Automated line scan analysis to quantify biosensor activity at the cell edge.

Biosensors are valuable tools used to image the subcellular localization and kinetics of protein activity in living cells. Signaling at the edge of motile cells that regulates cell protrusion and retraction is important in many aspects of cell physiology, and frequently studied using biosensors. However, quantitation and interpretation is limited by the heterogeneity of this signaling behavior; automated analytical approaches are required to systematically extract large data sets from biosensor studies for statistical analysis. Here we describe an automated analysis to relate the velocity at specific points along the cell edge with biosensor activity in adjoining regions. Time series of biosensor images are processed to interpolate a smooth edge of the cell at each time point. Profiles of biosensor activity ('line scans') are then calculated along lines perpendicular to the cell edge. An energy minimization method is used to calculate a velocity associated with each line scan. Sorting line scans by the proximal velocity has generated novel biological insights, as exemplified by analysis of the Src merobody biosensor. With the large data sets that can be generated automatically by this program, conclusions can be drawn that are not apparent from qualitative or 'manual' quantitative techniques. Our 'LineScan' software includes a graphical user interface (GUI) to facilitate application in other studies. It is available at hahnlab.com and is exemplified here in a study using the RhoC FLARE biosensor.

[Importance of the different i.v. iron generations for everyday medical practice]. / Bedeutung der verschiedenen i.v.-Eisengenerationen für den medizinischen Alltag.

BACKGROUND: Iron deficiency and anaemia occur in particular in women or as comorbid conditions to a varietyof chronic diseases. Besides oral preparations, parenteral iron therapies are also available for the treatment of iron deficiency or anaemia. In the light of the growing importance and increasing number of parenteral iron preparations, theirpharmacology and application as well as the chronology of their approvals and thecharacteristicsof the various preparations are presented herefor comparison. METHOD: Review. RESULTS: To date, there are three different generations of parenteral iron preparations, which differ in terms of stability, safety and dosage. In particular, the active substances of the third generation, ferric carboxymaltose, iron isomaltoside and ferumoxytol are characterised by high complex stability and comparable safety, also allowing rapid application of high doses of iron. CONCLUSIONS: High molecular weight iron dextran, as a representative of 1st generation iron preparations, should no longer be used if possible, as more recent i.v. iron preparations are available with considerably lower risk of serious anaphylactic reactions. Ferrous gluconate and iron sucrose, as representatives of the 2nd generation, are very efficient preparations, but they require frequent visits to the clinic or the doctor, as they may only be administered in low doses because of labile iron complexes. The three 3rd generation parenteral iron formulations have advantages in handling in everyday practice, since they offer comparably good safety profiles, high complex stability and thus the possibility of rapid application of high doses of iron up to the total cumulative dose. Furthermore, test doses are not required with these preparations, which also simplifies their use.

Effects of chronic hepatitis C infection on the treatment of breast cancer patients.

BACKGROUND: Although hepatitis C (HCV) is the most common blood-borne infection in the United States, little information exists about treatment of breast cancer in the setting of chronic HCV. PATIENTS AND METHODS: The databases of the University of Texas M.D. Anderson Cancer Center (MDACC) Tumor Registry, Department of Breast Medical Oncology, and Department of Laboratory Medicine were cross-referenced for patients with breast cancer, who were also identified as having HCV. Eligible patients had a diagnosis of invasive breast cancer, breast cancer treatment at MDACC, and a diagnosis of HCV. RESULTS: During chemotherapy, 25% of patients experienced elevations in aminotransferases and 44% of patients required dose reductions/delays in chemotherapy. More than 60% of the patients who received chemotherapy demonstrated a grade 2 or greater complication. However, 92% of patients were able to complete the number of cycles specified in the initial chemotherapy plan. CONCLUSIONS: As the majority of these breast cancer patients completed the initial chemotherapy plan, this study indicates that breast cancer patients with HCV can be treated with cytotoxic therapy. Comparison with historical controls showed similar rates of hepatic toxicity in the presence (or absence) of HCV, indicating that incidence of transaminitis may not be significantly affected by HCV.

Combined Atomic Force Microscope and Volumetric Light Sheet System for Correlative Force and Fluorescence Mechanobiology Studies.

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.

Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells.

We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h.

Localized Rac activation dynamics visualized in living cells.

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.

The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.

Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated.

A photocross-linking fluorescent indicator of mitochondrial membrane potential.

Ionic dyes that distribute across membranes according to electrical potential have proven valuable as fluorescent indicators of mitochondrial energetics in living cells. Applications have been limited, however, as potential-dependent staining is lost during cell fixation. We have produced a membrane potential indicator whose potential-dependent distribution can be made permanent, to enable correlation of membrane potential with cytochemical information from immunofluorescence. A carbocyanine dye was derivatized with a photoreactive nitrophenylazide moiety so that irradiation would induce nonspecific, covalent attachment to nearby molecules. Photo-induced cross-linking was observed in paper chromatography, when irradiation caused immobilization of the dye. The new dye, named PhoCy (photofixable cyanine), showed specific staining of mitochondria in living Swiss 3T3 fibroblasts. When living cells were stained, irradiated, and fixed with formaldehyde, mitochondrial staining was retained owing to cross-linking with mitochondrial components. Omission of irradiation eliminated mitochondrial staining in fixed cells. Labeling, irradiation, and fixation procedures were optimized to produce bright specific staining with minimal background. The indicator's sensitivity to mitochondrial potential was demonstrated by treating cells with 2,4-dinitrophenol, an uncoupler of mitochondrial electron transport, which decreased mitochondrial staining in living cells and in the corresponding fixed specimens.

