Live-cell Fluorescence Microscopy Research Articles

Local PI(4,5)P2 signaling inhibits fusion pore expansion during exocytosis.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live-cell total internal reflection fluorescence (TIRF) microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. Knockdown of phosphatidylinositol-phosphate-5-kinase-1c or optogenetic recruitment of 5-phosphatase reduces PI(4,5)P2 transients and accelerates fusion pore expansion, suggesting that acute PI(4,5)P2 synthesis is involved. Thus, local phospholipid signaling inhibits fusion pore expansion and peptide release through an unconventional endocytic mechanism.

Uncovering the leukemogenic mechanism of non-canonical NUP98 fusion oncoproteins.

Gene translocations involving NUP98 are associated with diverse pediatric leukemias, including acute myeloid leukemia (AML), with poor patient outcomes. The resulting gene fusions encode fusion oncoproteins (FOs) linking the disordered, phase-separation (PS) prone phenylalanine-glycine (FG) repeats of the NUP98 N-terminus to C-terminal DNA/chromatin-binding domains from >30 transcriptional/epigenetic regulators. NUP98 FOs form biomolecular condensates (puncta) that drive aberrant gene expression and transform hematopoietic stem and progenitor cells (HSPCs), promoting leukemogenesis. Interestingly, the C-terminal of the NUP98-LNP1 FO lacks a known DNA/chromatin-binding domain and is predicted to be disordered, yet this so-called “non-canonical” FO recapitulates the leukemogenic phenotype of other NUP98 FOs in HSPCs and in mice. Live cell fluorescence microscopy imaging showed that NUP98-LNP1 forms nuclear puncta that are larger and more sparce than those formed by other NUP98 FOs. Intriguingly, the parent LNP1 protein displays diffuse localization in cells. Our observations raise questions as to how NUP98-LNP1 alters gene expression and transforms HSPCs. We hypothesize that the C-terminal LNP1 region within the NUP98-LNP1 FO interacts with chromatin through an unknown mechanism, enabling the NUP98 FG-rich region to promote puncta formation and recruitment of the transcriptional machinery to drive aberrant gene expression and cell transformation. To test this hypothesis, I will determine whether NUP98-LNP1 induces the hallmark HOX gene expression program associated with AML in HEK293T cells and HSPCs. Further, I will determine protein interaction partners of NUP98-LNP1 by developing biochemical procedures to isolate NUP98-LNP1-driven puncta. Finally, I will identify genomic sites bound by NUP98-LNP1. Following these studies, I will extend my studies to a second non-canonical NUP98 FO, NUP98-RAP1GDS1, whose folded C-terminal interacts with GTPases. The proposed studies will uncover how non-canonical NUP98 FOs drive aberrant gene expression, expanding our knowledge of the role of PS in leukemogenesis by NUP98 FOs.

Elucidating the liquid structure within biomolecular condensates.

Biomolecular condensates are liquid-like membrane-less organelles where essential biochemistry occurs, including ribosome and spliceosome biogenesis. Many studies posit that to carry out biochemical processes, the condensate preferentially enriches or excludes specific molecules. Unlike gases, condensates are liquid-like phases and thus can have locally correlated structure. Such liquid structure may facilitate long-range conformational changes (i.e., allostery) between biomolecules, spatiotemporally coordinating interactions that direct binding, processing, and folding of biomolecules. Measurement of structure is classically obtained through diffraction-based methods, which is not conducive to condensates in cells. We instead turned to a different approach using live cell fluorescent microscopy to extract the strength driving molecules into condensates, quantified by their partitioning (i.e., free energy of transfer). To accomplish this, we employed a construct with two domains separated by a variable-sized linker. We surmised that its partitioning would depend on the exact length of the linker. For example, if the linker is too short or too long the transfer free energy would weaken as the domains are infrequently capable of “reaching” their preferred interaction partners within the condensate. Based on this intuition and other thermodynamic considerations, we converted these free energies into a measure of the degree of correlation between the two domains, known as the pair correlation function. Employing this scheme on common condensate driver proteins (e.g., NPM1 and G3BP1) allowed us to elucidate general principles underlying preferred molecular arrangements within biological condensates. Furthermore, by perturbing nucleolar composition and identifying how the degree of its structure is impacted, we gain insights into the connection between nucleolar form and ribosome biogenesis. Overall, this novel methodology opens the door to understanding the liquid structure of condensates, how they mechanistically facilitate function, and how they may change during development, aging, and disease.

