Newly synthesized procollagen type I (PC) assembles into 300 nm rigid, rod-like triple helices in the lumen of the endoplasmic reticulum. This oligomeric complex moves to the Golgi and forms large electron-dense aggregates. We have monitored the transport of PC along the secretory pathway. We show that PC moves across the Golgi stacks without ever leaving the lumen of the Golgi cisternae. During transport from the endoplasmic reticulum to the Golgi, PC is found within tubular-saccular structures greater than 300 nm in length. Thus, supermolecular cargoes such as PC do not utilize the conventional vesicle-mediated transport to traverse the Golgi stacks. Our results imply that PC moves in the anterograde direction across the Golgi complex by a process involving progressive maturation of Golgi cisternae.

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Collagen fibrils are the major tensile element in vertebrate tissues where they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess formation of the fibrils, predominantly by measuring fibril diameter. Here we describe an enhanced protocol for measuring fibril diameter as well as fibril-volume-fraction, mean fibril length, fibril cross-sectional shape, and fibril 3D organization that are also major determinants of tissue function. Serial section TEM (ssTEM) has been used to visualize fibril 3D-organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View®). We demonstrate the power of 3View® for studying collagen fibril organization in vivo and show how to find and track individual fibrils.Time scale: ~8 days from isolating the tissue to having a 3D image stack.

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.

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A–gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.

This study identifies HD-PTP as a central coordinator of the ESCRT pathway for EGFR. Based on these studies, we propose a model whereby the concerted recruitment of CHMP4B and UBPY to HD-PTP and the engagement of UBPY by STAM2 displaces ESCRT-0 from HD-PTP, deubiquitinates EGFR, and releases ESCRT-0 from cargo in favor of ESCRT-III.