Illuminating the secretory pathway: when do we need vesicles?

:Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300-500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60-80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane.

Stephens DJ ，Pepperkok R 《JOURNAL OF CELL SCIENCE》

Ultrastructural characterization of endoplasmic reticulum--Golgi transport containers (EGTC).

Recent observations made in live cells expressing green fluorescent protein (GFP)-tagged cargo markers have demonstrated the existence of large, mobile transport intermediates linking peripheral ER exit sites (ERES) to the perinuclear Golgi. Using a procedure of rapid ethane freezing, we examined ultrastructurally the intermediates involved in ER-Golgi transport of the vesicular stomatitis virus (VSV) G protein. When released at the permissive temperature of 32 degrees C, VSVG is first found to be concentrated in pleiomorphic, membrane-bound structures (of about 0.4 to 1 microm in diameter) with extensive budding profiles. These structures are devoid of COPII components and Golgi markers, but are enriched in COPI, the retrograde cargo ERGIC53, and the tethering protein p115. The structures appear to be able to undergo fusion with the Golgi stack and are tentatively referred to as ER-Golgi transport containers, or EGTCs. VSVG protein exiting the ERES at 15 degrees C is first found in clusters or strings of COPII-containing small vesicles, and morphological analysis indicates that these clusters and strings of COPII vesicles may coalesce by homotypic fusion to form the EGTCs. Together with the large transport containers mediating transport from the trans-Golgi network to the plasma membrane, EGTCs represents an emerging class of large membranous structures mediating anterograde transport between the major stations of the exocytic pathway.

Horstmann H ，Ng CP ，Tang BL ，Hong W ... -  《JOURNAL OF CELL SCIENCE》

Imaging Secretory Granule Budding from the Trans-Golgi Network Using Retention Using Selective Hook (RUSH).

The retention using selective hook (RUSH) system enables us to synchronize and visualize the movement of cargoes along the secretory pathway. A fluorescently tagged cargo of interest is retained in the endoplasmic reticulum and released in a biotin-dependent manner. Here, we report a detailed protocol describing the steps necessary to perform RUSH experiments to study secretory granule biogenesis at the trans-Golgi network (TGN).

Asensio CS 《-》

Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane.

Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington's disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER)-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous Htt(140Q/140Q) knock-in mice, which have the expansion inserted into the mouse huntingtin gene so lack wild-type huntingtin expression. Interestingly, heterozygous fibroblasts from a Huntington's disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.

Brandstaetter H ，Kruppa AJ ，Buss F 《-》

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.

Palmer KJ ，Watson P ，Stephens DJ 《biochemical society symposium》