Structure and dynamics of the Golgi complex at 15 degrees C: low temperature induces the formation of Golgi-derived tubules

Immunofluorescence and cryoimmunoelectron microscopy were used to examine the morphologic and functional effects on the Golgi complex when protein transport is blocked at the ERGIC (endoplasmic reticulum-Golgi intermediate compartment) in HeLa cells incubated at low temperature (15 degrees C). At this temperature, the Golgi complex showed long tubules containing resident glycosylation enzymes but not matrix proteins. These Golgi-derived tubules also lacked anterograde (VSV-G) or retrograde (Shiga toxin) cargo. The formation of tubules was dependent on both energy and intact microtubule and actin cytoskeletons. Conversely, brefeldin A or cycloheximide treatments did not modify the appearance. When examined at the electron microscope, Golgi stacks were long and curved and appeared connected to tubules immunoreactive to galactosyltransferase antibodies but devoid of Golgi matrix proteins. Strikingly, COPI proteins moved from membranes to the cytosol at 15 degrees C, which could explain the formation of tubules.     

Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C

We have used micromechanical tests to measure the thermoelastic properties of the liquid and gel phases of dimyristoylphosphatidylcholine (DMPC). We have found that the rippled P beta' phase is only formed when a vesicle is cooled to temperatures below the main acyl chain crystallization transition, Tc, under zero or very low membrane tension. We also found that the P beta' surface ripple or superlattice can be pulled flat under high membrane tension into a planar structure. For a ripple structure formed by acyl chains perpendicular to the projected plane, the projected area change that results from a flattening process is a direct measure of the molecular crystal angle. As such, the crystal angle was found to increase from about 24 degrees just below Tc to about 33 degrees below the pretransition. It was also observed that the P beta' superlattice did not form when annealed L beta' phase vesicles were heated from 5 degrees C to Tc; likewise, ripples did not form when the membrane was held under large tension during freezing from the L alpha phase. Each of these three procedures could be used to create a metastable planar structure which we have termed L*beta' since it is lamellar and plane-crystalline with acyl chains tilted to the bilayer plane. However, we show that this structure is not as condensed as the L beta' phase below 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

In the present study we have dissected the transport pathways between the ER and the Golgi complex using a recently introduced (Kuismanen, E., J. Jantti, V. Makiranta, and M. Sariola. 1992. J. Cell Sci. 102:505- 513) inhibition of transport by caffeine at 20 degrees C. Recovery of the Golgi complex from brefeldin A (BFA) treatment was inhibited by caffeine at reduced temperature (20 degrees C) suggesting that caffeine inhibits the membrane traffic between the ER and the Golgi complex. Caffeine at 20 degrees C did not inhibit the BFA-induced retrograde movement of the Golgi membranes. Further, incubation of the cells in 10 mM caffeine at 20 degrees C had profound effects on the distribution and the organization of the pre-Golgi and the Golgi stack membranes. Caffeine treatment at 20 degrees C resulted in a selective and reversible translocation of the pre- and cis-Golgi marker protein (p58) to the periphery of the cell. This caffeine-induced effect on the Golgi complex was different from that induced by BFA, since mannosidase II, a Golgi stack marker, remained perinuclearly located and the Golgi stack coat protein, beta-COP, was not detached from Golgi membranes in the presence of 10 mM caffeine at 20 degrees C. Electron microscopic analysis showed that, in the presence of caffeine at 20 degrees C, the morphology of the Golgi stack was altered and accumulation of numerous small vesicles in the Golgi region was observed. The results in the present study suggest that caffeine at reduced temperature (20 degrees C) reveals a functional interface between the pre-Golgi and the Golgi stack.     

Measurement and modeling of multiple substrate oxidation by methanotrophs at 20 degrees C

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 degrees C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 degrees C to examine the effect of mixtures of trichloroethylene, trans-dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Delta model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 degrees C. Finally, it was also discovered that at 20 degrees C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 degrees C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.     

Dielectric behavior of DNA solution at radio and microwave frequencies (at 20 degrees C).

