Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002440 ◽

1980 ◽

Vol 38 ◽

pp. 552-553

Author(s):

K.I. Pagh ◽

M.R. Adelman

Keyword(s):

Cell Shape ◽

Physarum Polycephalum ◽

Slime Mold ◽

Live Cells ◽

Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

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A Photoactivable Formaldehyde Donor with Fluorescence Monitoring Reveals Threshold to Arrest Cell Migration

10.26434/chemrxiv.11388321.v1 ◽

2019 ◽

Author(s):

Lukas P Smaga ◽

Nicholas W Pino ◽

Gabriela E Ibarra ◽

Vishnu Krishnamurthy ◽

Jefferson Chan

Keyword(s):

Wound Healing ◽

Cell Migration ◽

Fluorescence Enhancement ◽

Biological Effects ◽

Cellular Systems ◽

Live Cells ◽

First Report ◽

Cell Lysate ◽

Intracellular Release ◽

Biological Analytes

Controlled light-mediated delivery of biological analytes enables the investigation of highly reactivity molecules within cellular systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.

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α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

10.26434/chemrxiv.12078582.v2 ◽

2020 ◽

Author(s):

Lei Wang ◽

Louis Riel ◽

Bekim Bajrami ◽

Bin Deng ◽

Amy Howell ◽

...

Keyword(s):

Mass Spectra ◽

Live Cell ◽

Live Cells ◽

The Novel ◽

Proteomics Analysis ◽

Chemical Proteomics ◽

Tandem Mass Spectra ◽

Modified Peptides ◽

Covalent Probes ◽

Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412.v1 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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