| Popis: |
We examined the effect of purified immunoglobulins G (IgG) from amyotrophic lateral sclerosis (ALS) patients on the mobility and exocytotic release from Lysotracker-stained vesicles in cultured rat astrocytes. Time-lapse confocal images were acquired and vesicle mobility analyzed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. At rest, when mobility was monitored for 2 minutes in bath with Ca2+, two vesicle populations were discovered: i) non-mobile vesicles (6.1%) with total track length (TL) 1 μm, averaging at 3.03±0.01 μm (n=20200). ALS IgG (0.1 mg/ml) from 12 out of 13 patients increased the TL of mobile vesicles by ∼24% and maximal displacement (MD) by ∼26% within 4 minutes, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca2+, indicating that ALS-IgG vesicle mobility-enhancement involves changes in Ca2+ homeostasis. To examine, if enhanced mobility relates to elevated Ca2+ activity, cells were stimulated by 1 mM ATP, a cytosolic Ca2+ increasing agent, in the presence (2 mM) and in the absence of extracellular Ca2+. ATP stimulation triggered an increase in TL by ∼7% and ∼12%, and a decrease in MD by ∼11% and ∼1%, within 4 minutes respectively. Interestingly none of the stimuli triggered the release of vesicle cargo. It is concluded that ALS IgG-enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis. |
