Biology subjects

Pani, A. M.

Publications and source records attributed to Pani, A. M..

10 recordsLinked to original sources

bioRxiv2026

Understanding How Actomyosin Dynamics Drive Apical Constriction by Systematically Tagging and Mapping Actin-Binding Proteins

Apical constriction shapes tissues in fundamental events including gastrulation and neural tube formation. Diverse actin regulators play key roles in shaping cells, but the spatial distribution of most actin regulators in apical constriction is unknown. Here, we describe a systematic approach revealing the endogenous localization of 49 actin regulators in C. elegans gastrulation -- nearly every actin-binding protein (ABP) expressed in the early embryo -- and we use this atlas to test mechanistic hypotheses. We found that many actin regulators localized basolaterally, dependent on the Arp2/3 complex; that linear actin assembly factors enriched apically; and that apically constricting cells had an extra requirement for membrane-cortical linkers present in all cells. These findings answer some key questions about how ABPs function in apical constriction, and they establish a rich resource for investigating actin organization throughout early C. elegans development and a systematic in vivo protein localization reference for the actin cytoskeleton community.

cell biology↗

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↗

bioRxiv2023

Long-distance Wnt transport in axons highlights cell type-specific modes of Wnt transport in vivo

Wnt signaling performs critical functions in development, homeostasis, and disease states. Wnt ligands are secreted signaling proteins that often move between cells to activate signaling across a range of distances and concentrations. In different animals and developmental contexts, Wnts utilize distinct mechanisms for intercellular transport including diffusion, cytonemes and exosomes [1]. Mechanisms for intercellular Wnt dispersal remain controversial in part due to technical challenges with visualizing endogenous Wnt proteins in vivo, which has limited our understanding of Wnt transport dynamics. As a result, the cell-biological bases for long-range Wnt dispersal remain unknown in most instances, and the extent to which differences in Wnt transport mechanisms vary by cell type, organism, and/or ligand remain uncertain. To investigate processes underlying long-range Wnt transport in vivo, we utilized C. elegans as an experimentally tractable model where it is possible to tag endogenous Wnts with fluorescent proteins without disrupting signaling [2]. Live imaging of two endogenously tagged Wnt homologs revealed a novel mode for long-distance Wnt movement in axon-like structures that may complement Wnt gradients generated by diffusion and highlighted cell type-specific Wnt transport processes in vivo.

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↗

bioRxiv2023

Molecular characterization of nervous system organization in the hemichordate Saccoglossus kowalevskii

Hemichordates are an important group for investigating the evolution of bilaterian nervous systems. As the closest chordate outgroup with a bilaterally symmetric adult body plan, hemichordates are particularly informative for exploring the origins of chordates. Despite the importance of hemichordate neuroanatomy for testing hypotheses on deuterostome and chordate evolution, adult hemichordate nervous systems have not been comprehensively described using molecular techniques, and classic histological descriptions disagree on basic aspects of nervous system organization. A molecular description of hemichordate nervous system organization is important for both anatomical comparisons across phyla and for attempts to understand how conserved gene regulatory programs for ectodermal patterning relate to morphological evolution in deep time. Here, we describe the basic organization of the adult hemichordate Saccoglossus kowalevskii nervous system using immunofluorescence, in situ hybridization, and transgenic reporters to visualize neurons, neuropil, and key neuronal cell types. Consistent with previous descriptions, we found the S. kowalevskii nervous system consists of a pervasive nerve plexus that is concentrated in the anterior, along with nerve cords on both the dorsal and ventral sides. Neuronal cell types exhibited clear anteroposterior and dorsoventral regionalization in multiple areas of the body. We observed spatially demarcated expression patterns for many genes involved in synthesis or transport of neurotransmitters and neuropeptides but did not observe clear distinctions between putatively centralized and decentralized portions of the nervous system. In the trunk, there is a clear division of cell types between the dorsal and ventral cords suggesting differences in function. The plexus shows regionalized structure and is consistent with the proboscis base as a major site for information processing rather than the dorsal nerve cord. The absence of neural processes crossing the basement membrane into muscle and extensive axonal varicosities suggest that volumetric transmission may play an important role in neural function. These data now facilitate more informed neural comparisons between hemichordates and other groups and contribute to broader debates on the origins and evolution of bilaterian nervous systems.

evolutionary biology↗

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↗

bioRxiv2022

Transcriptional regulation and repressive condensates modulate a proliferative-invasive cellular switch in vivo

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of nhr-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

developmental biology↗

bioRxiv2019

Visualizing the metazoan proliferation-differentiation decision in vivo

Cell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live in C. elegans and zebrafish. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. We also derive a predictive model of future proliferation behavior in C. elegans and zebrafish based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell cycle control in a wide-range of developmental contexts.

developmental biology↗