Computational modeling of endocytic vesicle formation imaged by simultaneous two-wavelength axial ratiometry.

Clathrin-mediated endocytosis (CME) facilitates the internalization of extracellular cargoes. However, how clathrin-coated vesicles (CCVs) form remains unclear due to the limited resolution of live-cell fluorescence microscopy and the need for sample fixation in electron microscopy. To bridge this gap, our lab developed Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy that leverages the wavelength-dependent properties of Total Internal Reflection Fluorescence (TIRF) microscopy. Dual-tagging a protein of interest with two spectrally separated fluorophores allows STAR to measure the intensity ratio and retrieve the z-position of the protein. Although the exponential decay of the evanescent wave is critical for STAR, it results in an uneven excitation of fluorescently tagged proteins. This will bias STAR measurements when dual-tagged proteins are distributed on a 3D object such as CCVs. To understand the accuracy of STAR for studying vesicle formation, we used mathematical modeling. We represented the CCV as a monodisperse sphere and used the STAR equations to calculate vesicle height (∆z) and compared it to the “ground truth” center of mass (CM). We investigated the influence of vesicle formation, radius, the number and distribution of proteins, the distance of the vesicle from the plasma membrane, and Poisson noise on STAR. This mathematical model of STAR microscopy assesses how the ∆z measured by STAR compares to the ground truth. The theoretical findings will be used to relate experimental ∆z results to vesicle morphology and eventually to develop a 3D dynamic model of CCV formation from our experimental data.

Mitochondrial temporal network tracking for 4D live-cell fluorescence microscopy data.

Mitochondria form a network in the cell that rapidly changes through fission, fusion, and motility. This four-dimensional (4D, x, y, z, time) temporal network has only recently been made accessible through advanced imaging methods such as lattice light-sheet microscopy. Quantitative analysis tools for the resulting datasets however have been lacking. Here we present MitoTNT, the first-in-class software for Mitochondrial Temporal Network Tracking in 4D live-cell fluorescence microscopy data. MitoTNT uses spatial proximity and network topology to compute an optimal tracking. Tracking is >90% accurate in dynamic spatial mitochondria simulations and are in agreement with published motility results in vitro. Using MitoTNT, we reveal correlated mitochondrial movement patterns, local fission and fusion fingerprints, asymmetric fission and fusion dynamics, cross-network transport patterns, and network-level responses to pharmacological manipulations. MitoTNT is implemented in Python with a JupyterLab interface. The extendable and user-friendly design aims at making temporal network tracking accessible to the wider mitochondria community.

Spatio-temporal regulation of endocytic protein assembly by SH3 domains in yeast.

Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein-protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast Saccharomyces cerevisiae, we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation-promoting Las17-Vrp1 complex, and that the network of SH3 interactions coordinates both Las17-Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority use the SH3 domain to recruit other proteins and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology.

Confocal Microscopic Assays of Mitotically Active Proteins in an Agrobacterial Infiltration-Based, Cell Division-Enabled Leaf System of Tobacco (Nicotiana benthamiana).

