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.

Single-shot super-resolution total internal reflection fluorescence microscopy.

We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence.

The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.

Expression of the ORMDLS, modulators of serine palmitoyltransferase, is regulated by sphingolipids in mammalian cells.

The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.

Instant super-resolution imaging in live cells and embryos via analog image processing.

Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz. By using optical instead of digital image-processing operations, we removed the need to capture, store and combine multiple camera exposures, increasing data acquisition rates 10- to 100-fold over other super-resolution microscopes and acquiring and displaying super-resolution images in real time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to spinning-disk confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.

Visualizing S1P-directed cellular egress by intravital imaging.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that provides cellular signals through plasma membrane G protein-coupled receptors. The S1P receptor signaling system has a fundamental and widespread function in licensing the exit and release of hematopoietically derived cells from various tissues into the circulation. Although the outlines of the mechanism have been established through genetic and pharmacologic perturbations, the temporal and spatial dynamics of the cellular events involved have been unclear. Recently, two-photon intravital imaging has been applied to living tissues to visualize the cellular movements and interactions that occur during egress processes. Here we discuss how some of these recent findings provide a clearer picture regarding S1P receptor signaling in modulating cell egress into the circulation. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy.

Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

Detection of Protein Tyrosine Phosphatase Interacting Partners by Mass Spectrometry.

Unraveling interacting partners of protein tyrosine (Tyr) phosphatases is considered a key aspect in resolving the regulation of signaling cascades either in a pathological or in developmental context. Mass spectrometry (MS)-based protein identification has emerged as the major approach in this arena, complemented by the development of novel biochemical methodologies for sample preparation. In this chapter, we highlight two methods that, combined with mass spectrometry, may help the investigator create an interactome map for the phosphatase of interest within a specific biological context.

Imaging Approaches for the Study of Metabolism in Real Time Using Genetically Encoded Reporters.

Metabolism comprises of two axes in order to serve homeostasis: anabolism and catabolism. Both axes are interbranched with the so-called bioenergetics aspect of metabolism. There is a plethora of analytical biochemical methods to monitor metabolites and reactions in lysates, yet there is a rising need to monitor, quantify and elucidate in real time the spatiotemporal orchestration of complex biochemical reactions in living systems and furthermore to analyze the metabolic effect of chemical compounds that are destined for the clinic. The ongoing technological burst in the field of imaging creates opportunities to establish new tools that will allow investigators to monitor dynamics of biochemical reactions and kinetics of metabolites at a resolution that ranges from subcellular organelle to whole system for some key metabolites. This article provides a mini review of available toolkits to achieve this goal but also presents a perspective on the open space that can be exploited to develop novel methodologies that will merge classic biochemistry of metabolism with advanced imaging. In other words, a perspective of "watching metabolism in real time."

Compromise in mRNA processing machinery in senescent human fibroblasts: implications for a novel potential role of Phospho-ATR (ser428).

Ataxia-Telangiectasia and Rad3 related kinase (ATR) is a major gatekeeper of genomic stability and has been the subject of exhaustive study in the context of cell cycle progression and senescence as a DNA damage-induced response. Conditional knockout of the kinase in adult mice results in accelerated aging phenomena, such as such hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. In addition to that, recent reports strongly implicate signaling mediated by this kinase in the regulation of alternative splicing of certain, mostly cancer-associated transcripts. Interest to the function of mRNA synthesis and processing is constantly increasing as severe degenerative diseases, such as cancer, cystic fibrosis and Hutchinson-Gilford progeria syndrome are at least partly attributed to these abnormalities. In light of the above, we investigate the RNA processing machinery in senescent fibroblasts as opposed to young, either exponentially proliferating or quiescent, further focusing on the distribution and localization of active, phosphorylated ATR at ser428. This study implicates the spatiotemporal presence of the phosphorylated kinase in the regulation of mRNA splicing and polyadenylation. This function appears perturbed in senescent cells, accompanied by a distinct pattern of phospho-ATR in the senescent nucleus.

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.

Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.