Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

Abstract

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.

Figure 1. Two-photon imaging of exocytosis with SRB at the base of a PC12 cell

A, sequential images of SRB fluorescence (F) obtained from the base of a cell loaded with NPE-AM and immersed in a solution containing SRB. Photolysis by UV exposure of NPE was induced at a time between frames a1 and a2. The dye was washed out 50 s after stimulation, and frame a5 was obtained 13 s after the wash. Blue arrows in a5 indicate spots left after washout. B, the difference images (ΔF) shown in frames b1 to b4 were obtained by subtracting the resting image (frame a1) in A from frames a1 to a4, respectively. Fluorescent spots often became brighter over time. Blue arrows in b2–b4 labelled 1–10 represent the events that are referred in other figures. C and D, FWHM diameters of single event 1 in B, and massive event 8 at 2 s (black) and 20 s after (red) the UV stimulation, respectively. E, distributions of FWHM diameters for single (blue), and massive events at 2 s (black) and 20 s (red) after the UV stimulation, respectively.

Figure 2. Analysis of single- and multi-step events

Time courses of the fluorescence intensity of individual spots for single and multi-step events (A) and massive events (B). Traces labelled 1–9 correspond to events 1–9 in frames b2 to b4 in Fig. 3B. Arrow heads in A indicate individual exocytic events.

Figure 3. TEPIQ analyses of LVs in PC12 cells

A and C, histograms of vesicle diameter obtained by TEPIQ analyses of ΔV (A) and ΔS (C), respectively, in single events. B, multicolour TEP imaging of the exocytosis of a single LV. Shown are the background-subtracted images for SRB (ΔF_SRB) and FM1-43 (ΔF_FM1-43) fluorescence before and 3 s after NPE photolysis. D, time courses of ΔV and ΔS during single exocytic events simultaneously measured with SRB (continuous lines) and FM1-43 (dashed lines). Data for 6 vesicles are aligned at the onset of events. Thick lines show the mean time courses. E, time courses of vesicle diameter (6 ΔV/ΔS) for the data shown in D. F, a histogram of vesicle diameter obtained by TEPIQ analyses of ΔV/ΔS in single events. G and H, time courses of ΔV and ΔS (G) and of vesicle diameter (6 ΔV/ΔS) (H) for a massive event. I, a histogram of vesicle diameter obtained by TEPIQ analyses of ΔV/ΔS in massive events.

Figure 4. Behaviours of fusion pores of LVs in PC12 cells

A, C and E, time courses of fluorescence intensity of massive (A and C) and single (E) events before and after washout of SRB. Fluorescence was either eliminated (A) or left unwashed (C and E). These results indicate that the fusion pore was open in A but closed in C and E by the time of washout. Traces 9 and 10 correspond to respective spots in frames b3 and b4 of Fig. 3B, respectively. Dashed lines labelled BG indicate background fluorescence obtained from white squares in B, D and F after washout of SRB. B, D and F, fluorescence images for the data shown in A, C and E, respectively. Images on the left (ΔF) were obtained by subtraction of baseline images before washout of SRB. Images on the right (F) show those after the washout.

Figure 5. Relationship between numbers of LVs and FWHM diameters in multi-step and massive exocytic events in PC12 cells

A, relationship between actual diameters of vesicles (upper panels) and their FWHM diameters of fluorescence profiles (lower panels). B, the number of LVs involved in each exocytic event plotted against its FWHM diameter, which was estimated by dividing the volume of massive events by that of single events (0.005 μm³). The straight line denotes the maximal number of LVs that can be explained by surface vesicles [(d_E−d_F+d_A)/d_A]². The shaded area represents the events that cannot be accounted for by surface exocytosis at the plasma membrane, taking into account an error of 0.1 μm in the estimates of FWHM diameters. Open circles with a number denote data obtained from the events shown in Fig. 1B.

Figure 6. Time courses of LV exocytosis in PC12 cells

A and B, latency histograms for exocytosis of well-resolved individual LV exocytosis for single (A) and multi-step and massive events (B). C, single exocytic events stained by both SRB and 10 kDa FD. The background-subtracted images for SRB (ΔF_SRB) and 10 kDa FD (ΔF_10kDax) fluorescence before and 5 s after NPE photolysis are shown. D, time course of ΔV estimated by TEPIQ analysis with SRB (ΔV_SRB) and 10 kDa FD (ΔV_10kDax).

Figure 7. Ultrastructural identification of exocytic and endocytic vesicles in PC12 cells

Images were obtained at the base of the cells perpendicular to glass coverslips (black arrows). The surface of the glass appeared irregular due to deformation by tissue processing. A, a control cell without stimulation and photoconversion. B–E, cells immersed in FM1-43 for 20 s and stimulated by photolysis of NPE 10 s after the initial exposure to the tracer and fixed 10 s after photolysis. Photoconversion of DAB was induced by FM1-43 molecules remaining after tracer washout. LV and SEx indicate a single LV before and after exocytosis, respectively. CEx represents compound exocytosis by LVs. En denotes direct endocytic vesicles. The external scale bar (0.5 μm) in B applies to all panels.

Figure 8. Pre-stimulus docking and sequential exocytosis

Vesicles are clustered at the plasma membrane, and remained docked after fusion reaction, keeping their fusion pores open. Such primary exocytic vesicles become targets for subsequent exocytosis of LVs deep in the cytosol. Black bars denote ‘intervening strands’ connecting between docked vesicles and the plasma membrane. Open bars denote putative proteins involved in membrane fusions.