynamics and force transmission. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone. SummaryKLP-19 controls microtubule length in the spindle midzone of C. elegans, affecting spindle dynamics and force transmission during mitosis.

cell biology↗

bioRxiv2023

A simple method to dramatically increase C. elegans germline microinjection efficiency

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

genetics↗

bioRxiv2022

The C. elegans Anchor Cell Transcriptome: Ribosome Biogenesis Drives Cell Invasion through Basement Membrane

Cell invasion through basement membrane (BM) barriers is important in development, immune function, and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of cells actively transmigrating BM in vivo remains elusive. Using the stereotyped timing of C. elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including TCTP (Translationally Controlled Tumor Protein). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling, and ribosome biogenesis analysis revealed a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also strongly localize to the ACs endoplasmic reticulum (ER) and the endomembrane system expands prior to invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

cell biology↗