Photoconvertible Fluorescent Protein Research Articles

Kinetic isotope effect reveals rate-limiting step in green-to-red photoconvertible fluorescent proteins.

Photoconvertible fluorescent proteins (pcFPs) undergo a slow photochemical transformation when irradiated with blue light. Since their emission is shifted from green to red, pcFPs serve as convenient fusion tags in several cutting-edge biological imaging technologies. Here, a pcFP termed the Least Evolved Ancestor (LEA) was used as a model system to determine the rate-limiting step of photoconversion. Perdeuterated histidine residues were introduced by isotopic enrichment and chromophore content was monitored by absorbance. pH-dependent photoconversion experiments were carried out by exposure to 405-nm light followed by dark equilibration. The loss of green chromophore correlated well with the rise of red, and maximum photoconversion rates were observed at pH 6.5 (0.059 ± 0.001 min-1 for red color acquisition). The loss of green and the rise of red provided deuterium kinetic isotope effects (DKIEs) that were identical within error, 2.9 ± 0.9 and 3.8 ± 0.6, respectively. These data indicate that there is one rate-determining step in the light reactions of photoconversion, and that CH bond cleavage occurs in the transition state of this step. We propose that these reactions are rate-limited on the min time scale by the abstraction of a proton at the His62 beta-carbon. A conformational intermediate such as a twisted or isomerized chromophore is proposed to slowly equilibrate in the dark to generate the red form. Additionally, His62 may shuttle protons to activate Glu211 to serve as a general base, while also facilitating beta-elimination. This idea is supported by a recent X-ray structure of methylated His62.

Damage-repair events increase the instability of cortical microtubules in Arabidopsis.

Microtubules rely on dynamic assembly and disassembly for their functions. Increasing evidences support that the damage-repair of microtubule lattices can affect microtubule dynamics in vitro and in animal cells. Here we successfully established a way for visualizing damage-repair sites on microtubule lattices in plant cells, via labeling the tubulin proteins with the photoconvertible fluorescent protein mEOS3.2. We observed that the crossovers of the microtubule lattice were more prone to be damaged and repaired, with the frequency of damage-repair events positively correlated with the crossing angle between microtubules. The microtubules with damage-repair events displayed shorter lifespans and significantly increased severing frequency compared with the undamaged microtubules. These observations suggested that the damage-repair events promoted instability of cortical microtubules in plant cells.

The role of the correlated motion(s) of the chromophore in photoswitching of green and red forms of the photoconvertible fluorescent protein mSAASoti

Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.

Abstract 274: Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells

Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment, playing various roles through interactions with cancer cells and other stromal cell types. In a 3D co-culture system of CAFs and lung cancer cells, a specific subgroup of CAFs exhibited increased motility, while another subgroup of CAFs remained stationary. Using the photoconvertible fluorescent protein Dendra2, we isolated these distinct CAF subgroups and conducted RNA sequencing. Gene set enrichment and gene ontology analysis revealed that upregulated genes in motile CAFs are associated with hypoxia and glycolysis. Conditioned medium from mesenchymal-like cancer cells enhanced the expression of genes related to hypoxia and glycolysis in CAFs. Among these genes, Aldoa and Ldha were chosen for knockdown in CAFs, resulting in a reduction in CAF motility. We confirmed that mesenchymal-like cancer cells promoted CAF motility and intensified hypoxic conditions within the co-culture system, which was monitored using a hypoxia reporter. Ceruloplasmin (CP) was identified as a secretory protein from mesenchymal-like cancer cells through mass spectrometry. Knockdown of CP led to the inhibition of CAF motility. In conclusion, the motility of CAFs is enhanced under hypoxic conditions facilitated by the secretion of CP from mesenchymal-like cancer cells in the lung tumor microenvironment. Citation Format: Jihye Park, Sieun Lee, Jonathan M. Kurie, Young-Ho Ahn. Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 274.

Using ER-Targeted Photoconvertible Fluorescent Proteins in Living Plant Cells.

Photoconvertible fluorescent proteins (pcFPs) enable differential coloring of a single organelle. Several pcFP-based probes have been targeted to the endoplasmic reticulum (ER) and can serve as useful tools to study ER dynamics and interactions with other organelles. Here, we describe the procedure to conduct live-cell imaging experiments using ER-targeted pcFP-based probes. Potential problems that might occur during the experiments, their solutions, and several ways to improve the experiments are discussed.

Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties.

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62, altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

Capturing excited-state structural snapshots of evolutionary green-to-red photochromic fluorescent proteins.

Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies. A 3-methyl-histidine chromophore derivative, incorporated through amber suppression using orthogonal aminoacyl tRNA synthetase/tRNA pairs, displays more dynamic photoswitching but greatly reduced photoconversion versus the least-evolved ancestor (LEA). Excitation-dependent measurements of the green anionic chromophore reveal that the varying photoswitching efficiencies arise from both the initial transient dynamics of the bright cis state and the final trans-like photoswitched off state, with an exocyclic bridge H-rocking motion playing an active role during the excited-state energy dissipation. This investigation establishes a close-knit feedback loop between spectroscopic characterization and protein engineering, which may be especially beneficial to develop more versatile FPs with targeted mutations and enhanced functionalities, such as photoconvertible FPs that also feature photoswitching properties.

Primed conversion: The emerging player of precise and nontoxic photoconversion.

In 2015, we reported primed conversion, a novel way to convert green-to-red photoconvertible fluorescent proteins, which emerges as a powerful tool for precision optical imaging. Primed conversion uses the intercept of blue and red-to-far-red light instead of traditional violet or near-UV light illumination which offers a series of advantages. Here, we review the fundamental principles and applications of primed conversion with a focus on its use in single-cell labelling and lineage tracing. We provide a historical perspective of lineage tracing techniques, thereby covering basic principles of fluorescence, photoconvertible fluorescent proteins, and eventually primed conversion. We then present the molecular requirements for primed conversion to take place and showcase how it can be used for dual-colour high-fidelity lineage tracing. Further, we discuss potential future developments of the primed conversion imaging toolkit that can benefit the study of both development and disease progression.

A live-cell platform to isolate phenotypically defined subpopulations for spatial multi-omic profiling.

Numerous techniques have been employed to deconstruct the heterogeneity observed in normal and diseased cellular populations, including single cell RNA sequencing, in situ hybridization, and flow cytometry. While these approaches have revolutionized our understanding of heterogeneity, in isolation they cannot correlate phenotypic information within a physiologically relevant live-cell state with molecular profiles. This inability to integrate a live-cell phenotype-such as invasiveness, cell:cell interactions, and changes in spatial positioning-with multi-omic data creates a gap in understanding cellular heterogeneity. We sought to address this gap by employing lab technologies to design a detailed protocol, termed Spatiotemporal Genomic and Cellular Analysis (SaGA), for the precise imaging-based selection, isolation, and expansion of phenotypically distinct live cells. This protocol requires cells expressing a photoconvertible fluorescent protein and employs live cell confocal microscopy to photoconvert a user-defined single cell or set of cells displaying a phenotype of interest. The total population is then extracted from its microenvironment, and the optically highlighted cells are isolated using fluorescence activated cell sorting. SaGA-isolated cells can then be subjected to multi-omics analysis or cellular propagation for in vitro or in vivo studies. This protocol can be applied to a variety of conditions, creating protocol flexibility for user-specific research interests. The SaGA technique can be accomplished in one workday by non-specialists and results in a phenotypically defined cellular subpopulations for integration with multi-omics techniques. We envision this approach providing multi-dimensional datasets exploring the relationship between live cell phenotypes and multi-omic heterogeneity within normal and diseased cellular populations.

Characterization of recombinant photoconverting green fluorescent Akanes.

Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.

Nodal flow transfers polycystin to determine mouse left-right asymmetry

Left-dominant [Ca2+]i elevation on the left margin of the ventral node furnishes the initial laterality of mouse embryos. It depends on extracellular leftward fluid flow (nodal flow), fibroblast growth factor receptor (FGFR)/sonic hedgehog (Shh) signaling, and the PKD1L1 polycystin subunit, of which interrelationship is still elusive. Here, we show that leftward nodal flow directs PKD1L1-containing fibrous strands and facilitates Nodal-mediated [Ca2+]i elevation on the left margin. We generate KikGR-PKD1L1 knockin mice in order to monitor protein dynamics with a photoconvertible fluorescence protein tag. By imaging those embryos, we have identified fragile meshwork being gradually transferred leftward involving pleiomorphic extracellular events. A portion of the meshwork finally bridges over the left nodal crown cells in an FGFR/Shh-dependent manner. As PKD1L1 N-term is predominantly associated with Nodal on the left margin and that PKD1L1/PKD2 overexpression significantly augments cellular Nodal sensitivity, we propose that leftward transfer of polycystin-containing fibrous strands determines left-right asymmetry in developing embryos.