The production of tissues and organs in plants is brought about by mitotic cell divisions, starting from the zygote. Successful mitosis and cytokinesis harness the functional input of proteins that are expressed in cell cycle-dependent manners to regulate cytoskeletal reorganization and intracellular motility. Fluorescence microscopic assays of mitotically active proteins have been dependent on time-consuming transformation experiments in a host plant or cultured cells. To facilitate the detection and observation of cell cycle-dependent localization and dynamics of plant proteins, we demonstrate, in this chapter, a transiently induced cell division system in Nicotiana benthamiana, named the cell division-enabled leaf system (CDELS). Plasmid constructs which express the D-type cyclin along with a fluorescent fusion protein(s) of interestare delivered to the leaves of N. benthamiana by agrobacterial infiltration. Ectopic expression of cyclin D induces leaf epidermal cells to re-enter mitosis and subsequently cytokinesis, allowing the dynamic localization of fluorescent fusion protein(s) to be observed throughout the course of mitotic cell division using live-cell fluorescence microscopy. This effective approach not only allows one to detect mitotic activities of novel proteins but also record their dynamics and relationship with others during mitosis and cytokinesis in a greatly shortened period of time.

Molecular, morphological and functional properties of tunnelling nanotubes between normal and cancer urothelial cells: New insights from the in vitro model mimicking the situation after surgical removal of the urothelial tumor.

Tunnelling nanotubes (TNTs) are membranous connections that represent a unique type of intercellular communication in different cell types. They are associated with cell physiology and cancer pathology. The possible existence of tunnelling nanotubes communication between urothelial cancer and normal cells has not yet been elucidated. Therefore, we analyzed TNTs formed by T24 cells (human invasive cancer urothelial cells) and normal porcine urothelial (NPU) cells, which serve as surrogate models for healthy human urothelial cells. Monocultures and cocultures of NPU and T24 cells were established and analyzed using live-cell imaging, optical tweezers, fluorescence microscopy, and scanning electron microscopy. TNTs of NPU cells differed significantly from tunnelling nanotubes of T24 cells in number, length, diameter, lipid composition, and elastic properties. Membrane domains enriched in cholesterol/sphingomyelin were present in tunnelling nanotubes of T24 cells but not in NPU cells. The tunnelling nanotubes in T24 cells were also easier to bend than the tunnelling nanotubes in NPU cells. The tunnelling nanotubes of both cell types were predominantly tricytoskeletal, and contained actin filaments, intermediate filaments, and microtubules, as well as the motor proteins myosin Va, dynein, and kinesin 5B. Mitochondria were transported within tunnelling nanotubes in living cells, and were colocalized with microtubules and the microtubule-associated protein dynamin 2. In cocultures, heterocellular tunnelling nanotubes were formed between NPU cells and T24 cells and vice versa. The presence of connexin 43 at the end of urothelial tunnelling nanotubes suggests a junctional connection and the involvement of tunnelling nanotube in signal transduction. In this study, we established a novel urothelial cancer-normal coculture model and showed cells in the minority tend to form tunnelling nanotubes with cells in the majority. The condition with cancer cells in the minority is an attractive model to mimic the situation after surgical resection with remaining cancer cells and may help to understand cancer progression and recurrence. Our results shed light on the biological activity of tunnelling nanotubes and have the potential to advance the search for anticancer drugs that target tunnelling nanotubes.

Fission yeast cells mix parental mitochondria in a progressive manner during meiosis.

Mitochondria in many fungi are inherited uniparentally during meiosis. It has remained unclear whether parental mitochondria in the fission yeast Schizosaccharomyces pombe are inherited uniparentally or biparentally. Here, we assessed the mixing of parental mitochondria carefully by live-cell microscopy and developed an algorithm to determine the degree of mitochondrial mixing in a quantitative manner. We found that parental mitochondria in fission yeast cells were mixed progressively as meiosis progressed. Moreover, we established that mitochondrial fission and the size of the conjugation neck are the limiting factors in restricting the mixing of parental mitochondria. We further employed a combination of quantitative polymerase chain reaction, fluorescent live-cell microscopy, and transmission electron microscopy approaches to examine the mitochondrial inheritance of progeny cells derived from a cross between wild-type and Rho0 (mitochondrial DNA absent) cells. The results show that all progeny cells of the cross carry mitochondrial DNA. Hence, our data support the model in which parental mitochondria in the fission yeast S. pombe are inherited biparentally during meiosis.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Abstract 15779: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons as Models for Toxoplasma Gondii Infection

