Clare E. Futter

Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell Extrinsic Function of CTLA-4

An inhibitory T cell receptor acts by stripping activating ligands off dendritic cells. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4–expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28–CTLA-4 system.

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase‐binding (CRIB) motif. The C‐terminal regions of the proteins are essentially unrelated. Using yeast two‐hybrid analysis and in vitro dot‐blots, we show that MLK2 and MLK3 interact with the activated (GTP‐bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c‐Jun N–terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal‐regulated kinase) and p38. When expressed in fibroblasts, MLK2 co‐localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two‐hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation

Here we show that EGF and EGF receptor (EGFR) are trafficked through a subpopulation of multivesicular endosomes/bodies (MVBs) that are distinct from morphologically identical vacuoles that label for the late endosomal marker lyso‐bisphosphatidic acid (LBPA). EGF stimulation increases both MVB biogenesis and inward vesiculation within EGFR‐containing MVBs. Deletion of annexin 1, a substrate of EGFR tyrosine kinase, abolishes the effect of EGF stimulation on inward vesiculation. This phenotype is reversible by transfection with wild‐type but not Y21F phosphorylation mutant annexin 1. Deletion of annexin 1 has no effect on EGF‐stimulated MVB biogenesis, suggesting that MVB biogenesis and inward vesiculation within MVB are mediated by separate mechanisms. Loss or depletion of annexin 1 has no effect on EGF degradation and causes only a small delay in EGFR degradation, indicating that annexin 1 operates downstream of Hrs‐ and ESCRT‐mediated sorting and is required solely for EGF‐stimulated inward vesiculation. Annexin 1 accumulates on internal vesicles of MVB after EGF‐stimulated inward vesiculation, suggesting that it may be required for a late stage in inward vesiculation.

EGF receptor trafficking: consequences for signaling and cancer

Highlights • EGF receptor endocytic traffic can regulate signaling and cell survival.• Signaling from activated EGFR occurs at the endosome as well as the cell surface.• Endocytosis can have positive and negative effects on signaling and tumorigenesis.• EGFR traffic promoted by antineoplastic therapy is important in tumor resistance.

Human VPS34 is required for internal vesicle formation within multivesicular endosomes

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3′-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3′-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3′-kinase activity, whereas vesicle formation requires PI 3′-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Membrane contacts between endosomes and ER provide sites for PTP1B–epidermal growth factor receptor interaction

The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR–PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles.

Rab27b regulates number and secretion of platelet dense granules

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27aash/ash Rab27b−/−) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet α-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for δ-storage pool deficiency in humans.

Rab11-FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling compartment.

Several protein families control intracellular transport processes in eukaryotic cells. Here, we show that the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) directly interacts with the dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1) subunit of the cytoplasmic dynein 1 motor protein complex. We show that Rab11a, FIP3 and DLIC-1 form a ternary complex and that DLIC-1 colocalises with endogenous FIP3 and Rab11a in A431 cells. We demonstrate that association between FIP3 and DLIC-1 at the cell periphery precedes minus-end-directed microtubule-based transport, that FIP3 recruits DLIC-1 onto membranes, and that knockdown of DLIC-1 inhibits pericentrosomal accumulation of key endosomal-recycling compartment (ERC) proteins. In addition, we demonstrate that expression of a DLIC-1-binding truncation mutant of FIP3 disrupts the ability of ERC proteins to accumulate pericentrosomally. On the basis of these and other data, we propose that FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport of material from peripheral sorting endosomes to the centrally located ERC.

Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations

Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome–lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome–lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations.

Multivesicular bodies: co-ordinated progression to maturity

Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB.