The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles. We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography. We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites. This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles. Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features). These findings provide an alternative framework for analysis of ER-to-Golgi traffic.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.Viruses are now considered to be an important component of all aquatic microbial communities. The reevaluation of the role of viruses in marine ecosystems is due to the discovery of very high virus abundance (see reference 13 for a review). Recent studies have stressed the ecological implication of viruses in the release of dissolved organic matter, nutrient recycling (18), and the pathways of organic carbon utilization, with cascade effects on marine microbial food webs and organicmatter cycling (12). The available methods for the determination of virus abundance in aquatic environments include counting by transmission electron microscopy (TEM) (1, 2, 16, 21), by flow cytometry (17), and by epifluorescence microscopy (EFM) (9,14,20,26,29). The last technique allows an accurate and easily performed enumeration, avoiding the use of expensive and bulky equipment (13). In addition, EFM is reported to be up to seven times more efficient than TEM for counting viruses (15,28).Available information dealing with benthic virus ecology is scant. This is due to the lack of adequate protocols for easily determining their abundance and distribution in marine sediments (6). The main objective of this work was the optimization of procedures to enumerate viruses in different marine sediments. We focused our attention on EFM counting using SYBR Green I as a stain (20) in order to address the following issues: (i) virus dislodgment from sediment particles (using surfactant and ultrasound treatments), (ii) the efficiency of virus extraction from bulk sediment (by the number of postsonication washings), and (iii) stain-counting accuracy and efficiency (by removing possible interferences due to extracellular DNA in virus counting and by comparison with TEM counts). In addition, we tested the effects of preservatives on virus abundance in long-time-course experiments carried out on fixed sediment samples.Sediment sampling and selection of sediment. In order to make the protocol for virus counting suitable for the widest variety of sediment samples, two different sediment types were selected in this study: shallow sands and deep-sea muds. As deep-sea samples are not generally analyzed immediately, the effects of long-term storage with preservat...

Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. We identified the nucleo-cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP I as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 39 untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing. In eukaryotic cells, transcription represents the first step of gene expression. However, a variety of nuclear and cytoplasmic post-transcriptional events determine the final fate of RNAs and thus regulate gene product diversity as well as the spatio-temporal pattern of gene expression.In recent years, subcellular targeting of mRNAs, coupled to localized translation, has emerged as a powerful mechanism to spatially and temporally restrict protein synthesis. Furthermore, a genome-wide in situ hybridization analysis in Drosophila embryos suggests that RNA localization could represent a general mechanism for the establishment of cell polarity (Lecuyer et al. 2007). Consistent with this, functional studies have shown that local translation of asymmetrically enriched mRNAs is used by differentiated cells to generate functionally distinct compartments, or by developing organisms to partition cell fate determinants (St Johnston 2005;Du et al. 2007). In several species, the asymmetric distribution in unfertilized eggs of maternal RNAs encoding cytoplasmic determinants controls embryonic body axis specification. In Drosophila, oskar mRNA encodes the posterior determinant and is transported to the posterior pole of the oocyte, where it is specifically translated. This precise spatio-temporal control is critical for embryonic patterning, as mutant oocytes in which oskar is not expressed at the posterior pole develop into embryos lacking abdominal structures and germ cells (Ephrussi et al. 1991;Kim-Ha et al. 1991). Conversely, ectopic translation of unlocalized oskar causes patterning defects characterized by a loss of anterior structures, and in extreme cases, duplication of posterior structures (Ephrussi and...

The Golgi complex in mammalian cells forms a continuous ribbon of interconnected stacks of flat cisternae. We show here that this distinctive architecture reflects and requires the continuous input of membranes from the endoplasmic reticulum (ER), in the form of pleiomorphic ER-to-Golgi carriers (EGCs). An important step in the biogenesis of the Golgi ribbon is the complete incorporation of the EGCs into the stacks. This requires the Golgi-matrix protein GM130, which continuously cycles between the cis-Golgi compartments and the EGCs. On acquiring GM130, the EGCs undergo homotypic tethering and fusion, maturing into larger and more homogeneous membrane units that appear primed for incorporation into the Golgi stacks. In the absence of GM130, this process is impaired and the EGCs remain as distinct entities. This induces the accumulation of tubulovesicular membranes, the shortening of the cisternae, and the breakdown of the Golgi ribbon. Under these conditions, however, secretory cargo can still be delivered to the Golgi complex, although this occurs less efficiently, and apparently through transient and/or limited continuities between the EGCs and the Golgi cisternae.

The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex.

In the most widely accepted version of the cisternal maturation/progression model of intra-Golgi transport, the polarity of the Golgi complex is maintained by retrograde transport of Golgi enzymes in COPI-coated vesicles. By analyzing enzyme localization in relation to the three-dimensional ultrastructure of the Golgi complex, we now observe that Golgi enzymes are depleted in COPI-coated buds and 50-to 60-nm COPI-dependent vesicles in a variety of different cell types. Instead, we find that Golgi enzymes are concentrated in the perforated zones of cisternal rims both in vivo and in a cell-free system. This lateral segregation of Golgi enzymes is detectable in some stacks during steady-state transport, but it was significantly prominent after blocking endoplasmic reticulum-to-Golgi transport. Delivery of transport carriers to the Golgi after the release of a transport block leads to a diminution in Golgi enzyme concentrations in perforated zones of cisternae. The exclusion of Golgi enzymes from COPI vesicles and their transport-dependent accumulation in perforated zones argues against the current vesicle-mediated version of the cisternal maturation/progression model.