Biology subjects

Goldstein, B.

Publications and source records attributed to Goldstein, B..

25 records · Page 2Linked to original sources

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↗

bioRxiv2021

In vivo analysis of LEA-1 in C. elegans desiccation tolerance

BackgroundCells and organisms typically cannot survive in the absence of water. However, there are some notable exceptions, including animals such as nematodes, tardigrades, rotifers, and some arthropods. One class of proteins known to play a role in desiccation resistance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear. Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection. ResultsTo study the endogenous function of an LEA protein in an animal, we created a true null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutant animals were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress. We identified minimal motifs within C. elegans LEA-1 that are sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1s in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans survived mild desiccation and osmotic stress at the same levels as worms with the full-length protein. ConclusionsOur results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection.

genetics↗

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

Differential Expression Gene Explorer (DrEdGE): A tool for generating interactive online data visualizations for exploration of quantitative transcript abundance datasets

As the scientific community becomes increasingly interested in data sharing, there is a growing need for tools that facilitate the querying of public data. Mining of RNA-seq datasets, for example, has value to many biomedical researchers, yet is often effectively inaccessible to non-genomicist experts, even when the raw data are available. Here we present DrEdGE (dredge.bio.unc.edu), a free Web-based tool that facilitates data sharing between genomicists and their colleagues. The DrEdGE software guides genomicists through easily creating interactive online data visualizations, which colleagues can then explore and query according to their own conditions to discover genes, samples, or patterns of interest. We demonstrate DrEdGEs features with three example websites we generated from publicly available datasets--human neuronal tissue, mouse embryonic tissue, and a C. elegans embryonic series. DrEdGE increases the utility of large genomics datasets by removing the technical obstacles that prevent interested parties from exploring the data independently.

genomics↗

bioRxiv2018

Molecular aspects of multivalent engagement between Syk and FcϵRIγ

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as the tetrameric ({beta}{gamma}2) Fc{varepsilon}RI The simplest model assumes that Syk activation occurs through cis binding of its tandem SH2 domains to dual phosphotyrosines within immunoreceptor tyrosine-based activation motifs of individual {gamma} chains. In this model, Syk activity is modulated by phosphorylation occurring between adjacent Syk molecules docked on {gamma} homodimers and by Lyn molecules bound to Fc{varepsilon}RI{beta}. However, the mechanistic details of Syk docking on {gamma} homodimers are not fully resolved, particularly the possibility of trans binding orientations and the impact of Y130 autophosphorylation within Syk interdomain A. Analytical modeling shows that multivalent interactions lead to increased WT Syk cis-oriented binding by three orders of magnitude. Molecular dynamics (MD) simulations show increased inter-SH2 flexibility in a Y130E phosphomimetic form of Syk, associated with reduced overall helicity of interdomain A. Hybrid MD/worm-like chain polymer models show that the Y130E substitution reduces cis binding of Syk. We report computational models and estimates of relative binding for all possible cis and trans 2:2 Syk:Fc{varepsilon}RI{gamma} complexes. Calcium imaging experiments confirm model predictions that cis binding of WT Syk is strongly preferred for efficient signaling, while trans conformations trigger weak but measurable responses.

biophysics↗

bioRxiv2018

Optogenetic dissection of mitotic spindle positioning in vivo

The position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of G-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through G-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the G regulators RIC-8Ric-8A and RGS-7Loco/RGS3, and replacement of G with a light-inducible membrane anchor demonstrated that G-GTP signaling is dispensable for pulling force generation. In the absence of G, cortical recruitment of GPR-1/2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment of LIN-5 overruled normal cell-cycle and polarity regulation, and provided experimental control over the spindle and cell cleavage plane. Our results define G*GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle positioning forces. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.

cell biology↗

bioRxiv2017

LITE microscopy: a technology for high numerical aperture, low photobleaching fluorescence imaging

Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

cell biology↗