Endosomal EEA1 Research Articles

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro.

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments.

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

IQGAP1 critically promotes CXCR4 chemokine receptor expression and signaling by regulating CXCR4 intracellular trafficking through EEA-1 endosomes (CCR3P.212)

Abstract IQGAP1 is a ubiquitous, cytoskeletal-interacting scaffold protein frequently overexpressed in cancer. CXCR4 is a chemokine receptor that signals upon binding stromal cell-derived factor 1 (SDF-1, CXCL12). CXCR4 regulates lymphocyte migration and development and cancer cell proliferation and metastasis. Here, we show that IQGAP1 potently enhances CXCR4’s expression, trafficking, and function in the Jurkat T cell line and epithelial cancer cells. The SDF-1-induced cell migration and activation of ERK MAPK were impaired in IQGAP1-deficient cells, even after forced CXCR4 overexpression. Exploring the mechanism, we found that SDF-1 treatment induced CXCR4 endocytosis and caused IQGAP1 to bind a-tubulin and localize to EEA-1 endosomes containing endocytosed CXCR4. In IQGAP1-deficient cells, CXCR4-containing EEA-1 endosomes accumulated at the distal ends of microtubules instead of moving towards the centrosome. This inhibited CXCR4’s signaling and cell-surface expression by preventing CXCR4 from trafficking into the Rab11 endosomes previously shown to be a critical site of CXCR4 signaling and recycling to the cell-surface. In contrast, IQGAP1 depletion had no effect on TCR trafficking, which does not require EEA-1 endosomes. Together, these results characterize a novel function of IQGAP1 as a regulator of EEA-1 endosomes, and in addition identify IQGAP1 as a critical enhancer of CXCR4 cell-surface expression and signaling in lymphocytes and epithelial cancer cells.

ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.

ABSTRACTEndosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.

Mammalian CORVET is required for fusion and conversion of distinct early endosome subpopulations.

Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.

Gαs promotes EEA1 endosome maturation and shuts down proliferative signaling through interaction with GIV (Girdin)

The organization of the endocytic system into biochemically distinct subcompartments allows for spatial and temporal control of the strength and duration of signaling. Recent work has established that Akt cell survival signaling via the epidermal growth factor receptor (EGFR) occurs from APPL early endosomes that mature into early EEA1 endosomes. Less is known about receptor signaling from EEA1 endosomes. We show here that EGF-induced, proliferative signaling occurs from EEA1 endosomes and is regulated by the heterotrimeric G protein Gαs through interaction with the signal transducing protein GIV (also known as Girdin). When Gαs or GIV is depleted, activated EGFR and its adaptors accumulate in EEA1 endosomes, and EGFR signaling is prolonged, EGFR down-regulation is delayed, and cell proliferation is greatly enhanced. Our findings define EEA1 endosomes as major sites for proliferative signaling and establish that Gαs and GIV regulate EEA1 but not APPL endosome maturation and determine the duration and strength of proliferative signaling from this compartment.

GIPC Is Recruited by APPL to Peripheral TrkA Endosomes and Regulates TrkA Trafficking and Signaling

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.