The dielectric constant and conductivity of calf thymus DNA were investigated at frequencies between 0.1 MHz and 70 GHz. This work is to investigate the dielectric properties of DNA in low gigahertz region and also to study whether the dielectric behavior of the water is affected by the presence of highly charged DNA. The results of these measurements indicate the presence of two anomalous dispersions, the one between 1 MHz and 1 GHz and the second one above 1 GHZ. The dispersion at low frequencies is likely to arise from polar groups in the DNA molecule. The relaxation behavior of unbound water in DNA solution is only slightly affected by the presence of DNA at concentrations below 1%.     

DNA replication in Escherichia coli 15T- growing at 20 degrees C

Escherichia coli 15T⁻ grows slowly in succinate or aspartate-M9 media. In both media, a gap in DNA replication is observed at 37 °C which is either not present at 20 °C or of very much shorter duration than at 37 °C. However, dichotomous replication is not observed in glucose M9 at 20 °C. The results suggest that initiation of replication in glucose is different from that in aspartate or succinate cultures.     

Conductivity values of tissue culture medium from 20 degrees C to 40 degrees C

Few studies are available that relate conductivity and temperature in solutions commonly used in tissue culture media. The purpose of this paper is to provide equations relating conductivity and temperature (in the range 20 degrees C-40 degrees C) for five solutions: 0.9% saline, MEM (Minimum Essential Media), horse serum, MEM with 1% horse serum, and MEM with 10% horse serum.     

Organization of the yeast Golgi complex into at least four functionally distinct compartments

Pro-alpha-factor (pro-alphaf) is posttranslationally modified in the yeast Golgi complex by the addition of alpha1,6-, alpha1,2-, and alpha1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the alpha1,6- and alpha1,3-mannosylation and Kex2p-dependent processing of pro-alphaf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that alpha1,2-mannosylation of pro-alphaf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-alpha1,6-D-mannanase (endoM) were used to quantitate the amount of each pro-alphaf intermediate during transport through the Golgi complex. We found that alpha1,6-, alpha1,2-, and alpha1,3-mannose were sequentially added to pro-alphaf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-alphaf that accumulated in brefeldin A-treated cells received only alpha1,6-mannosylation as did approximately 50% of pro-alphaf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an alpha1,6-mannosyltransferase but lacks alpha1,2-mannosyltransferase activity in vivo. We propose that the alpha1,6-, alpha1,2-, and alpha1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial, trans, and trans-Golgi network of the yeast Golgi complex, respectively.     

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.     

Muscle glycogen depletion during exercise at 9 degrees C and 21 degrees C

This study compared glycogen depletion in active skeletal muscle after light and moderate exercise in both cold and comfortable ambient conditions. Twelve male subjects (Ss) were divided into two groups equally matched for the submaximal exercise intensity corresponding to a blood lactate concentration of 4 mM (W4) during cycle exercise. On two separate days Ss rested for 30 min at ambient temperatures of either 9 degrees C or 21 degrees C, with the order of temperature exposure being counter-balanced among Ss. Following rest a tissue specimen was obtained from the m. vastus lateralis with the needle biopsy technique. Six Ss then exercised on a cycle ergometer for 30 min at 30% W4 (range = 50 - 65 W) while the remaining group exercised at 60% W4 (range = 85 - 120 W). Another biopsy was taken immediately after exercise and both samples were assayed for glycogen content. Identical procedures were repeated for the second environmental exposure. No significant glycogen depletion was observed in the Ss exercising at 30% W4 in 21 degrees C, but a 23% decrease (p = 0.04) was observed when the same exercise was performed at 9 degrees C. A 22% decrease (p = 0.002) in glycogen occurred in the 60% W4 group at 21 degrees C, which was not significantly different from that observed during the same exercise at 9 degrees C. The results suggest that muscle substrate utilization is increased during light exercise in a cold environment as compared to similar exercise at a comfortable temperature, probably due to shivering thermogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved fluorometric assay of histamine: condensation with O-phthalaldehyde at -20 degrees C

Histamine reacts with OPT at an alkaline pH giving rise to fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 10 hr under nitrogen at ?20°C, i.e., in the frozen state. After acidification with sulfuric acid to pH 2.5 the resulting fluorescence, read at room temperature, was stable for hours. The procedure now measures as little as 1 ng histamine/ml and is much more specific than the conventional fluorometric assay. Spermidine did not interfere with the assay of histamine, and histidine only if present in great excess over histamine. It could be shown that with deproteinized extracts of rat gastric mucosa, histamine could be estimated without further purification, which means saving a lot of time and labor.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.