Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.

Abstract

ABSTRACT"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or 8 ,uM 5-fluoro-2'-deoxyuridine, gentamicin at 50 ,ug/ml, 10 ,uM cytosine arabinofuranoside, Fungizone at 2.5 ,ug/ml, penicillin at 25 units/ml, and streptomycin at 25 jig/ml and plated at a density of 100,000 cells per ml on glass coverslips in 35-mm-diameter Petri dishes. Cells were cultured in a humidified atmosphere at 37°C in the presence of 5% CO2 for 1-5 days prior to use. For experiments, cells were washed in an extracellular medium comprising 120 mM NaCl, 20 mM Hepes, 4 mM MgCl2, 2 mM CaCl2, 0.4% glucose, and 1 ,M tetrodotoxin at pH 7.2 (adjusted with NaOH). The patch-pipette solution contained 120 mM cesium D-glutamate, 30 mM Hepes, 8 mM NaCl, 1 mM MgCl2, 2 mM ATP, and 0.3 mM GTP at pH 7.2, with either 100 ,tM EGTA or, for imaging studies, 0.4 mM rhod-2 (triammonium salt). Experiments were carried out at room temperature.

Trafficking of MHC molecules to the cell surface creates dynamic protein patches

“…The size of observed patches at steady state is thus determined by a combination of delivery rate and escape rate over barriers to lateral diffusion. We also present evidence that, as is found in neuronal cells (Robinson et al, 1995), vesicles fuse with the surface membrane at specific hotspots.…”

Measurement of Intracellular Calcium

“…A pulsed-laser imaging system was developed by Kinoshita and colleagues (154,194) and was applied to the investigations of rapid Ca 2ϩ signaling in excitation-secretion or excitation-contraction coupling by Fernandez and co-workers (104,262,327). The concept of this system is that high temporal resolution can be obtained by piling up single images obtained at various time lags after repeated stimulation of an event if the event can be reproduced consistently.…”

Confinement of intracellular calcium signaling in secretory and steroidogenic cells

“…The latter mechanisms are not yet entirely elucidated but require relatively high concentrations of Ca 2+ , of the order of 100 mmol/l (34). Such high concentrations of Ca 2+ can be theoretically reached in microdomains, in the proximity of the mouth of the channels (32,35), and have been detected in presynaptic terminals (36) or in adrenal chromaffin cells, in which Ca 2+ 'hot spots' were co-localized with sites of catecholamine release (37). Recently, targeting of the Ca 2+ -sensitive luminescent protein aequorin to the plasma membrane inner surface in A7r5 cells was performed by fusion of the protein with SNAP-25 (38).…”