Traffic最新文献

The ciliate phylum is a group of protists noted for their unusual membrane trafficking system and apparent environmental ubiquity; as highly successful microbial predators, they are found in all manner of environments and the ability for specific species to adapt to extremely challenging conditions makes them valued as bioindicators. Ciliates have also been used for many years as cell biological models because of their large cell size and ease of culturing, and for many fundamental cell structures, particularly membrane-bound organelles, ciliates were some of the earliest organisms in which these were observed via microscopy. In this study, we carried out a comparative genomic survey of selected membrane trafficking proteins in a pan-ciliate transcriptome and genome dataset. We observed considerable loss of membrane trafficking system (MTS) proteins that would indicate a loss of machinery that is generally conserved across eukaryotic diversity, even after controlling for potentially incomplete genome representation. In particular, the complete DSL1 complex was missing in all surveyed ciliates. This protein complex has been shown as involved in peroxisome biogenesis in some model systems, and a paucity of DSL1 components has been indicative of degenerate peroxisome. However, Tetrahymena thermophila (formerly Tetrahymena pyroformis) was one of the original models for visualizing peroxisomes. Conversely, the AP3 complex essential for mucocyst maturation in T. thermophila, is poorly conserved despite the presence of secretory lysosome-related organelles across ciliate diversity. We discuss potential resolutions for these apparent paradoxes in the context of the heterogenous distribution of MTS machinery across the diversity of ciliates.

Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

The extremely dynamic life cycle of gap junction connections requires highly efficient intracellular trafficking system especially designed for gap junction proteins, but the underlying mechanisms are largely unknown. Here, we identified that the COPII-associated proteins ERGIC2 (ER-Golgi intermediate compartment) and ERGIC3 are specifically required for the efficient intracellular transport of gap junction proteins in both Caenorhabditis elegans and mice. In the absence of Ergic2 or Ergic3, gap junction proteins accumulate in the ER and Golgi apparatus and the size of endogenous gap junction plaques is reduced. Knocking out the Ergic2 or Ergic3 in mice results in heart enlargement and cardiac malfunction accompanied by reduced number and size of connexin 43 (Cx43) gap junctions. Invertebrates' gap junction protein innexins share no sequence similarity with vertebrates' connexins. However, ERGIC2 and ERGIC3 could bind to gap junction proteins in both worms and mice. Characterization of the highly specialized roles of ERGIC2 and ERGIC3 in metazoans reveals how the early secretory pathway could be adapted to facilitate the efficient transport for gap junction proteins in vivo.

The intracellular trafficking of β-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-β production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aβ production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4.