Ferroptosis is an emerging cancer suppression strategy. However, how to select cancer patients for treating with ferroptosis inducers remains challenging. Here, we develop photochemical activation of membrane lipid peroxidation (PALP), which uses targeted lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation for reporting ferroptosis sensitivity in cells and tissues. PALP captured by BODIPY-C11 can be suppressed by lipophilic antioxidants and iron chelation, and is dependent on PUFA-lipid levels. Moreover, we develop PALPv2, for studying lipid peroxidation on selected membranes along the z axis in live cells using two-photon microscopes. Using PALPv1, we detect PUFA-lipids in multiple tissues, and validate a PUFA-phospholipid reduction during muscle aging as previously reported. Patterns of PALPv1 signals across multiple cancer cell types in vitro and in vivo are concordant with their ferroptosis susceptibility and PUFA-phospholipid levels. We envision that PALP will enable rapid stratification of ferroptosis sensitivity in cancer patients and facilitate PUFA-lipid research.

ABSTRACT Polyunsaturated phospholipids are essential for multiple cellular functions; however, their uncontrolled peroxidation leads to ferroptosis. Here we describe p hotochemical a ctivation of membrane l ipid p eroxidation (PALP), which uses localized laser pulses to induce lipid peroxidation photochemically. While PALP bypasses enzymatic requirements for lipid peroxidation, the resulting BODIPY-C11-based signal is largely correlated with local polyunsaturated phospholipid concentration on membranes. This technique enables non-invasive reporting of lipid unsaturation levels and sensitivity to ferroptosis in live cells.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

We investigate solid-immersion fluorescence microscopy suitable for super-resolution nanotechnology and biological imaging, and have observed limit of resolution as small as 15 nm with microspheres, mitochondria, and chromatin fibers. We have further observed that fluorescence efficiency increases with excitation power density, implicating appreciable stimulated emission and increased resolution. We discuss potential advantages of the solid-immersion microscopy, including combined use with previously established super-resolution techniques for reaching deeper beyond the conventional diffraction limit.

INTRODUCTIONCharged-coupled device (CCD) cameras are commonly used to acquire wide-field, spinning-disk confocal, and total internal reflection fluorescence (TIRF) microscopy images. Collecting digital images for a live cell fluorescence microscopy experiment requires balancing the desire to acquire sufficiently high-quality images with the need to minimize exposure of the specimen to light to reduce phototoxicity and photobleaching. The choice of CCD camera and the camera acquisition settings are critical determinants in the quality of a digital image that one can acquire from a given fluorescent specimen. In this article, we discuss the characteristics of CCD cameras, how they affect digital-image quality, and how to properly use a CCD camera for fluorescence live cell imaging experiments.

Changes in intracellular calcium are necessary for the successful progression of mitosis in many cells. Both elevation and reduction in intracellular calcium can disrupt mitosis by mechanisms that remain ill defined. In this study we explore the role of transmembrane voltage-gated calcium channels (CaV channels) as regulators of mitosis in the mouse corticotroph cell line (AtT-20). We report that the nifedipine-sensitive isoform CaV1.2 is localized to the “poleward side” of kinetechores during metaphase and at the midbody during cytokinesis. A second nifedipine-sensitive isoform, CaV1.3, is present at the mid-spindle zone in telophase, but is also seen at the midbody. Nifedipine reduces the rate of cell proliferation, and, utilizing time-lapse microscopy, we show that this is due to a block at the prometaphase stage of the cell cycle. Using Fluo-4 we detect calcium fluxes at sites corresponding to the mid-spindle zone and the midbody region. Another calcium dye, Fura PE3/AM, causes an inhibition of mitosis prior to anaphase that we attribute to a chelation of intracellular calcium. Our results demonstrate a novel, isoform-specific localization of CaV1 channels during cell division and suggest a possible role for these channels in the calcium-dependent events underlying mitotic progression in pituitary corticotrophs.

There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance.

Despite the general importance of Ca2+ signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca2+ (Ca-i(2+)) has not been reported. In this article, we describe the results of experiments measuring Ca-i(2+) in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S) and phorbol 12-myristate 13-acetate (PMA). Ca2+ signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca-i(2+) transient from a basal activity of 110 nM to a peak response of 260.1 +/- 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 +/- 0.2 nM) between 10 and 15 min. The rise in Ca-i(2+) appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca2+. Loading goblet cells with BAPTA inhibited the ATP gamma S-induced Ca2+ transient by 86.0 +/- 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 +/- 7% of the ATP gamma S control peak), brief rise in Ca-i(2+). This diminutive signal likely denotes a local Ca2+ gradient, which may be associated with the mucin granule exocytotic process.

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

Fluorescent speckle microscopy (FSM) is a new imaging technique with the potential for simultaneous visualization of translocation and dynamic turnover of polymer structures. However, the use of FSM has been limited by the lack of specialized software for analysis of the positional and photometric fluctuations of hundreds of thousand speckles in an FSM time-lapse series, and for translating this data into biologically relevant information. In this paper we present a first version of a software for automated analysis of FSM movies. We focus on mapping the assembly and disassembly kinetics of a polymer meshwork. As a model system we have employed cortical F-actin meshworks in live newt lung epithelial cells. We lay out the algorithm in detail and present results of our analysis. The high spatial and temporal resolution of our maps reveals a kinetic cycling of F-actin, where phases of polymerization alternate with depolymerization in a spatially coordinated fashion. The cycle rates change when treating cells with a low dose of the drug latrunculin A. This shows the potential of this technique for future quantitative screening of drugs affecting the actin cytoskeleton. Various control experiments demonstrate that the algorithm is robust with respect to intensity variations due to noise and photobleaching and that effects of focus plane drifts can be eliminated by manual refocusing during image acquisition.

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.

BACKGROUND:In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS:We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS:We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.

We have discovered a new method, Fluorescent Speckle Microscopy (FSM), for analyzing the dynamic movement and turnover of macromolecular protein assemblies, such as the cytoskeleton, in living cells (Waterman-Storer et al., 1998). FSM compliments or replaces the techniques of fluorescence recovery after photobleaching or photoactivation of fluorescence for measuring protein dynamics in vivo. For FSM, cells are microinjected with a very low fraction of fluorescently labeled subunits that co-assemble with unlabeled subunits to give a structure with a fluorescent speckled appearance in diffraction-limited wide-field or confocal digital fluorescence images. At low fractions of fluorescent subunits relative to unlabeled subunits, fluorescent speckles vary randomly in intensity according to the number of fluorescent subunits within a diffraction-limited region. in time-lapse FSM image series, movement of the fluorescent speckle pattern indicates translocation of structures, while changes in speckle intensity indicate subunit turnover. We have used FSM to study microtubule and actin behavior in interphase and mitotic cells. We use kymograph analysis to quantitate the movement of speckled structures (Fig 1) and are currently developing analysis procedures to quantify subunit turnover in structures.We have applied these methods to the study of microtubule and actin cytoskeletal dynamics in migrating vertebrate cells in culture. Interactions between the microtubule and actin cytoskeletons underlie fundamental cellular processes such as cytokinesis and cell locomotion, but are poorly understood.