Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Abstract

Optical imaging of individual vesicle exocytosis is providing new insights into the mechanism and regulation of secretion by cells. To study calcium-triggered secretion from astrocytes, we used acridine orange (AO) to label vesicles. Although AO is often used for imaging exocytosis, we found that imaging vesicles labeled with AO can result in their photolysis. Here, we define experimental and analytical approaches that permit us to distinguish unambiguously between fusion, leakage, and lysis of individual vesicles. We have used this approach to demonstrate that lysosomes undergo calcium-triggered exocytosis in astrocytes.acridine orange ͉ imaging ͉ secretion ͉ evanescent wave microscopy ͉ ATP E xocytosis, the process by which a vesicle inside of a cell fuses to the cell surface, serves a plethora of important functions in all eukaryotic cells. These include insertion of membrane proteins such as ion channels and receptors, addition of new membranes during neuronal development, and secretion of neurotransmitter at the synapse.Over the past few decades, much has been learned about the physiology of exocytosis in neurons. Through the use of biochemistry, electrophysiology, and genetics, a considerable body of knowledge has been gathered about the molecular participants in exocytosis. However, much remains to be learned about the dynamics of these molecules. Our knowledge has been limited in part by our ability to image single molecules, or even the fusion of single vesicles. Fortunately, the use of total internal reflection fluorescence microscopy (TIR-FM) allows imaging fusion of single vesicles (1-6).Ca 2ϩ -triggered exocytosis, considered a hallmark of excitable cells, has also been reported in nonexcitable cells such as fibroblasts (5,(7)(8)(9). In all nonexcitable cells, Ca 2ϩ -triggered exocytosis is believed to serve a role in membrane repair (10). Additionally, some cells, such as hematopoietic cells, use Ca 2ϩ -triggered exocytosis for specialized functions such as release of cytolytic molecules (11). Astrocytes are nonexcitable cells of the nervous system that were previously thought to have only a protective role for neurons. However, astrocytes have now been shown to be more intricately involved in regulating neuronal physiology (12)(13)(14). This is believed in part to be due to the ability of astrocytes to exocytose a variety of molecules (15).Exocytosis of synaptic vesicles (16), dense core granules (3, 17), and even astrocytic vesicles that undergo Ca 2ϩ -triggered exocytosis (18) have been imaged by using the fluorescence of acridine orange (AO), a weak base that accumulates in acidified vesicles (19). Accumulation at high concentrations causes quenching of AO fluorescence. On release from the vesicle, AO fluorescence is unquenched and thus increases rapidly. Such rapid increase in AO fluorescence followed by its lateral diffusion is called an ''AO flash'' and has been used as a hallmark of exocytosis (3,(20)(21)(22).To examine potential Ca 2ϩ -regulated exocytic vesicles in astrocytes, we incubate...

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

“…11,12,20,44 We show that astrocyte cell membrane injury causes a greater [Ca 2+ ] c increase, triggering more robust lysosome exocytosis. Receptor stimulation causes a sustained and low rate of exocytosis where lysosomes release a part of their luminal content and none of their membrane proteins (Figures 2 and 6).…”

“…Fluorescently labeling lysosomes with fluorescein isothicyanate (FITC) dextran allows imaging without photodamage. 12 [Ca 2+ ] c increase by purinergic signaling triggered sustained FITC dextran-labeled lysosome exocytosis that peaked within a minute, and most fusions occurred within the first 90 s of ATP addition (Figure 1f; Supplementary Video 1). Each cell exocytosed 10.5 ± 2.5 lysosomes in a minute ( Figure 1j); however, many membrane proximal lysosomes did not exocytose (Figure 1f, inset).…”

“…We have shown that monitoring CD63-GFP fluorescence by TIRF allows distinguishing between lysosome exocytosis and lysis. 12 As the CD63-GFP-labeled lysosome enters the TIRF field, there is a rapid increase in vesicle-associated fluorescence intensity ('peak intensity'). Exocytosis and lysis both decrease the peak fluorescence.…”

“…Thus, integrated TIRF fluorescence ('total intensity') in the cell membrane plane decreases during lysis but remains constant during exocytosis. 12,35,36 Upon ionomycin treatment, the 'total intensity' of lysosomal CD63-GFP increased and then remained constant ( Figure 2f inset and corresponding plot in Figure 2h), demonstrating that CD63-GFP dispersed freely into the plasma membrane. This established that the lysosomes underwent exocytosis.…”

Supportive or information-processing functions of the mature protoplasmic astrocyte in the mammalian CNS? A critical appraisal

“…This approach failed to produce SICs in neurons in the hippocampus, leading to the idea that changes seen using other means of increasing astrocytic Ca 2+ are of a pathological rather than physiological nature. Also some of the astrocytic fusion events appear to involve lysosomes (Jaiswal et al, 2007;Zhang et al, 2007), which is unlikely to be physiological. The question also arises of what it means if there is bona fide vesicular release from astrocytes.…”