Then, membrane fusion of the two apposing bilayers is accomplished by a special set of proteins called SNAREs, and the two previously separate organelles now have a direct community. We focused on the involvement of Ann5 in endomembrane trafficking. Ann5 likely promotes vesicle transport following BFA stimulation by binding to negatively charged phospholipids in a Ca2+-dependent manner and to actin filaments that served as molecular tracks in pollen grains and tubes. Annexin A2 is involved in the biogenesis of multivesicular transport intermediates that are L-Ascorbyl 6-palmitate destined for late endosomes in mammals. In Arabidopsis, AnnAt3, 4, 5 and 8 are more closely related to human annexins based on phylogenetic analysis. RNA interference–mediated knockdown of the annexin AnnAt3 caused the trans-Golgi network and multivesicular body markers to colocalize and block vacuolar transport in tobacco mesophyll protoplasts. It has been shown that AnnAt3 is required for the final step of MVBs as a transport carrier from the TGN to the vacuole. The Ann5-overexpressing pollen cell exhibited a higher velocity for cytoplasmic streaming and vesicle transport than control pollen cells following BFA treatment,BMS-378806 further suggesting that Ann5 promoted endomembrane trafficking. Cytoplasmic streaming is fundamentally under the control of myosin motors moving along actin filament bundles that are involved in the intracellular transport of organelles and vesicles. In addition to the fact that actin filaments serve as molecular tracks for Ann5 in vesicle trafficking, Ann5 can bind to F-actin in vitro and may be involved in the dynamic rearrangement of actin through an unknown mechanism. Alterations in the arrangement of actin cables greatly influence the pattern and velocity of cytoplasmic streaming. Ann5 binds to actin and associates with phospholipid membranes in a Ca2+-dependent manner, making it an ideal regulator of endomembrane trafficking where the cell membrane and cytoskeleton interact and are modulated by Ca2+ within pollen cells. The growth rates of pollen tubes and the cytosolic Ca2+ concentration also synchronously oscillate with the same period and phase.