An ER clamp for endosome fission

Abstract

Endosomes are known to undergo budding and fission reactions that separate regions destined for lysosomal degradation from carriers to be recycled to the plasma membrane. A recent paper (Rowland et al, 2014) shows that contact sites between endosomes and the endoplasmic reticulum (ER) define the position and timing for fission. This uncovers an unanticipated role for the ER in controlling endosomal sorting and maturation.

See also: AA Rowland et al (November 2014)

Eukaryotic cells internalize portions of their plasma membrane and molecules associated with it by endocytosis, a process that entails invagination and budding of defined regions of the plasma membrane (Conner & Schmid, 2003). Endocytic vesicles fuse with early endosomes, from where cargoes can be sorted along three main routes—back to the plasma membrane (known as recycling), to the trans-Golgi network, or to lysosomes. The latter pathway involves maturation from early to late endosomes by piecemeal changes of the endosome membrane before the late endosomes ultimately fuse with dense-core lysosomes (Huotari & Helenius, 2011).

Live microscopy has revealed that endosomes are dynamic organelles that undergo frequent fusion and fission reactions, and there is evidence that such events are crucial for both endosomal sorting and maturation (Puthenveedu et al, 2010; Huotari & Helenius, 2011). Central in regulating endosome dynamics are the Rabs, members of a large family of small GTPases that control membrane traffic through interactions with various effector proteins (Stenmark, 2009). On early endosomes, Rab5 resides on vacuolar regions and promotes membrane fusion, whereas Rab4 resides on tubular regions and promotes recycling (Sonnichsen et al, 2000). While Rab5-containing endosome subdomains involved in membrane fusion have been well characterized (Sonnichsen et al, 2000), it has remained more enigmatic how the exact sites of membrane fission are determined. The work by Rowland et al now shows that tubular regions of the ER play a central role in defining such sites.

The ER forms contact sites with multiple cellular membranes, including endosomes (van der Kant & Neefjes, 2014), and ER–endosome contacts become more frequent as endosomes mature (Friedman et al, 2013). Such contact sites have previously been implicated in receptor dephosphorylation in-trans, negative regulation of microtubule motor association, and cholesterol and Ca²⁺ exchange (Rocha et al, 2009; Eden et al, 2010; van der Kant & Neefjes, 2014). The new function of ER–endosome contact sites in defining points of endosome fission shows a striking parallel to sites of mitochondrial fission, which are also marked by contacts with ER tubules (Friedman et al, 2011). Thus, interactions between the ER and endosomes, the main organelles of the exocytic and endocytic pathways, respectively, are even more important than previously appreciated.

In order to facilitate studies of endosome fission, Rowland et al treated cells with dynasore, a drug that promotes the presence of tubular early endosomes through inhibition of dynamin GTPases (Macia et al, 2006). The authors noted that sites of membrane diffusion-limited constriction and fission for early and late endosomes correlated in space and time with formation of ER–endosome contacts. Interestingly, overexpression of the ER shaping protein Reticulon 4, which induces long ER tubules, was found to inhibit endosome fission, suggesting that ER structure and dynamics indeed control endosome fission (Rowland et al, 2014).

But how does the ER define endosomal fission sites? ER–endosome contacts were found to form immediately prior to fission, and the contact sites correlated spatially and temporally with the presence of FAM21, a protein that interacts with the cargo sorting retromer complex (Gomez & Billadeau, 2009). Retromer is known to be recruited to tubular parts of endosomes and contains subunits that promote membrane tubulation (Cullen & Korswagen, 2012). FAM21 is a subunit of the WASH complex that promotes actin nucleation, and actin has been shown to play an essential role in stabilizing endosome domains that promote cargo recycling (Puthenveedu et al, 2010). However, Rowland et al showed that the ER is recruited to endosome fission sites independent of WASH complex assembly or WASH-mediated actin nucleation, suggesting that the retromer-binding function of FAM21 might be most relevant in this case.

One possible function for ER–endosome contact sites is to serve as stations for recruitment of factors that mediate membrane scission, among which FAM21 and retromer are possible candidates. Other, not mutually exclusive possibilities, are that the contact sites could mediate lipid or Ca²⁺ exchange that might trigger membrane fission (Rowland et al, 2014).

ER–endosome contact site-dependent endosome fission results in products that typically consist of one vacuolar Rab5-positive vesicle that contains an endocytosed cargo destined for lysosomal degradation (epidermal growth factor) and one tubular Rab4-positive element that contains a cargo destined for recycling (transferrin) (Rowland et al, 2014) (Fig1). These fission products are relatively large, but it is worth noting that much of the recycling from early endosomes is thought to occur via small vesicles and tubules that are below the resolution of the light microscope (Stoorvogel et al, 1996). In future studies, it will be important to identify the molecular composition and regulation of the ER–endosome membrane contact sites that define endosome fission, to investigate the exact functions of the fission products in cargo trafficking and endosome maturation, and to understand which molecular forces drive the fission reaction.

Figure 1. ER tubules define sites of endosome scission.

An early endosome that contains endocytosed epidermal growth factor (EGF) and transferrin (Tf) is contacted by a tubular ER element that defines the site of constriction of the tubule neck, to which FAM21(red) is recruited. Constriction is followed by fission into two endosomal elements, a Rab5-positive vacuole that contains EGF and a Rab4-positive tubule that contains Tf.