, 2001 and Sundborger et al., 2011) and from studies of endophilin (Rvs167) in budding yeast (Kaksonen et al., 2005). Further interest in a potential role of endophilin in fission was elicited by the proposal that synaptojanin-dependent PI(4,5)P2 dephosphorylation is directly implicated

in the fission reaction by generating a line tension between the PI(4,5)P2-rich plasma membrane and a PI(4,5)P2-depleted, deeply invaginated coated bud (Liu et al., 2009; Selleck CT99021 see also Chang-Ileto et al., 2011). Thus, endophilin could participate in fission via its interaction with both dynamin and synaptojanin. However, an essential action of endophilin and synaptojanin in fission contrasts with the prominent accumulation of CCVs,

but not of CCPs, in synaptojanin 1 knockout (KO) mice (Cremona et al., 1999 and Hayashi et al., 2008). Finally, and surprisingly, a recent find protocol study suggested that the interactions of endophilin with dynamin and synaptojanin are not required for the role of endophilin in endocytic SV recycling (Bai et al., 2010). To help dissect the role of endophilin at synapses, in particular in SV fission and uncoating, we have carried out an analysis of the effects produced by deletion of all three endophilin genes in mice. The most striking change observed at synapses without endophilin is an accumulation of CCVs without a change in the number of CCPs, supporting the idea that the major function of endophilin is to couple fission to uncoating in partnership the with synaptojanin and Hsc70/auxilin. We also show that synaptojanin is recruited before fission and independently of dynamin, suggesting that depletion of PI(4,5)P2 from the vesicle bud may precede fission. Collectively, these findings advance our understanding of the sequence of events underlying SV recycling and, more generally, of the process of clathrin-mediated endocytosis. The accumulation of endophilin on the tubular stalks of the arrested endocytic CCPs in fibroblasts that lack dynamin (dynamin

1,2 double KO cells) proves that the recruitment of endophilin to the pits occurs before fission and independently of dynamin (Ferguson et al., 2009). However, it is unknown whether synaptojanin 1, in particular the synaptojanin 1 isoform that lacks clathrin and AP-2 binding sites (synaptojanin 1-145), is recruited upstream of dynamin as well. To address this question, we expressed fluorescently tagged synaptojanin 1-145, endophilin 2, and clathrin light chain (LC) in pairwise combinations in dynamin double KO cells, which we then imaged by confocal microscopy. In control cells, both endophilin and synaptojanin 1-145 had a primarily cytosolic distribution, with only a few transient puncta that coincided with a subpopulation of late-stage CCPs (Perera et al., 2006) (Figure 1A and insets).