Dynamic Cellular Events Research Articles

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters.

Hyperspectral imaging (HSI) technologies have enabled a range of experimental techniques and studies in the fluorescence microscopy field. Unfortunately, a drawback of many HSI microscope platforms is increased acquisition time required to collect images across many spectral bands, as well as signal loss due to the need to filter or disperse emitted fluorescence into many discrete bands. We have previously demonstrated that an alternative approach of scanning the fluorescence excitation spectrum can greatly improve system efficiency by decreasing light losses associated with emission filtering. Our initial system was configured using an array of thin-film tunable filters (TFTFs, VersaChrome, Semrock) mounted in a tiltable filter wheel (VF-5, Sutter) that required ~150-200 ms to switch between wavelengths. Here, we present a new configuration for high-speed switching of TFTFs to allow rapid time-lapse HSI microscopy. A TFTF array was mounted in a custom holder that was attached to a piezoelectric rotation mount (ThorLabs), allowing high-speed rotation. Switching between adjacent filters was achieved using the internal optics of a DG-4 lightsource (Sutter Instrument), including a pair of off-axis parabolic mirrors and galvanometers. Output light was coupled to a liquid lightguide and into an inverted widefield fluorescence microscope (TI-2, Nikon Instruments). Initial tests indicate that the HSI system provides a 15-20 nm bandwidth tunable excitation band and ~10-20 ms wavelength switch time, allowing for high-speed HSI imaging of dynamic cellular events. This work was supported by NIH P01HL066299, R01HL169522, NIH TL1TR003106, and NSF MRI1725937.

Biomarkers of chemotherapy-induced cardiotoxicity: toward precision prevention using extracellular vesicles.

Detrimental side effects of drugs like doxorubicin, which can cause cardiotoxicity, pose barriers for preventing cancer progression, or treating cancer early through molecular interception. Extracellular vesicles (EVs) are valued for their potential as biomarkers of human health, chemical and molecular carcinogenesis, and therapeutics to treat disease at the cellular level. EVs are released both during normal growth and in response to toxicity and cellular death, playing key roles in cellular communication. Consequently, EVs may hold promise as precision biomarkers and therapeutics to prevent or offset damaging off-target effects of chemotherapeutics. EVs have promise as biomarkers of impending cardiotoxicity induced by chemotherapies and as cardioprotective therapeutic agents. However, EVs can also mediate cardiotoxic cues, depending on the identity and past events of their parent cells. Understanding how EVs mediate signaling is critical toward implementing EVs as therapeutic agents to mitigate cardiotoxic effects of chemotherapies. For example, it remains unclear how mixtures of EV populations from cells exposed to toxins or undergoing different stages of cell death contribute to signaling across cardiac tissues. Here, we present our perspective on the outlook of EVs as future clinical tools to mitigate chemotherapy-induced cardiotoxicity, both as biomarkers of impending cardiotoxicity and as cardioprotective agents. Also, we discuss how heterogeneous mixtures of EVs and transient exposures to toxicants may add complexity to predicting outcomes of exogenously applied EVs. Elucidating how EV cargo and signaling properties change during dynamic cellular events may aid precision prevention of cardiotoxicity in anticancer treatments and development of safer chemotherapeutics.

Functionalized Phytochemicals-Embedded Carbon Dots Derived from Medicinal Plant for Bioimaging Application.

Precise visualization of biological processes necessitates reliable coloring technologies, and fluorescence imaging has emerged as a powerful method for capturing dynamic cellular events. Low emission intensity and solubility of intrinsic fluorescence are still challenging, hindering their application in the biomedical field. The nanostructurization and functionalization of the insoluble phytochemicals, such as chlorophyll and curcumin, into carbon dots (CDs) were conducted to address these challenges. Due to their unique fluorescence characteristics and biocompatibility, CDs derived from medicinal plants hold promise as bioimaging agents. Further, the nitrogen in situ functionalization of the as-synthesized CDs offered tunable optical properties and enhanced solubility. The surface modification aims to achieve a more positive zeta potential, facilitating penetration through biological membranes. This work provides valuable insights into utilizing functionalized phytochemical-embedded carbon dots for bioimaging applications. The doping of nitrogen by adding urea showed an alteration of surface charge, which is more positive based on zeta potential measurement. The more positive CD particles showed that Andrographis paniculata-urea-based CDs were the best particles to penetrate cells than others related to the alteration of the surface charge and the functional group of the CDs, with the optimum dose of 12.5 μg/mL for 3 h of treatment for bioimaging assay.

