Figure 5

DAPI stains polyP in distinct punta along axon-like projections. A) At 456 nm (emission) DAPI stains nucleic acids primarily in the nucleus but also in puncta. B) At 560 nm (emission) DAPI stains polyP. PolyP is in the perinuclear space and cytoplasm of the cell body, and is found in distinct puncta along axon-like projections. C) Overlay of A&B, showing polyP puncta on axon-like filaments across from dendritic structures. D) Gel electrophoreses demonstrating presence of and release from synaptic vesicles of polyP. Lanes: M - DNA marker; 1 - long polyP (average 130 chain length); 2 - medium polyP (average 60 chain length); 3 - short polyP (average 15 chain length); 4 - Synaptic vesicles + 70 mM KCl; 5 - Synaptic vesicles alone; 6 - Synaptic vesicles + 70 mM KCl + phosphatase (sample from lane 4); 7 - Synaptic vesicles + phosphatase (sample from lane 5). 70 mM KCl evoked vesicular depolarization. Phosphatase digested polyP. Live imaging of in vitro polyP release from and uptake into distinct puncta - E) T = 0. The arrows indicate distinct puncta to follow through F &G. F) Following a 10 second stimulation at 60Hz, a subpopulation (16% +/-5, SEM, n = 6) of polyP puncta disappear. Adjacent polyP puncta remain, indicating that there are no confounding focal plane shifts or spatial movements. G) 30 seconds later the diminished puncta have begun to refill with polyP.