Ultrastructural organization of bovine chromaffin cell cortex-analysis by cryofixation and morphometry of aspects pertinent to exocytosis

Abstract

We have analyzed ultrathin sections from isolated bovine chromaffin cells grown on plastic support, after fast freezing, by quantitative electron microscopy. We determined the size and intracellular distribution of dense core vesicles (DVs or chromaffin granules) and of clear vesicles (CVs). The average diameter of DVs is 356 nm, and that of CVs varies between 35-195 nm (average 90 nm). DVs appear randomly packed inside cells. When the distance of the center of DVs to the cell membrane (CM) is analyzed, DV density is found to decrease as the CM is approached. According to Monte Carlo simulations performed on the basis of the measured size distribution of DVs, this decay can be assigned to a "wall effect." Any cortical barrier, regardless of its function, seems to not impose a restriction to a random cortical DV packing pattern. The number of DVs closely approaching the CM (docked DVs) is estimated to be between 364 and 629 (average 496), i.e., 0.45 to 0.78 DVs/micron2 CM. Deprivation of Ca2+, priming by increasing [Ca2+]i, or depolarization by high [K+]e for 10 s (the effect of which was controlled electrophysiologically and predicted to change the number of readily releasable granules [RRGs]) does not significantly change the number of peripheral DVs. The reason may be that (a) structural docking implies only in part functional docking (capability of immediate release), and (b) exocytosis is rapidly followed by endocytosis and replenishment of the pool of docked DVs. Whereas the potential contribution of DVs to CM area increase by immediate release can be estimated at 19-33%, that of CVs is expected to be in the range of 5.6-8.0%.

Figure 7

Relative frequency of DVs in cortical regions of chromaffin cells, evaluated by DV center to CM distance. 100% = plateau value inside cell that corresponds on average to a granule density of 6.4 granules/μm³, if related to the evaluated membrane length and converted to density according to Weibel et al. (1966). It should be pointed out that this value is lower than the one obtained from volume fraction (see text). (A) control + Ca²⁺, (B) primed state, (C) stimulation by depolarization, and (D) control − Ca²⁺ (see Materials and Methods). Superimposed on all graphs (continuous curve) is the expected distribution, calculated from the cumulative distribution of granule radii according to Eq. 3.

Figure 4

Size distribution histogram of DVs (n = 219).

Figure 1

Results from Monte Carlo simulations. Top panels show simulated cross-sections. (A) Three cross-sections through a cell, one half of which was filled with granules, as described in the text. (C) Histogram for the number of granule centers found at a certain distance from the membrane. (B) The same for a smaller cell that was carved out of the cell in A as described in the text. (D) Corresponding distance histogram.

Figure 2

Survey of a nonstimulated chromaffin cell (control + Ca²⁺) after the preparation was applied (see Materials and Methods). Note (a) abundance of DVs with compact or spongy contents depending on individual cryofixation, (b) homogenous distribution of DVs in cell interior, (c) decay of apparent DV packing density close to CM, (d) occurrence of small CVs in the cortex, and (e) rare occurrence of some other organelles, such as a mitochondrion (m) in the cytoplasm; n indicates the nucleus. Bar, 1 μm.

Figure 3

Magnification from top right portion of Fig. 2. Note tapering of CM, occasionally with a funnel-like depression towards an approaching DV (arrowhead), and decreasing apparent density of DVs close to CM. Bar, 1 μm.

Figure 5

Cell region attached to the thermanox support. Note occurrence of a filamentous cortical region with apparent DV exclusion. Bar, 1 μm.

Figure 6

Cell region containing exceptionally few DVs as an example of heterogenous DV distribution (compare with Figs. 2 and 5). Note centriole (c), nucleus (n), and Golgi areas (g). Bar, 1 μm.