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics.

Successful B cell activation, which is critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodeling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through the AKT and ERK1/2 axes.

Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy.

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies-in this case, the study of the second messenger Ca2+. Time-lapse excitation-scanning HSI data of Ca2+ signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca2+ and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals.

Algorithm for biological second messenger analysis with dynamic regions of interest.

Physiological function is regulated through cellular communication that is facilitated by multiple signaling molecules such as second messengers. Analysis of signal dynamics obtained from cell and tissue imaging is difficult because of intricate spatially and temporally distinct signals. Signal analysis tools based on static region of interest analysis may under- or overestimate signals in relation to region of interest size and location. Therefore, we developed an algorithm for biological signal detection and analysis based on dynamic regions of interest, where time-dependent polygonal regions of interest are automatically assigned to the changing perimeter of detected and segmented signals. This approach allows signal profiles to be rigorously and precisely tracked over time, eliminating the signal distortion observed with static methods. Integration of our approach with state-of-the-art image processing and particle tracking pipelines enabled the isolation of dynamic cellular signaling events and characterization of biological signaling patterns with distinct combinations of parameters including amplitude, duration, and spatial spread. Our algorithm was validated using synthetically generated datasets and compared with other available methods. Application of the algorithm to volumetric time-lapse hyperspectral images of cyclic adenosine monophosphate measurements in rat microvascular endothelial cells revealed distinct signal heterogeneity with respect to cell depth, confirming the utility of our approach for analysis of 5-dimensional data. In human tibial arteries, our approach allowed the identification of distinct calcium signal patterns associated with atherosclerosis. Our algorithm for automated detection and analysis of second messenger signals enables the decoding of signaling patterns in diverse tissues and identification of pathologic cellular responses.

Programmable Aperture Light‐Field Microscopy

AbstractA computational three‐dimensional (3D) microscopy technique, termed programmable aperture light‐field microscopy (PALFM), for motion‐free, high‐resolution volumetric imaging of fluorescent or self‐luminous samples is proposed. The well‐known Fourier slice theorem is extended to incoherent tomographic imaging, which states that a detected image under an ideal aperture corresponds to a central slice in the 3D object spectrum, so the spectrum coverage can be accomplished based on the motion‐free aperture modulation. When further considering frequency extension and coverage, a hybrid aperture modulation scheme is designed consisting of non‐centrosymmetric circular and annular apertures for high‐efficiency, non‐ambiguous depth discrimination. A PALFM system with an easy‐to‐build programmable aperture module attached to an off‐the‐shelf inverted fluorescence microscope is constructed, where annular apertures can manipulate Bessel‐like beams for spectrum modulation. Experimental results on near‐diffraction‐limited imaging of a resolution target across a large depth range and high‐resolution, multi‐color 3D imaging of a mouse kidney section verify the validity and effectiveness of PALFM. High‐speed, long‐term time‐lapse volumetric imaging of HeLa cells in vitro further demonstrates that PALFM is a promising tomographic imaging tool for studying dynamic cellular processes and events without requiring complicated sample rotation or beam scanning.

Molecular recorders to track cellular events.

DNA tapes could be used to record dynamic molecular and cellular events in animals.

Landmark-based retrieval of inflamed skin vessels enabled by 3D correlative intravital light and volume electron microscopy

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM).

The Hippo pathway drives the cellular response to hydrostatic pressure

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context‐dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well‐understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label‐free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co‐transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin‐dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD‐mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

Vangl as a Master Scaffold for Wnt/Planar Cell Polarity Signaling in Development and Disease.

