COPI selectively drives maturation of the early Golgi

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.

Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes.

Adverse Effect of Heparin on Antithrombin Action during Endotoxemia: Microhemodynamic and Cellular Mechanisms

SummaryA recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin+AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH+AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPStreated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p <0.01), whereas Heparin+AT and LMWH+AT animals showed microcirculatory alterations comparable to that in controls. In additional in vitro chemotaxis assays, AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH. Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia. These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.

Intracellular calcium oscillations induced by ATP in airway epithelial cells

In airway epithelial cells, extracellular ATP (ATPo) stimulates an initial transient increase in intracellular Ca2+ concentration that is followed by periodic increases in intracellular Ca2+ concentration (Ca2+ oscillations). The characteristics and mechanism of these ATP-induced Ca2+ responses were studied in primary cultures of rabbit tracheal cells with digital video fluorescence microscopy and the Ca2+-indicator dye fura 2. The continual presence of ATPo at concentrations of 0.1–100 μM stimulated Ca2+ oscillations that persisted for 20 min. The frequency of the Ca2+ oscillations was found to be dependent on both ATPoconcentration and intrinsic sensitivity of each cell to ATPo. Cells exhibited similar Ca2+ oscillations to extracellular UTP (UTPo), but the oscillations typically occurred at lower UTPoconcentrations. The ATP-induced Ca2+ oscillations were abolished by the phospholipase C inhibitor U-73122 and by the endoplasmic reticulum Ca2+-pump inhibitor thapsigargin but were maintained in Ca2+-free medium. These results are consistent with the hypothesis that in airway epithelial cells ATPo and UTPo act via P2U purinoceptors to stimulate Ca2+ oscillations by the continuous production of inositol 1,4,5-trisphosphate and the oscillatory release of Ca2+ from internal stores. ATP-induced Ca2+oscillations of adjacent individual cells occurred independently of each other. By contrast, a mechanically induced intercellular Ca2+ wave propagated through a field of Ca2+-oscillating cells. Thus Ca2+ oscillations and propagating Ca2+ waves are two fundamental modes of Ca2+signaling that exist and operate simultaneously in airway epithelial cells.

Genome replication in early mouse embryos follows a defined temporal and spatial order

The spatial and temporal organisation of replication sites during early mouse embryogenesis was analysed using high resolution confocal and video fluorescence microscopy. The results show that distinct replication patterns occur in the transcriptionally inactive pronuclei of 1-cell embryos as well as in the transcriptionally active nuclei from 2- and 16/32-cell embryos. This indicates that specific chromatin regions are replicated at different times during S-phase and provides the first evidence that mechanisms controlling the temporal and spatial replication of DNA are already present in the haploid pronuclei of the mammalian zygote. Furthermore the data demonstrate that the male and female pronuclei in one-cell embryos replicate their genomes asynchronously. Finally, we observe changes in the dynamics of embryonic genome replication during early development which correlate with gross chromatin structure transitions detected at the electron microscope level. Taken together these results indicate that DNA synthesis in the mouse zygote follows a defined four-dimensional order which may evolve during development and differentiation.

Nitric oxide inhibits calcium release from sarcoplasmic reticulum of porcine tracheal smooth muscle cells

In the present study, effects of the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), on sarcoplasmic reticulum (SR) Ca2+ release were examined in freshly dissociated porcine tracheal smooth muscle (TSM) cells. Fura 2-loaded TSM cells were imaged using video fluorescence microscopy. SR Ca2+ release was induced by acetylcholine (ACh), which acts principally through inositol 1,4,5-trisphosphate (IP3) receptors, and by caffeine, which acts principally through ryanodine receptors (RyR). SNAP inhibited ACh-induced SR Ca2+ release at both 0 and 2.5 mM extracellular Ca2+. Degraded SNAP had no effect on ACh-induced SR Ca2+ release. SNAP also inhibited caffeine-induced SR Ca2+ release. ACh-induced Ca2+ influx was not affected by SNAP when SR reloading was blocked by thapsigargin. SNAP also did not affect SR Ca2+ reuptake. The membrane-permeant analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromo-cGMP, mimicked the effects of SNAP. These results suggest that, in porcine TSM cells, SNAP reduces the intracellular Ca2+ response to ACh and caffeine by inhibiting SR Ca2+ release through both IP3 and RyR, but not by inhibiting influx or repletion of the SR Ca2+ stores. These effects are likely mediated via cGMP-dependent mechanisms.

GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.

Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1-7) neurons

1. Individual immortalized gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons in semiconfluent cultures showed spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) as measured by video fluorescence microscopy and fura-2. In parallel experiments, GT1-7 neurons also showed spontaneous bursts of action potentials that were recorded as action currents from intact cells. The bursts of action currents occurred in characteristic patterns, suggesting an underlying rhythmic oscillation in membrane potential. 2. Depolarization with increased extracellular K+ evoked a concentration-dependent increase in the frequency of Ca2+ oscillations or a sustained plateau of increased [Ca2+]i in GT1-7 neurons. Increased extracellular K+ (30 mM) caused an initial increase in the frequency of action currents, after which they were reversibly abolished. 3. The Ca2+ channel blockers Ni2+ and nimodipine abolished Ca2+ oscillations, whereas nifedipine, gadolinium, omega-conotoxin and omega-agatoxin had no effect on Ca2+ oscillations. These results indicate that Ca2+ oscillations are generated by influx of Ca2+ through voltage-gated Ca2+ channels that are not sensitive to nifedipine and are not N-type or P-type channels. 4. Thapsigargin caused a small, transient rise in baseline [Ca2+]i but had no effect on Ca2+ oscillations. Caffeine and ryanodine had no effect on baseline [Ca2+]i or Ca2+ oscillations. These results indicate that the release of Ca2+ from inositol 1,4,5-trisphosphate (IP-3)-sensitive or caffeine sensitive intracellular stores does not play a major role in Ca2+ oscillations in GT1-7 neurons.(ABSTRACT TRUNCATED AT 250 WORDS)