1749-P: Influence of the Biomechanical Environment on Islet Function and Maturity

It is well-established that cells respond to mechanical cues via cytoskeletal remodeling. However, it is not known whether the mechanical environment influences β-cell maturity and function. To examine how mechanical cues influence the β-cell, we used biocompatible substrates with tunable mechanical properties synthesized with elastic moduli ranging from ~3kPa-33 kPa. The surface was coated with extracellular matrix and MIN6 β-like cells were seeded. To independently examine a link between β-cell function and maturity, we examined islets from mice in which β-cells express CaMPARI, a photoconvertible fluorescent protein that in the presence of high Ca2+ activity changes from green to red. For all conditions in MIN6 cells and CAMPARI-expressing islets, we imaged Ca2+ dynamics via Fluo4 at low (2mM) and high glucose (11, 20mM). qPCR was conducted on MIN6 cells (6, 22 kPa) and flow-sorted CaMPARI-expressing β-cells (green, red) to examine expression of genes linked to β-cell function and maturity. With increased substrate stiffness, MIN6 cell Ca2+ oscillations were more robust, including a significant increase in duty cycle (p<0.01) and were more coordinated, consistent with increased excitability. With increasing stiffness, there was significantly decreased expression of Sur1, Kir6.2, Ins2, Pdx1, Fltp, and Ecad (p<0.01). Thus, the dynamics and coordination of Ca2+ activity is mechanoresponsive and may result from altered β-cell maturity. In CaMPARI islets, Ca2+ oscillations were more robust and coordinated for photo-converted red cells which marks cells with higher Ca2+, compared to green cells. In these red cells that show higher Ca2+, qPCR showed significantly increased expression of cFos, but decreased expression of Pdx1 and Ins2, again showing more excitable cells showed altered maturity. In conclusion, our results suggest there may be an inverse relationship between β-cell maturity and function. Further, that the maturity and function of β-cells is responsive to the mechanical environment. Disclosure K.Vazquez: None. R.K.Benninger: None. Funding National Institutes of Health (R01DK102950, R01DK106412)

Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell–cell interactions

Comprehensive screenings to clarify indirect cell–cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells’ effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell–cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell–cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell–cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell–cell interactions by revealing live phenotype-inducible cells, and it could be a platform for discovering new targets of drugs for conventional chemotherapies.

Recent advances inin-resin correlative light andelectron microscopy ofEpon-embedded cells.

Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.

Photophysics of the red-form Kaede chromophore.

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.

Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts

Emission fluorescence is one of the most versatile and powerful biophysical techniques used in several scientific subjects. It is extensively applied in the studies of proteins, their conformations, and intermolecular contacts, such as in protein-ligand and protein-protein interactions, allowing qualitative, quantitative, and structural data elucidation. This review, aimed to outline some of the most widely used fluorescence techniques in this area, illustrate their applications and display a few examples. At first, the data on the intrinsic fluorescence of proteins is disclosed, mainly on the tryptophan side chain. Predominantly, research to study protein conformational changes, protein interactions, and changes in intensities and shifts of the fluorescence emission maximums were discussed. Fluorescence anisotropy or fluorescence polarization is a measurement of the changing orientation of a molecule in space, concerning the time between the absorption and emission events. Absorption and emission indicate the spatial alignment of the molecule's dipoles relative to the electric vector of the electromagnetic wave of excitation and emitted light, respectively. In other words, if the fluorophore population is excited with vertically polarized light, the emitted light will retain some polarization based on how fast it rotates in solution. Therefore, fluorescence anisotropy can be successfully used in protein-protein interaction investigations. Then, green fluorescent proteins (GFPs), photo-transformable fluorescent proteins (FPs) such as photoswitchable and photoconvertible FPs, and those with Large Stokes Shift (LSS) are disclosed in more detail. FPs are potent tools for the study of biological systems. Their versatility and wide range of colours and properties allow many applications. Finally, the application of fluorescence in life sciences is exposed, especially the application of FPs in fluorescence microscopy techniques with super-resolution that enables precise in vivo photolabeling to monitor the movement and interactions of target proteins.

In Vivo Tracking of Dendritic Cell Migration.

Dendritic cells (DCs) in peripheral tissue serve as a sentinel to invasion and maintain tolerance. They ingest and carry antigens to the draining lymph nodes and present antigens to antigen-specific T cells to initiate acquired immune responses. Thus, understanding DC migration from peripheral tissues and function is critical for understanding DCs' roles in immune homeostasis. Here, we introduced the KikGR in vivo photolabeling system, an ideal tool for monitoring precise cellular movements and related functions in vivo under physiological conditions and during various immune responses that occur in pathologic condition. Using a mouse line expressing photoconvertible fluorescent protein KikGR, we can label DCs in peripheral tissues by changing the color of KikGR from green to red after exposure to violet light and accurately track DC migration from each peripheral tissue to its respective draining lymph nodes.

To twist or not to twist: From chromophore structure to dynamics inside engineered photoconvertible and photoswitchable fluorescent proteins.

Green-to-red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super-resolution imaging. A unique Kaede-type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady-state and time-resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green-to-red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA-A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π-π stacking interaction between the chromophore phenolate (P-)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60-67 ps are attributed to P-ring twist as the onset for photoswitching in LEA (major) and LEA-A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side-chain twist in ALL-GFP/ALL-Q62H, indicative of the light-induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA-A69T capable of positive photoswitching was revealed by time-resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green-to-red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances.

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.