Clathrin has been previously reported with myosins -V and -VI in synaptosomes prepared from honey bee brains and fractionated in a Percoll gradient (Silva et al., 2002), and myosin-Va has been immunolocalized by Calabria et al. (2010). In this study, we obtained a honey bee brain P2 fraction using the same protocol used to purify myosin-Va from chicken brains. In the vertebrate brain, a similar P2 fraction showed that myosin-Va is associated with selleckchem actin and fragments of the Golgi apparatus, mitochondria, endoplasmic reticulum and synaptic vesicle membrane (Evans et al., 1998). Our results showed that the P2

fraction of the honey bee brain contains myosins -Va and -VI, DYNLL1/LC8, CaMKII, synaptotagmin and clathrin. These data provide new directions for future studies on the interactions between honey bee brain myosin-Va and other target proteins associated with its function. Vertebrate myosin-Va is found in synaptic vesicle preparations and forms stable complexes between synaptic vesicle proteins, such as synaptobrevin II, synaptophysin and syntaxin (Mani et al., 1994, Prekeris and

Terrian, 1997 and Watanabe et al., 2005). While the direct mechanisms of melittin-induced myosin-Va overexpression have yet to be defined, a study has shown that this bee toxin binds to a myriad of calmodulin-binding proteins (Jarrett and Madhavan, 1991). Interestingly, melittin affects the Ion Channel Ligand Library calmodulin-dependent ATPase activity of chick brain myosin-Va (unpublished results). A more recent study demonstrates melittin attacks the plasma membrane of blood cells and induces death by loss of cytoplasmic contents. However, it remains to be determined whether this permeabilization allows release of higher molecular complexes like myosin-Va itself or whether a pro-survival

response could induce protein overexpression. Similarly, the mechanisms underlying NMDA effects remain to be elucidated. A previous study showed myosin-Va levels increased in mammalian cell cultures treated with PJ34 HCl NMDA (Alavez et al., 2004). It is possible that this increase reflect a higher demand of vesicle and organelle trafficking to allow neuronal plasticity in response to NMDA. Finally, like kinesin, myosins -IIb and -Vb (Amparan et al., 2005, Hirokawa et al., 2010, Lei et al., 2001 and Wang et al., 2008), it is also possible that myosin-Va be involved in trafficking of NMDA receptor subunits. Mammals express the DYNLL1 and DYNLL2 isoforms that interact with myosin-Va and cytoplasmic dynein (Naisbitt et al., 2000 and Pfister et al., 2006). DYNLL proteins are highly conserved throughout evolution, and more than 94% sequence identity exists between D. melanogaster and mammals ( Patel-King and King, 2009 and Wilson et al., 2001).