scholarly journals Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

2018 ◽  
Author(s):  
H M York ◽  
A Patil ◽  
U K Moorthi ◽  
A Kaur ◽  
A Bhowmik ◽  
...  
Keyword(s):  
Signal Processing ◽  
Cell Imaging ◽  
Light Sheet ◽  
Endosomal Trafficking ◽  
Egf Receptors ◽  
Whole Cell ◽  
Cellular Processes ◽  
Light Sheet Microscopy ◽  
Signaling Receptors ◽  
Cell Surface Signaling

ABSTRACTMulticellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

scholarly journals Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
H. M. York ◽  
A. Patil ◽  
U. K. Moorthi ◽  
A. Kaur ◽  
A. Bhowmik ◽  
...  
Keyword(s):  
Signal Processing ◽  
Growth Factor ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Endosomal Trafficking ◽  
Egf Receptors ◽  
Ph Domain ◽  
Cell Level ◽  
Whole Cell ◽  
Light Sheet Microscopy

AbstractThe endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

scholarly journals Deformation Measurements of Neuronal Excitability Using Incoherent Holography Lattice Light-Sheet Microscopy (IHLLS)

Photonics ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 383
Author(s):  
Mariana Potcoava ◽  
Jonathan Art ◽  
Simon Alford ◽  
Christopher Mann
Keyword(s):  
Cell Membrane ◽  
Structural Changes ◽  
Neuronal Excitability ◽  
Light Sheet ◽  
Phase Imaging ◽  
Excitable Cells ◽  
Full Field ◽  
Cellular Processes ◽  
Light Sheet Microscopy ◽  
Fluorescent Markers

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective.

scholarly journals Digital Spindle: A New Way to Explore Mitotic Functions by Whole Cell Data Collection and a Computational Approach

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1255
Author(s):  
Norio Yamashita ◽  
Masahiko Morita ◽  
Hideo Yokota ◽  
Yuko Mimori-Kiyosue
Keyword(s):  
Cell Biology ◽  
Information Science ◽  
Three Dimensional ◽  
Light Sheet ◽  
Live Imaging ◽  
Three Dimensions ◽  
Complex Data ◽  
Spatiotemporal Resolution ◽  
Whole Cell ◽  
Light Sheet Microscopy

From cells to organisms, every living system is three-dimensional (3D), but the performance of fluorescence microscopy has been largely limited when attempting to obtain an overview of systems’ dynamic processes in three dimensions. Recently, advanced light-sheet illumination technologies, allowing drastic improvement in spatial discrimination, volumetric imaging times, and phototoxicity/photobleaching, have been making live imaging to collect precise and reliable 3D information increasingly feasible. In particular, lattice light-sheet microscopy (LLSM), using an ultrathin light-sheet, enables whole-cell 3D live imaging of cellular processes, including mitosis, at unprecedented spatiotemporal resolution for extended periods of time. This technology produces immense and complex data, including a significant amount of information, raising new challenges for big image data analysis and new possibilities for data utilization. Once the data are digitally archived in a computer, the data can be reused for various purposes by anyone at any time. Such an information science approach has the potential to revolutionize the use of bioimage data, and provides an alternative method for cell biology research in a data-driven manner. In this article, we introduce examples of analyzing digital mitotic spindles and discuss future perspectives in cell biology.

scholarly journals Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

2016 ◽  
Vol 27 (22) ◽  
pp. 3418-3435 ◽  
Author(s):  
François Aguet ◽  
Srigokul Upadhyayula ◽  
Raphaël Gaudin ◽  
Yi-ying Chou ◽  
Emanuele Cocucci ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Imaging ◽  
Single Cells ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Bottom Surface ◽  
Light Sheet Microscopy ◽  
Dividing Cells ◽  
Entire Cell ◽  
Membrane Bound

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

scholarly journals Reflected Beam Light-Sheet Microscopy for Whole-Cell 3D Super-Resolution Imaging

