Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl− serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium

A visual pigment-like protein, referred to as peropsin, has been identified by large-scale sequencing of cDNAs derived from human ocular tissues. The corresponding mRNA was found only in the eye, where it is localized to the retinal pigment epithelium (RPE). Peropsin immunoreactivity, visualized by light and electron microscopy, localizes the protein to the apical face of the RPE, and most prominently to the microvilli that surround the photoreceptor outer segments. These observations suggest that peropsin may play a role in RPE physiology either by detecting light directly or by monitoring the concentration of retinoids or other photoreceptor-derived compounds.

Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis.

Hypoxia/reoxygenation is an important cause of tissue injury in a variety of organs and is classically considered to be a necrotic form of cell death. We examined the role of endonuclease activation, considered a characteristic feature of apoptosis, in hypoxia/reoxygenation injury. We demonstrate that subjecting rat renal proximal tubules to hypoxia/reoxygenation results in DNA strand breaks and DNA fragmentation (both by an in situ technique and by agarose gel electrophoresis), which precedes cell death. Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kDa on a substrate gel. This DNA-degrading activity was entirely calcium dependent and was blocked by the endonuclease inhibitor aurintricarboxylic acid. The protein extract from tubules subjected to hypoxia/reoxygenation cleaved intact nuclear DNA obtained from normal proximal tubules into small fragments, which further supports the presence of endonuclease activity. Despite unequivocal evidence of endonuclease activation, the morphologic features of apoptosis, including chromatin condensation, were not observed by light and electron microscopy. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, provided complete protection against DNA damage induced by hypoxia/reoxygenation but only partial protection against cell death. Taken together, our data provide strong evidence for a role of endonuclease activation as an early event, which is entirely responsible for the DNA damage and partially responsible for the cell death that occurs during hypoxia/reoxygenation injury. Our data also indicate that in hypoxia/reoxygenation injury endonuclease activation and DNA fragmentation occur without the morphological features of apoptosis.

The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography

Surmises of how myosin subfragment 1 (S1) interacts with actin filaments in muscle contraction rest upon knowing the relative arrangement of the two proteins. Although there exist crystallographic structures for both S1 and actin, as well as electron microscopy data for the acto–S1 complex (AS1), modeling of this arrangement has so far only been done “by eye.” Here we report fitted AS1 structures obtained using a quantitative method that is both more objective and makes more complete use of the data. Using undistorted crystallographic results, the best-fit AS1 structure shows significant differences from that obtained by visual fitting. The best fit is produced using the F-actin model of Holmes et al. [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature (London) 347, 44–49]. S1 residues at the AS1 interface are now found at a higher radius as well as being translated axially and rotated azimuthally. Fits using S1 plus loops missing from the crystal structure were achieved using a homology search method to predict loop structures. These improved fits favor an arrangement in which the loop at the 50- to 20-kDa domain junction of S1 is located near the N terminus of actin. Rigid-body movements of the lower 50-kDa domain, which further improve the fit, produce closure of the large 50-kDa domain cleft and bring conserved residues in the lower 50-kDa domain into an apparently appropriate orientation for close interaction with actin. This finding supports the idea that binding of ATP to AS1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50-kDa cleft of S1.

Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

Cryo-electron microscopy studies of empty capsids of human parvovirus B19 complexed with its cellular receptor.

The three-dimensional structures of human parvovirus B19 VP2 capsids, alone and complexed with its cellular receptor, globoside, have been determined to 26 resolution. The B19 capsid structure, reconstructed from cryo-electron micrographs of vitrified specimens, has depressions on the icosahedral 2-fold and 3-fold axes, as well as a canyon-like region around the 5-fold axes. Similar results had previously been found in an 8 angstrom resolution map derived from x-ray diffraction data. Other parvoviral structures have a cylindrical channel along the 5-fold icosahedral axes, whereas density covers the 5-fold axes in B19. The glycolipid receptor molecules bind into the depressions on the 3-fold axes of the B19:globoside complex. A model of the tetrasaccharide component of globoside, organized as a trimeric fiber, fits well into the difference density representing the globoside receptor. Escape mutations to neutralizing antibodies map onto th capsid surface at regions immediately surrounding the globoside attachment sites. The proximity of the antigenic epitopes to the receptor site suggests that neutralization of virus infectivity is caused by preventing attachment of viruses to cells.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

PV-1 is a component of the fenestral and stomatal diaphragms in fenestrated endothelia

PV-1 is a novel endothelial protein shown by immunocytochemical tests to be specifically associated with the stomatal diaphragms of caveolae in lung endothelium. Although the highest expression levels of both mRNA and protein are in the lung, PV-1 also has been found to be expressed in other organs. Using a specific antibody to the extracellular domain of PV-1, we have extended the survey on the presence of this protein at light and electron microscope level in several rat organs. Here we show that by immunofluorescence the antibody recognizes with high specificity the endothelium of the fenestrated peritubular capillaries of the kidney and those of the intestinal villi, pancreas, and adrenals. By immunolocalization at electron microscope level, the antibody recognizes specifically the diaphragms of the fenestrae and the stomatal diaphragms of caveolae and transendothelial channels in the endothelia of these vascular beds. No signal was detected in the continuous endothelium of the heart, skeletal muscle, intestinal muscularis, or brain capillaries or the nondiaphragmed fenestrated endothelium of kidney glomeruli. Taken together, our findings define the only antigen to be localized thus far in fenestral diaphragms. They also show that the stomatal diaphragms of caveolae and transendothelial channels and the fenestral diaphragms might be biochemically related, in addition to being morphologically similar structures.

Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene

We have used Mössbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-N-alkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme (“green CPO”) displayed a nearly pure low-spin ferric EPR signal with g = 1.94, 2.15, 2.31. The Mössbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three 2T2 levels of the (3d)5 configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the 6A1 states follow the 2T2 ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green CPO to 22°C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S = ½ ferric species. One of these species displayed g = 1.82, 2.25, 2.60 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g = 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this “quasi-native CPO,” which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.