Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics

Keil, Wolfgang W.; Kutscher, Lena M; Shaham, Shai; Siggia, Eric D.

doi:10.1016/j.devcel.2016.11.022

Cited by 84 publications

(105 citation statements)

References 48 publications

(73 reference statements)

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“…Nuclei were outlined using the 240 threshold tool in Fiji or for animals with no detectable GFP signal, the corresponding DIC 241 image was utilized to identify the nucleus. Images of L3 larvae were captured in a C. 242 elegans larvae-specific microfluidic device (Keil et al 2017). To quantify AID::GFP 243 degradation, animals were loaded into the microfluidic chamber and fed NA22 bacteria.…”

Section: Image Acquisition 210mentioning

confidence: 99%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

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As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system, allows for spatial and temporal control of protein degradation, functioning through the activity of a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID method, utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 minutes of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 interacts with C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the Ftz-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

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Section: Image Acquisition 210mentioning

confidence: 99%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

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“…While no other microfluidic devices have been published for studying mechanobiology of worm embryos and larvae, other devices for sorting (Cornaglia et al, 2015; Sofela et al, 2018; Yang et al, 2017) and observing (Cornaglia et al, 2015; Hulme et al, 2010; Keil, Kutscher, Shaham, & Siggia, 2017) embryos and larvae have been presented. These devices have the potential to be used for studying mechanobiology if additional features are added to apply mechanical stimuli and observe the resulting information processing (Fig.…”

Section: Microfluidics For Mechanobiology Of Model Organismsmentioning

confidence: 99%

Microfluidics for mechanobiology of model organisms

Kim

Nekimken

Fechner

et al. 2018

Methods in Cell Biology

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Mechanical stimuli play a critical role in organ development, tissue homeostasis, and disease. Understanding how mechanical signals are processed in multicellular model systems is critical for connecting cellular processes to tissue- and organism-level responses. However, progress in the field that studies these phenomena, mechanobiology, has been limited by lack of appropriate experimental techniques for applying repeatable mechanical stimuli to intact organs and model organisms. Microfluidic platforms, a subgroup of microsystems that use liquid flow for manipulation of objects, are a promising tool for studying mechanobiology of small model organisms due to their size scale and ease of customization. In this work, we describe design considerations involved in developing a microfluidic device for studying mechanobiology. Then, focusing on worms, fruit flies, and zebrafish, we review current microfluidic platforms for mechanobiology of multicellular model organisms and their tissues and highlight research opportunities in this developing field.

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“…In particular, a recent device where worms are gently immobilized by flow and a gradual increase in pressure 63 , allows for high temporal resolution of imaging for extended durations. The downsides to microfluidic approaches are the complexity and cost of the systems.…”

Section: Introductionmentioning

confidence: 99%

Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans

et al. 2017

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Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory.

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Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics

Cited by 84 publications

References 48 publications

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Microfluidics for mechanobiology of model organisms

Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans

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