2015 ◽  
Vol 108 (2) ◽  
pp. 35a
Author(s):  
Marjolein B.M. Meddens ◽  
Sheng Liu ◽  
Conrad D. James ◽  
Keith A. Lidke
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Whole Cell ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

scholarly journals The ‘super-resolution’ revolution

2009 ◽  
Vol 37 (5) ◽  
pp. 1042-1044 ◽  
Author(s):  
Ilan Davis
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cell ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Light Sheet Microscopy ◽  
Resolution Imaging ◽  
New Instruments

We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.

scholarly journals Single Objective Light-Sheet Microscopy for High-Speed Whole-Cell 3D Super-Resolution

2017 ◽  
Vol 112 (3) ◽  
pp. 187a ◽  
Author(s):  
Marjolein B.M. Meddens ◽  
Sheng Liu ◽  
Patrick S. Finnegan ◽  
Thayne L. Edwards ◽  
Conrad D. James ◽  
...  
Keyword(s):  
High Speed ◽  
Super Resolution ◽  
Light Sheet ◽  
Whole Cell ◽  
Light Sheet Microscopy ◽  
Single Objective

scholarly journals u-track 3D: measuring and interrogating intracellular dynamics in three dimensions

2020 ◽  
Author(s):  
Philippe Roudot ◽  
Wesley R. Legant ◽  
Qiongjing Zou ◽  
Kevin M. Dean ◽  
Erik S. Welf ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Region Of Interest ◽  
Confidence Score ◽  
Light Sheet ◽  
Three Dimensions ◽  
Dense Particle ◽  
Cellular Processes ◽  
Light Sheet Microscopy ◽  
Large Sets ◽  
Complete Cell

AbstractParticle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes by live microscopy. Light-sheet microscopy has recently opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges with three algorithmic innovations. Building on the established framework of globally optimal particle association in space and time implemented in the u-track package and recent advances in gaining association robustness in the case of erratic motion, we first report a complete and versatile pipeline for particle tracking. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability, which provides for every trajectory a confidence score, thereby overcoming the challenges of visual validation of trajectories in dense particle fields. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.

scholarly journals Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

2016 ◽  
Vol 7 (6) ◽  
pp. 2219 ◽  
Author(s):  
Marjolein B. M. Meddens ◽  
Sheng Liu ◽  
Patrick S. Finnegan ◽  
Thayne L. Edwards ◽  
Conrad D. James ◽  
...  
Keyword(s):  
High Speed ◽  
Super Resolution ◽  
Light Sheet ◽  
Whole Cell ◽  
Light Sheet Microscopy ◽  
Single Objective

scholarly journals Global morphogenetic flow is accurately predicted by the spatial distribution of myosin motors

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sebastian J Streichan ◽  
Matthew F Lefebvre ◽  
Nicholas Noll ◽  
Eric F Wieschaus ◽  
Boris I Shraiman
Keyword(s):  
Light Sheet ◽  
Global Perspective ◽  
Spatial Modulation ◽  
Drosophila Embryo ◽  
Cellular Processes ◽  
Light Sheet Microscopy ◽  
Spatio Temporal ◽  
Global Coordination ◽  
Myosin Motors ◽  
Temporal Flow

During embryogenesis tissue layers undergo morphogenetic flow rearranging and folding into specific shapes. While developmental biology has identified key genes and local cellular processes, global coordination of tissue remodeling at the organ scale remains unclear. Here, we combine in toto light-sheet microscopy of the Drosophila embryo with quantitative analysis and physical modeling to relate cellular flow with the patterns of force generation during the gastrulation process. We find that the complex spatio-temporal flow pattern can be predicted from the measured meso-scale myosin density and anisotropy using a simple, effective viscous model of the tissue, achieving close to 90% accuracy with one time dependent and two constant parameters. Our analysis uncovers the importance of a) spatial modulation of myosin distribution on the scale of the embryo and b) the non-locality of its effect due to mechanical interaction of cells, demonstrating the need for the global perspective in the study of morphogenetic flow.

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