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7.

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.

Synthesis and evaluation of 2-diazo-3,3,3-trifluopropanoyl derivatives of colchicine and podophyllotoxin as photoaffinity labels: reactivity, photochemistry, and tubulin binding.

Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2-diazo-3,3,3-trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, and N-DTFP-deacetylthiocolchicine was shown to competitively inhibit tubulin-colchicine binding with a Ki of 4-5 microM. The tubulin off-rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein-labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of photoaffinity labels.

Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.

When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells.

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.

A stable diazo photoaffinity label with high absorptivity and effective photoactivation beyond 300 nm.

The sulfosuccinimidyl active ester of 3-(3-carbethoxy-4-diazo-5-oxo-2-pyrrolin-1-yl)propanoic acid (DIAZOPY-SE) has been synthesized for use as a photoaffinity labeling reagent. This compound was obtained from commercial chemicals by a four-step synthesis requiring no complex procedures or special apparatus. The active ester efficiently derivatizes protein amino groups with the chromophore 3-carbethoxy-4-diazo-5-oxo-2-pyrroline (DIAZOPY, epsilon 8800 M-1 cm-1 at lambda max 330 nm), which on irradiation yielded products expected from formation of a reactive carbene intermediate. Brief irradiation of DIAZOPY in 2-propanol using wavelengths greater than 300 nm for photolysis yielded mainly an isopropyl ether resulting from insertion of the carbene into the O-H bond of the alcohol. Formed concurrently and to a somewhat lesser extent was an isopropyl ester, resulting from a ring-contracting Wolff rearrangement of the carbene and subsequent reaction with isopropanol. Analogous products were produced by photolysis in 2-propanol of DIAZOPY-PA (for DIAZOPY propanoic acid), the carboxylic acid precursor of DIAZOPY-SE. Facile protein derivatization by DIAZOPY-SE was demonstrated using actin and sheep IgG. Actin labeled with DIAZOPY-SE and irradiated while in the F-actin (reversibly polymerized) form was crosslinked to yield a covalently-linked dimer, illustrating the potential of the reagent in photoaffinity applications. Advantages of DIAZOPY-SE as a photoaffinity labeling reagent include ease of synthesis, chemical and photostability, efficient photolysis at wavelengths greater than 300 nm, and a capacity for crosslinking by carbene insertion processes.

A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore.

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.

Changes in the cytoplasmic structure of CTLs during target cell recognition and killing.

CTL play a critical role in immune defense by recognizing and killing virally infected or tumor cells. In this report, the structure of cytoplasm within living CTL was monitored during CTL killing of target cells. Living CTL were simultaneously loaded with fluorescent 70,000- and 10,000-kDa dextran particles. The relative distribution of the large and small dextrans within CTL revealed subcellular heterogeneities in the submicroscopic structure of cytoplasm. Localized alterations in cytoplasmic structure correlated with specific events during CTL killing. Recognition of target cells was accompanied by a transient increase in large dextran accessibility over a broad front near the interface between CTL and target cells. This region narrowed to a smaller area from which pseudopodia were extended toward the target. During extension, there was a large difference between regions of high dextran accessibility within the pseudopod and more structured cytoplasm within the cell body. Areas undergoing structural changes showed localized foci of high dextran accessibility. During retraction, cytoplasmic structure became gradually more uniform throughout the protrusion and cell body. These observations revealed subcellular regions undergoing major changes during early stages of the killing response, and addressed the role of cytoplasmic solation in controlling CTL morphology. They support mechanisms of pseudopod extension driven by hydrostatic pressure and demonstrate a precise regulation of cortical structure to control the direction of pseudopod extension.

Fluorescent protein biosensors: measurement of molecular dynamics in living cells.

A new generation of reagents that report on specific molecular events in living cells, called fluorescent protein biosensors, has evolved from in vitro fluorescence spectroscopy and fluorescent analogue cytochemistry. Creative designs of fluorescent protein biosensors to measure the molecular dynamics of macromolecules, metabolites, and ions in single cells emerge from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. Future advances in fluorescent probe design, computer-driven optical instrumentation, and software will allow us to engineer endogenous cellular components that localize and function as reporters of their activities, thus moving molecular measurement beyond the single cell to living tissues and the whole organism.

Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development.

The presynaptic plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa) has been implicated as one of several neural-specific components that direct constitutive fusion mechanisms to the regulated vesicle trafficking and exocytosis of neurotransmitter release. There exist two alternatively spliced isoforms of SNAP-25, a and b, which differ in a putative membrane-interacting domain. We show that these two isoforms have distinct quantitative and anatomical patterns of expression during brain development, in neurons, and in neuroendocrine cells and that the proteins localize differently in neurites of transfected PC12 pheochromocytoma cells. These findings indicate that alternative isoforms of SNAP-25 may play distinct roles in vesicular fusion events required for membrane addition during axonal outgrowth and for release of neuromodulatory peptides and neurotransmitters.

Galpha i3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

Galphai3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Galphai3 and Ca(2+) and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Galphai3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Galphai3 and calnuc in COS-7 cells expressing Galphai3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Galphai3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when Galphai3 is coexpressed with cytosolic calnuc(DeltaN2-25)-CFP lacking its signal sequence. No FRET signal is detected when Galphai3(DeltaC12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Galphai3-YFP and calnuc(DeltaEF-1,2)-CFP, which is unable to bind Galphai3, are coexpressed. Galphai3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to Galphai3 on Golgi membranes in living cells and that Galphai3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca(2+) sensor protein capable of binding to and potentially regulating interactions of Galphai3 on Golgi membranes.