Introduction: Toxoplasma gondii infects all nucleated cells in warm-blooded vertebrates. Infection may be asymptomatic in healthy people but may result in the lifelong chronic persistence of cysts containing bradyzoites in specific tissues such as muscle and neurons. Hypothesis: Stem cell-derived neurons and cardiomyocytes will recapitulate the tissue-specific parasite-host interaction underlying chronic persistence in Toxoplasma infection. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (iCMs), cardiac fibroblasts (iCFs) and neurons (iNEs) were used as biologically relevant models to study the interactions of T. gondii with human hosts in vitro . iCM, iCF and iNE were infected with T. gondii TypeI/III EGS strain (dual fluorescent stage-specific reporter strain: mCherry driven by SAG1 promoter and sfGFP driven by the LDH2 promoter). Results: We observed higher rate of bradyzoite formation in iCM and iNE, compared to iCF from the same donor at 72 hours post-infection by fluorescence microscopy of live cells and flow cytometry. Furthermore, we have analyzed the changes in the gene expression of host cells during Toxoplasma infection using high-throughput RNA-seq. Compared to uninfected iCMs, iCMs infected for 24 hours showed 1090 upregulated and 753 downregulated differentially expressed genes (DEGs); iCMs infected for 72 hours showed 1956 upregulated and 1253 downregulated DEGs. Compared to uninfected iCFs, iCFs infected for 24 hours showed 1654 upregulated and 1971 downregulated DEGs; iCFs infected for 72 hours showed 2742 upregulated and 1916 downregulated DEGs. In comparison to uninfected iNEs, iNEs infected for 24 hours showed 190 upregulated and 321 downregulated DEGs; iNEs infected for 72 hours showed 244 upregulated and 91 downregulated DEGs. Enrichment analysis of gene sets regulated by infection will be presented. Conclusions: Our in vitro generated human iCMs, iCF and iNEs have provided us a valuable tool for better understanding the impact on the host cells during Toxoplasma infection. A detailed analysis of validation and intervention of the top dysregulated genes in all different cell types will inform the design of potential therapeutic targets.

Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.

Super-resolution fluorescence blinking imaging using compressive sensing

Super-resolution optical fluctuation imaging (SOFI) is an extensively used super-resolution (SR) imaging technique. The sample condition and imaging system are simpler than most SR systems, and the cusp-artifacts limit the resolution of high-order SOFI. To improve the resolution of SOFI, an alternative method is to improve the reconstruction algorithm. Here, compressive sensing (CS) is used in SOFI to improve its resolution. The detailed CS reconstruction algorithm is chosen as multiple measurement vector model sparse Bayesian learning (MSBL). We demonstrate that MSBL can achieve higher than threefold resolution improvement in simulation under certain conditions. The SOFI experiment analysis demonstrates that MSBL can improve the resolution about 2.5-fold compared with the diffraction limit. All results prove that MSBL has a higher resolving ability compared with other algorithms. Our results prove that CS is a broad applicability tool for most of the existing SR microscopy techniques and can be applied in 3D and live-cell SR fluorescence microscopy imaging in the future.

An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells.

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells.

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy.

Visualizing Yeast Organelles with Fluorescent Protein Markers.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Bromodomains regulate dynamic targeting of the PBAF chromatin-remodeling complex to chromatin hubs

Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA polymerase II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single-molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF’s bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome-binding kinetic profiles that mimic chromatin stability. Removal of PBAF’s bromodomains stabilizes H3.3 binding within chromatin, indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF’s dynamic bromodomain-mediated engagement of a nucleosome may reflect the chromatin-remodeling potential of differentially bound chromatin states.