The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.

The early endosome (EE), also known as the sorting endosome (SE) is a crucial station for the sorting of cargoes, such as receptors and lipids, through the endocytic pathways. The term endosome relates to the receptacle-like nature of this organelle, to which endocytosed cargoes are funneled upon internalization from the plasma membrane. Having been delivered by the fusion of internalized vesicles with the EE or SE, cargo molecules are then sorted to a variety of endocytic pathways, including the endo-lysosomal pathway for degradation, direct or rapid recycling to the plasma membrane, and to a slower recycling pathway that involves a specialized form of endosome known as a recycling endosome (RE), often localized to the perinuclear endocytic recycling compartment (ERC). It is striking that 'the endosome', which plays such essential cellular roles, has managed to avoid a precise description, and its characteristics remain ambiguous and heterogeneous. Moreover, despite the rapid advances in scientific methodologies, including breakthroughs in light microscopy, overall, the endosome remains poorly defined. This Review will attempt to collate key characteristics of the different types of endosomes and provide a platform for discussion of this unique and fascinating collection of organelles. Moreover, under-developed, poorly understood and important open questions will be discussed.

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)-and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.

β1 integrins bind to the extracellular matrix and stimulate signaling pathways leading to crucial cellular functions, including proliferation, apoptosis, cell spreading and migration. Consequently, control of β1 integrin function depends upon its subcellular localization, and recent studies have begun to unravel the complex regulatory mechanisms involved in integrin trafficking. We report that the C-terminal Eps15-homology (EH) domain-containing protein EHD1 plays an important role in regulating β1 integrin transport. Initially, we demonstrated that RNAi-knockdown of Ehd1 results in impaired recycling of β1 integrins and their accumulation in a transferrin-containing endocytic recycling compartment. Mouse embryonic fibroblast (MEF) cells derived from EHD1-knockout mice (Ehd1–/– MEF) exhibited lower overall levels of β1 integrins on the plasma membrane, but higher cell-surface-expressed activated β1 integrins, and larger, more prominent focal adhesions resulting from slower kinetics of focal adhesion disassembly. In addition, both migration and cell spreading on fibronectin were impaired in Ehd1–/– MEF cells, and these defects could be similarly induced by EHD1-RNAi treatment of normal Ehd1+/+ MEF cells. They could also be rescued by transfection of wild-type EHD1 into Ehd1–/– MEF cells. Our data support a role for EHD1 in β1 integrin recycling, and demonstrate a requirement for EHD1 in integrin-mediated downstream functions.

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotidebinding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1 -/-embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.