"Late" macroendosomes and acidic endosomes in vertebrate motor nerve terminals.

Processing of spent vesicles in motor nerve terminals often involves creation of large endosomal intermediates, which we call macroendosomes (MEs). In early processing models, similar structures (vacuoles or cisternae) were thought to result from fusion of 50-nm vesicles internalized by clathrin-mediated endocytosis (CME; Heuser and Reese, 1973). Although vesicle fusion with permanent endosomes remains a possibility (e.g., Hoopmann et al., 2010; Uytterhoeven et al., 2011), MEs as we define them originate directly from the plasma membrane via infolding and subsequent clathrin-independent fission (Kadota et al., 1994; Koenig and Ikeda, 1996). This bulk endocytosis has been reported in association with high-intensity stimulation in a variety of nerve terminals, precisely the conditions under which CME from the plasma membrane alone might not keep pace with exocytosis (Miller and Heuser, 1984; Takei et al., 1996; Teng et al., 1999; Richards et al., 2000; Gaffield et al., 2011; reviewed by Clayton and Cousin, 2009). However, we have found that MEs appear after even very brief stimulation in a bath containing the endocytic probes sulforhodamine (Teng et al., 1999) or FM1-43 (Teng et al., 2007). Thus bulk endocytosis occurs along with CME under all conditions of use, including response to low-intensity stimulation, under which CME alone would seem adequate for vesicle reformation. Moreover, although some MEs undergo clathrin-mediated budding into vesicles, others, which we call “ late” MEs, remain intact well after compensatory endocytosis is complete (Teng et al., 2007). If not used exclusively for compensatory endocytosis and vesicle reformation, bulk endocytosis must serve some additional function. One possibility is that MEs participate in a constitutive process not directly related to recycling. Sorting and degradative functions are two possibilities. Both are associated with receptor-mediated endocytosis in the soma of neurons and other cell types. Analogues of these processes within motor terminals have already been described (Weible et al., 2001; Qualmann and Kelly, 2000; Hoopmann et al., 2010; Uytterhoeven et al., 2011). Sorting in particular might be required for long-term homeostasis within motor terminals, which are isolated from the cell body due to transit times required for axonal transport. One function, for which the existence of sorting endosomes has long been inferred, is correction of errors made in reformation of vesicles by CME (reviewed by Shupliakov and Brodin, 2010). Regardless of its function, bulk endocytosis challenges the nerve terminal with a set of unique homeostatic problems. Vesicle membrane proteins are known to reside within permanent pools at the presynaptic membrane of the central nervous system (CNS) (Fernandez-Alfonso and Ryan, 2004) and motor terminals (Tabares et al., 2007), even after completion of compensatory endocytosis following activity. Endosomes internalized from the cleft contain not only these proteins but plasma membrane proteins and high (extracellular) levels of Ca2+ as well. After vesicle budding from MEs is complete, what ultimately happens to these various “leftover” internalized components of the extracellular milieu is unclear. To investigate the function of MEs and the disposition of their contents, we have imaged living late MEs over time. We find evidence for two processing and degradation pathways—fusion of MEs with the plasma membrane, and fusion (or extremely close association) of MEs with a second class of endosomes within the terminal. Both occur after brief periods of low-level synaptic activity. Part of this work has appeared in abstract form (Stewart and Wilkinson, 2010).