Biology subjects

Matus, D. Q.

Publications and source records attributed to Matus, D. Q..

At least 19 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↗

bioRxiv2026

mRNAbow: A versatile gene expression system for multiplexed fluorescent imaging using optimized in vitro transcribed mRNA

Messenger RNA (mRNA) transfection enables rapid, transient protein expression without nuclear entry, providing a powerful alternative to DNA or viral delivery in post-mitotic and otherwise difficult-to-transfect cells. Although in vitro transcribed (IVT) mRNAs have revolutionized therapeutic applications, their adoption in experimental biology remains limited by challenges in synthesis, variability across cell types, and concerns about cytotoxicity. Here, we define design principles that maximize IVT mRNA performance across diverse cellular and organismal systems. Through systematic comparison of capping strategies and base modifications, including N1-methyl-pseudouridine, 5-methylcytidine, and 5-methoxyuridine, we identify modifications that enhance translation while minimizing activation of cellular stress responses. Optimized transcripts drive robust protein expression within four hours, persist for up to one week, and support multiplexed expression of structurally and functionally distinct proteins in mammalian cells, including cancer cell lines, iPSC-derived systems, primary cells, and organoids, as well as in vivo in zebrafish embryos and in less genetically tractable models such as Danionella cerebrum and sea urchin embryos. To further expand accessibility for community use, we developed mRNAbow, a platform for generating low-toxicity mRNAs encoding organelle-targeted fluorescent proteins and biosensors for multiplex imaging, with corresponding plasmids made publicly available. Together, these advances establish a generalizable framework for IVT mRNA design and expand experimental access to synthetic mRNA technologies for dissecting cellular architecture and dynamics.

cell biology↗

bioRxiv2025

A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

cell biology↗

bioRxiv2023

A NanoDam toolkit for tissue-specific transcription factor profiling in C. elegans

During development of multicellular organisms, cells must execute precise molecular decisions to achieve cell fate specification and differentiation. These decisions are orchestrated by networks of transcription factors (TFs) which act to regulate gene expression of specific cohorts of genes to ultimately confer identity. Depending on the cellular context, TF expression can vary dramatically both spatially and temporally. These differences in expression patterns can result in tissue-specific differences in TF binding to downstream targets. To identify targets on a tissue-specific basis, Targeted DamID (TaDa) has been recently introduced to generate TF binding profiles in various models including C. elegans. However, TaDa suffers from portability such that a new promoter-TF fusion transgene must be constructed for every new experimental condition of interest. Here, we adapt NanoDam for usage in C. elegans, which relies on endogenous TF-GFP knock-ins, a plethora of which have already been generated by the community. We report that NanoDam single copy transgenes consisting of lowly expressed, tissue-specific GFP nanobody-Dam fusions, when combined with endogenous GFP-tagged alleles of TFs, results in robust, tissue-specific profiling. Using an endogenous GFP-tagged allele of EGL-43/EVI1, we performed NanoDam profiling of two disparate tissue types, the anchor cell (AC) and dopaminergic neurons, and identify targets unique to each and shared by both cell types. We also identify two GATA TFs, ELT-6 and EGL-18, as novel regulators of AC invasion. Together, we demonstrate that NanoDam is capable of profiling endogenous GFP-tagged TFs to identify novel downstream targets in specific cell types of C. elegans.

genetics↗

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

Breaking the C. elegans invasion/proliferation dichotomy

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state, initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators--including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 ({beta}-TrCP)--resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.

developmental 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

An expandable FLP-ON::TIR1 system for precise spatiotemporal protein degradation in C. elegans

The auxin-inducible degradation system has been widely adopted in the C. elegans research community for its ability to empirically control the spatiotemporal expression of target proteins. This system can efficiently degrade auxin-inducible degron (AID)-tagged proteins via the expression of a ligand-activatable AtTIR1 protein derived from A. thaliana that adapts target proteins to the endogenous C. elegans proteosome. While broad expression of AtTIR1 using strong, ubiquitous promoters can lead to rapid degradation of AID-tagged proteins, cell type-specific expression of AtTIR1 using spatially restricted promoters often results in less efficient target protein degradation. To circumvent this limitation, we have developed a FLP/FRT3-based system that functions to reanimate a dormant, high-powered promoter that can drive sufficient AtTIR1expression in a cell type-specific manner. We benchmark the utility of this system by generating a number of tissue specific FLP-ON::TIR1 drivers to reveal genetically separable cell type-specific phenotypes for several target proteins. We also demonstrate that the FLP-ON::TIR1 system is compatible with enhanced degron epitopes. Finally, we provide an expandable toolkit utilizing the basic FLP-ON::TIR1 system that can be adapted to drive optimized AtTIR1expression in any tissue or cell type of interest.

developmental biology↗

bioRxiv2022

A reevaluation of the relationship between EGL-43 (EVI1/MECOM) and LIN-12 (Notch) during C. elegans anchor cell invasion