The establishment of polarity within tissues and dynamic cellular morphogenetic events are features common to both developing and adult tissues, and breakdown of these programs is associated with diverse human diseases. Wnt/Planar cell polarity (Wnt/PCP) signaling, a branch of non-canonical Wnt signaling, is critical to the establishment and maintenance of polarity in epithelial tissues as well as cell motility events critical to proper embryonic development. In epithelial tissues, Wnt/PCP-mediated planar polarity relies upon the asymmetric distribution of core proteins to establish polarity, but the requirement for this distribution in Wnt/PCP-mediated cell motility remains unclear. However, in both polarized tissues and migratory cells, the Wnt/PCP-specific transmembrane protein Vangl is required and appears to serve as a scaffold upon which the core pathway components as well as positive and negative regulators of Wnt/PCP signaling assemble. The current literature suggests that the multiple interaction domains of Vangl allow for the binding of diverse signaling partners for the establishment of context- and tissue-specific complexes. In this review we discuss the role of Vangl as a master scaffold for Wnt/PCP signaling in epithelial tissue polarity and cellular motility events in developing and adult tissues, and address how these programs are dysregulated in human disease.

Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals.

Polyubiquitination signal deliver diverse cellular signal, which have been recognized as a sophisticated ubiquitin code. The perception and transduction of ubiquitination signal depend on the specificity and sensitivity of the ubiquitin-binding domain. Accurate and sensitive detection of polyubiquitination signal is crucial for revealing the dynamic cellular ubiquitin-regulated events. Western blotting (WB) and immunohistochemistry (IHC) are the most widely used biochemical strategies to detect ubiquitination signal on substrates under diverse physiological and pathological conditions. However, anti-ubiquitin antibodies fail to reflect polyubiquitination signal unbiasedly because of their strong preference for K63-linked ubiquitin chains. Herein, we demonstrated that our previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (ThUBD-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection. This advanced method was named TUF-WB Plus (TUF-WB+). The TUF-WB+ method significantly increases the sensitivity and accuracy of ubiquitin detection and requires a shorter experimental operation time. Furthermore, it enables the ThUBD-HRP probe to function as a powerful tool for spatial in situ polyubiquitination detection in cells by immunohistochemistry. Our newly developed ThUBD-HRP probe and TUF-WB+ method provide a robust and powerful tool for ubiquitination signal detection with hypersensitivity in an unbiased manner.

Invivo dynamic 3D imaging of oocytes and embryos in the mouse oviduct.

SUMMARYDevelopmental biologists have always relied on imaging to shed light on dynamic cellular events. However, processes such as mammalian fertilization and embryogenesis are generally inaccessible for direct imaging. In consequence, how the oviduct (fallopian tube) facilitates the transport of gametes and preimplantation embryos continues to be unanswered. Here we present a combination of intravital window and optical coherence tomography for dynamic, volumetric, in vivo imaging of oocytes and embryos as they are transported through the mouse oviduct. We observed location-dependent circling, oscillating, and long-distance bi-directional movements of oocytes and embryos that suggest regulatory mechanisms driving transport and question established views in the field. This in vivo imaging approach can be combined with a variety of genetic and pharmacological manipulations for live functional analysis, bringing the potential to investigate reproductive physiology in its native state.

Watching the embryo: Evolution of the microscope for the study of embryogenesis.

Embryos and microscopes share a long, remarkable history and biologists have always been intrigued to watch how embryos develop under the microscope. Here we discuss the advances in microscopy which have greatly influenced our current understanding of embryogenesis. We highlight the evolution of microscopes and the optical technologies that have been instrumental in studying various developmental processes. These imaging modalities provide mechanistic insights into the dynamic cellular and molecular events which drive lineage commitment and morphogenetic changes in the developing embryo. We begin the journey with a brief history of microscopy to study embryos. First, we review the principles and optics of light, fluorescence, confocal, and electron microscopy which have been key techniques for imaging cellular and molecular events during embryonic development. Next, we discuss recent key imaging modalities such as light-sheet microscopy, which are suitable for whole embryo imaging. Further, we highlight imaging techniques like multiphoton and super resolution microscopy for beyond light diffraction limit, high resolution imaging. Lastly, we review some of the scattering-based imaging methods and techniques used for imaging human embryos.

