Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Nguyen, Alyson; Battle, Keith; Paudel, Sunita S.; Xu, Ningyong; Bell, Jessica; Ayers, Linn; Chapman, Cassandra M.; Singh, Ajay Pratap; Паланки, Сринивас; Rich, Thomas C.; Alvarez, Diego F.; Stevens, Troy; Tambe, Dhananjay

doi:10.3791/63095

Cited by 3 publications

(1 citation statement)

References 7 publications

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“…Monolayer stress microscopy image analysis was performed using the Analysis and Visualization Module (AnViM) of the Integrative Toolkit to Analyze Cellular Signals (iTACS), as described in 21 . In brief, single wavelength and unmixed images were organized into separate folders corresponding to each label (Cal-590, Big Beads, and Tiny Beads).…”

Section: Msm Image Analysismentioning

confidence: 99%

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics

Leavesley

Johnson²,

Paudel³

et al. 2023

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XXI

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Second messenger signals, e.g., Ca 2+ and cyclic nucleotides, orchestrate a wide range of cellular events. The methods by which second messenger signals determine specific physiological responses are complex. Recent studies point to the importance of temporal and spatial encoding in determining signal specificity. Studies also indicate the importance of mechanical stimuli, substrate stiffness, and mechanical responses -the "mechanosome"in regulating physiology. Hence, approaches that probe both chemical and mechanical signals are needed. Here, we report preliminary efforts to combine hyperspectral imaging for second messenger signal measurements, monolayer stress microscopy for mechanical force measurements, and S8 analysis software for quantifying localized signalsspecifically, Ca 2+ dynamics and mechanical forces in human airway smooth muscle cells (HASMCs). HASMCs were prepared as confluent monolayers on 11 kPa gels with embedded fluorescent microparticles that serve as fiducial markers as well as smaller microparticles to measure deformation (strain). Imaging was performed using a custom excitation-scanning hyperspectral microscope. Hyperspectral images were unmixed to identify signals from cellular fluorescent labels (e.g., CAL 590-AM) and fluorescent microparticles. Images were analyzed to quantify localized force dynamics through monolayer stress microscopy. S8 software was used to identify, track, and quantify spatially-localized Ca 2+ activity. Results indicate that localized and transient cellular signals and forces can be quantified and mapped within cell populations. Importantly, these results establish a method for simultaneous interrogation of cellular signals and mechanical forces that may play synergistic roles in regulating downstream cellular physiology in confluent monolayers. This work was supported by NIH P01HL066299, R01HL137030, R01HL058506, and NSF MRI1725937. Drs. Leavesley and Rich disclose financial interest in a university start-up company, SpectraCyte LLC, to commercialize spectral imaging technologies.

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Section: Msm Image Analysismentioning

confidence: 99%

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics

Leavesley

Johnson²,

Paudel³

et al. 2023

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XXI

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Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals

Johnson,

Annamdevula,

Leavesley

et al. 2024

Biochemical Society Transactions

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Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger known to orchestrate a myriad of cellular functions over a wide range of timescales. In the last 20 years, a variety of single-cell sensors have been developed to measure second messenger signals including cAMP, Ca2+, and the balance of kinase and phosphatase activities. These sensors utilize changes in fluorescence emission of an individual fluorophore or Förster resonance energy transfer (FRET) to detect changes in second messenger concentration. cAMP and kinase activity reporter probes have provided powerful tools for the study of localized signals. Studies relying on these and related probes have the potential to further revolutionize our understanding of G protein-coupled receptor signaling systems. Unfortunately, investigators have not been able to take full advantage of the potential of these probes due to the limited signal-to-noise ratio of the probes and the limited ability of standard epifluorescence and confocal microscope systems to simultaneously measure the distributions of multiple signals (e.g. cAMP, Ca2+, and changes in kinase activities) in real time. In this review, we focus on recently implemented strategies to overcome these limitations: hyperspectral imaging and adaptive thresholding approaches to track dynamic regions of interest (ROI). This combination of approaches increases signal-to-noise ratio and contrast, and allows identification of localized signals throughout cells. These in turn lead to the identification and quantification of intracellular signals with higher effective resolution. Hyperspectral imaging and dynamic ROI tracking approaches offer investigators additional tools with which to visualize and quantify multiplexed intracellular signaling systems.

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Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers

Paudel

Deweever

Sayner

et al. 2022

American Journal of Physiology-Cell Physiology

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The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify motion of the peripheral membrane of cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, stiffness consistent with a healthy pulmonary artery. Unlike these large length scale morphological changes, the baseline of PMFs was largely insensitive to substrate stiffness on which cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel - a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate: (i) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; (ii) PMFs increase in response to calcium influx through store-operated calcium entry channels; and (iii) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

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Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Cited by 3 publications

References 7 publications

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics

Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers

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