Development of the C. elegans reproductive tract is orchestrated by the anchor cell (AC). Among other things, this occurs through a cell invasion event that connects the uterine and vulval tissue. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. EGL-43 is the C. elegans homolog of the human EVI1/MECOM proto-oncogene, and recently, a mechanistic connection has been made between its loss and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling is mitogenic in the absence of EGL-43. To reevaluate the relationship between EGL-43 and LIN-12, we designed and implemented a heterologous co-expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating the AIDHB approach using AID-tagged GFP, we sought to test this approach using AID-tagged alleles of egl-43 and lin-12. Auxin-inducible degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively. This combined approach revealed that LIN-12 is not required for AC proliferation following loss of EGL-43, which contrasts with a double RNAi experiment directed against these same targets. The addition of AIDHB to the C. elegans transgenic toolkit should facilitate functional in vivo imaging of cell-cycle associated phenomena.

developmental 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↗

bioRxiv2022

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function

SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are a family of chromatin remodellers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here we identify an alanine to valine causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in C. elegans nematodes harbouring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating this specific subset of genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 might be viable therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits.

developmental biology↗

bioRxiv2022

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.

developmental biology↗

bioRxiv2022

The Caenorhabditis elegans centrosome is surrounded by a membrane reticulum, the centriculum, that affects centrosome size and function

Centrosomes are membraneless organelles that nucleate microtubules. At their core is a pair of centrioles that recruit pericentriolar material (PCM), a phase-separated condensate. In many cell types, including human cells, centrosomes are surrounded by endoplasmic reticulum-derived membranes of unknown structure and function. Using volume electron microscopy, we show that the C. elegans centrosome is surrounded by a membrane reticulum that we call the centriculum, for centrosome-associated membrane reticulum. Increasing centriculum size by genetic means led to expansion of the PCM and increased microtubule nucleation capacity, an unexpected finding given that the PCM is a membraneless condensate. We provide evidence that the centriculum serves as a microtubule "filter" by limiting the number of microtubules that can elongate fully. We also show the centriculum fuses with the nuclear envelope during mitosis. We propose that this fusion contributes to nuclear envelope breakdown by transducing forces from the elongating spindle to the nuclear membranes.

cell biology↗

bioRxiv2021

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex (SKR-1/2-CUL-1-F-box (SCF)), targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin-derivative/mutant AtTIR1 pair (C. elegans AID version 2 (C.e.AIDv2)) that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID targets, addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expands the utility of the AID system and broadens the number of proteins that can be effectively targeted with it. ARITCLE SUMMARYImplementation of the auxin induced degradation (AID) system has increased the power if the C. elegans model through its ability to rapidly degrade target proteins in the presence of the plant hormone auxin (IAA). The current C.e.AID system is limited in that a substantial level of target degradation occurs in the absence of ligand and full levels of target protein degradation require high levels of auxin inducer. In this manuscript, we modify the AID system to solve these problems.

molecular biology↗

bioRxiv2021

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis. SUMMARY STATEMENTCellular invasion through the basement membrane by the C. elegans anchor cell requires both BAF and PBAF SWI/SNF assemblies to arrest the cell cycle and promote the expression of pro-invasive genes.

developmental biology↗

bioRxiv2020

A laboratory module that explores RNA interference and codon optimization through fluorescence microscopy using Caenorhabditis elegans

Scientific research experiences are beneficial to students allowing them to gain laboratory and problem-solving skills, as well as foundational research skills in a team-based setting. We designed a laboratory module to provide a guided research experience to stimulate curiosity, introduce students to experimental techniques, and provide students with foundational skills needed for higher levels of guided inquiry. In this laboratory module, students learn about RNA interference (RNAi) and codon optimization using the research organism Caenorhabditis elegans (C. elegans). Students are given the opportunity to perform a commonly used method of gene downregulation in C. elegans where they visualize gene depletion using fluorescence microscopy and quantify the efficacy of depletion using quantitative image analysis. The module presented here educates students on how to report their results and findings by generating publication quality figures and figure legends. The activities outlined exemplify ways by which students can improve their critical thinking, data interpretation, and technical skills, all of which are beneficial for future laboratory classes, independent inquiry-based research projects, and careers in the life sciences and beyond.

scientific communication and education↗

bioRxiv2020

Expanding the Caenorhabditis elegans auxin-inducible degron system toolkit with internal expression and degradation controls and improved modular constructs for CRISPR/Cas9-mediated genome editing

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell-types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying an equimolar co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. We have also constructed a set of plasmids to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These templates can be produced through frequently used cloning systems (Gibson assembly or SapTrap) or through ribonucleoprotein complex-mediated insertion of PCR-derived, linear repair templates. We have generated a set of sgRNA plasmids carrying modifications shown to boost editing efficiency, targeting standardized transgene insertion sites on chromosomes I and II. Together these reagents should complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID* tagging of factors of interest. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for facile assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

genetics↗

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↗