Cell-ECM contact-guided intracellular polarization is mediated via lamin A/C dependent nucleus-cytoskeletal connection

Cell polarization plays a crucial role in dynamic cellular events, such as cell proliferation, differentiation, and directional migration in response to diverse extracellular and intracellular signals. Although it is well known that cell polarization entails highly orchestrated intracellular molecular reorganization, the underlying mechanism of repositioning by intracellular organelles in the presence of multiple stimuli is still unclear. Here, we show that front-rear cell polarization based on the relative positions of nucleus and microtubule organizing center is precisely controlled by mechanical interactions including cellular adhesion to extracellular matrix and nucleus-cytoskeletal connections. By modulating the size and distribution of fibronectin-coated adhesive spots located in the polarized cell shape mimicking micropatterns, we monitored the alterations in cell polarity. We found that the localization of individual adhesive spots is more dominant than the cell shape itself to induce intracellular polarization. Further, the degree of cell polarization was diminished significantly by disrupting nuclear lamin A/C. We further confirm that geometrical cue-guided intracellular polarization determines directional cell migration via local activation of Cdc42. These findings provide novel insights into the role of nucleus-cytoskeletal connections in single cell polarization under a combination of physical, molecular, and genetic cues, where lamin A/C acts as a critical molecular mediator in ECM sensing and signal transduction via nucleus-cytoskeletal connection.

Multiscale In Vivo Imaging of Collective Cell Migration in Drosophila Embryos.

Coordinated cell movements drive embryonic development and tissue repair, and can also spread disease. Time-lapse microscopy is an integral part in the study of the cell biology of collective cell movements. Advances in imaging techniques enable monitoring dynamic cellular and molecular events in real time within living animals. Here, we demonstrate the use of spinning disk confocal microscopy to investigate coordinated cell movements and epithelial-to-mesenchymal-like transitions during embryonic wound closure in Drosophila. We describe image-based metrics to quantify the efficiency of collective cell migration. Finally, we show the application of super-resolution radial fluctuation microscopy to obtain multidimensional, super-resolution images of protrusive activity in collectively moving cells in vivo. Together, the methods presented here constitute a toolkit for the modern analysis of collective cell migration in living animals.

A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells.

Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazocoumarin compounds that can be photoactivated by visible (430-490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo an insertion reaction with concomitant fluorescence "turned on". With these new molecules in hand, we have developed a new approach for rapid, selective, and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia-induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with "click chemistries". We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photoirradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.

Endothelial Cell Dynamics in Vascular Development: Insights From Live-Imaging in Zebrafish.

The formation of the vertebrate vasculature involves the acquisition of endothelial cell identities, sprouting, migration, remodeling and maturation of functional vessel networks. To understand the cellular and molecular processes that drive vascular development, live-imaging of dynamic cellular events in the zebrafish embryo have proven highly informative. This review focusses on recent advances, new tools and new insights from imaging studies in vascular cell biology using zebrafish as a model system.

Photo‐SNAP‐Tag, a Light‐Regulated SNAP‐Tag Chemical Labeling System

Methods that allow labelling and tracking of proteins during dynamic cellular events have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self‐labeling chemical tagging systems such as SNAP‐Tag or Halo‐Tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest though a small fusion tag. In this work, we sought to improve the versatility of self‐labeling chemical‐tagging systems by regulating their activity with light. We used light‐inducible dimerizers to reconstitute a split SNAP‐Tag (modified human O6‐alkylguanine‐DNA‐alkyltransferase, hAGT) protein, allowing tight light‐dependent control of chemical labeling. In addition, we generated a split SNAP‐Tag version that can efficiently self‐assemble, showing high labeling efficacy. We envision these tools will extend the versatility and utility of the SNAP‐Tag chemical system for protein labeling applications.