Function and regulation of the endosomal fusion and fission machineries

Abstract

Organelles within the endomembrane system are connected via vesicle flux. Along the endocytic pathway, endosomes are among the most versatile organelles. They sort cargo through tubular protrusions for recycling or through intraluminal vesicles for degradation. Sorting involves numerous machineries, which mediate fission of endosomal transport intermediates and fusion with other endosomes or eventually with lysosomes. Here we review the recent advances in our understanding of these processes with a particular focus on the Rab GTPases, tethering factors, and retromer. The cytoskeleton has also been recently recognized as a central player in membrane dynamics of endosomes, and this review covers the regulation of the machineries that govern the formation of branched actin networks through the WASH and Arp2/3 complexes in relation with cargo recycling and endosomal fission.

Figure 1.

Roles of tethering factors within the endolysosomal pathway. (A–D) Sequence of events requiring the different tethering factors. Fusion of early endosomes requires Rab5 and different Rab5 effectors for tethering. EEA1 and Rabenosyn5, which are long coiled-coil proteins, are both required for early endosomal fusion, whereas the multiprotein complex CORVET is required for homotypic fusion of endosomes. How these fusion events differ is not yet clear. Rab7 gradually replaces Rab5 along maturation toward late endosomes. Rab7 promotes both recycling through membrane tubule, a process that requires the retromer, and degradation through the fusion with lysosomes. The latter event depends on the HOPS complex to fuse two Rab7-positive membranes.

Figure 2.

Architecture of CORVET and HOPS complexes. CORVET and HOPS complexes share four subunits (depicted in gray). Vps3 and Vps8 connect the CORVET complex to Rab5, whereas Vps39 and Vps41 connect the HOPS complex to Rab7. The EM density of the HOPS complex, shown next to its diagram, displays a large head, which contains Vps41, and a small tail, which contains Vps39 (Bröcker et al. 2012).

Figure 3.

WASH complex generation of branched actin network at the surface of an endosome. The WASH complex labels a restricted domain of endosome, which corresponds to a location where endosomal tubules are elongated. The WASH complex interacts with the Arp2/3 complex and activates it. The activated Arp2/3 complex nucleates new actin filaments branching off preexisting filaments. The branched actin network has been shown to be instrumental first for the elongation of the endosomal tubule, in tight coordination with microtubule motors, and then to promote endosomal fission. Fission, which occurs at the base of the tubule, requires the activity of the large GTPase dynamin. All these steps are necessary to generate an intermediate of transport-containing sorted cargoes.

Figure 4.

Regulation of WASH within a stable multiprotein complex and associated activities. The WASH complex is composed of five core subunits and of the peripheral capping protein (CP) heterodimer. The core complex regulates the activity of the VCA output domain harbored by WASH. This ouput region interacts with the Arp2/3 complex, activates it, and thus induces the formation of a new actin filament branching of a preexisting filament. The exposure of the VCA output domain is regulated by WASH polyubiquitination catalyzed by the E3 ubiquitin ligase TRIM27 bound to its enhancer MAGE-L2. Recruitment of the WASH complex at the surface of endosomes and recruitment of the E3 ubiquitin ligase require the cargo recognition complex of retromer, composed of Vps35, Vps26, and Vps29. There are multiple weak binding sites along the FAM21 tail for Vps35, suggesting that these machineries form a fluid sorting platform with the abilities to elongate tubules and to fission them. In this context, the role of CP, which can block the dynamics of newly created actin filaments, is not yet understood.

Figure 5.

Endosome clumping in patients affected with mental retardation. In a family of patients where the SWIP subunit of the WASH complex harbors a single point mutation, the whole WASH complex is destabilized (Ropers et al. 2011). In lymphoblastoid cell lines derived from these patients, endosomes, labeled here with transferrin, are clumped (E Derivery and A Gautreau, unpubl.), suggesting that actin polymerization prevents unspecific aggregation of organelles. Neurons appear particularly sensitive to defective endosomal sorting associated with impairment of WASH activity. Scale bar, 10 μm.