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Figure 1. Typical fluorescent micrographs of rhodamine labeled AChR distributions after stimulation with agrin or laminin-1 alone or in combination. See Table 2 for stimulus protocols. Note that the analysis is restricted to the perimeter of the images (Materials and Methods) A, no stimulus. There is a subtle presence of small aggregates about the cell perimeter (arrows). B, Agrin. C, laminin. D, Agrin and Laminin. E, Agrin followed by laminin. F, laminin followed by Agrin. All of these latter conditions consistently display larger, more intense receptor aggregates than the control cells in A.
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Figure 2. Quantitative analysis of AChR aggregate parameters. The stimulus protocols for all panels are shown at the bottom: C control (no stimulus), A agrin, L laminin-1, AL agrin and laminin, A>L agrin followed by laminin, L>A laminin followed by agrin. See Table 2 for protocol details. A-D, numbers at the top indicate statistical groupings based on one factor ANOVA F tests, p < .05. A, expression of receptors (arbitrary units). Both agrin and LN1 increased the label intensity of AChRs significantly, and agrin clearly more so than LN1. In all combinations assayed the effects of agrin and LN1 on receptor expression were additive. B, number of aggregates detected per cell. On this basis LN1 alone was similar to control cells, while agrin alone or in combination gave similarly increased numbers of aggregates. C, aggregate size in micrometers. In this case both agrin and laminin, alone or in combination, produced similarly enhanced aggregate size compared to control cells. D, aggregate density (arbitrary units). With regard to this metric agrin alone was indistinguishable from control cells, while laminin alone or in combination showed a significant increase in density. E, the mean Z corresponding to the average separation between aggregates. For random distributions this metric has an expected value of 0. Significant deviations from random separation (p < .01) are marked with *. When significant, negative values indicate that aggregates are closer together than random distributions, and positive values would indicate aggregates farther apart than average (see Materials and Methods). In this set of experiments controls showed a small but significant deviation from randomness, and agrin alone showed a large deviation. In both cases the aggregates were found to be further apart than expected at random.
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Figure 3. Typical fluorescent micrographs of double labeled cell pairs following the stimulus protocols. Images on the left were fluorescein labeled for AChRs, while the corresponding images on the right were rhodamine labeled for α-DG . Note that the analysis is restricted to the perimeter of the images (Materials and Methods) A, B, no stimulus. The arrows illustrate a lack of contamination of the fluorescein (left) signal by the rhodamine (right) image. C, D, Agrin; arrows illustrate the absence of contamination of the rhodamine (right) image by the fluorescein label on the left. E, F, laminin; the arrows illustrate modest correlation between the two labels. G, H, Agrin and Laminin. The arrows indicate regions of substantial correlation between the two molecular distributions. The examples of correlation, and the lack thereof, are for purposes of illustration only and not representative of the respective stimulus conditions. See text for quantitative measures of correlation between the two molecular distributions.
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Figure 4. Quantitative analysis of α-DG aggregate parameters. The stimulus protocols for all panels are shown at the bottom: C control (no stimulus), A agrin, L laminin-1, AL agrin and laminin. A-D, numbers at the top indicate statistical groupings based on one factor ANOVA F tests, p < .05. A, density of α-DG (arbitrary units). Agrin clearly increased the expression of α-DG on the cell surface. The effects of LN1 cannot be assessed, because it competes for the binding of the immunological probe for α-DG. B, number of aggregates detected per cell. On this basis LN1, alone or in combination with agrin, significantly increased the number of aggregates; agrin by itself had no effect on aggregate number. C, aggregate size in micrometers. These results parallel those for aggregate number; laminin alone or in combination increased aggregate size. D, aggregate density (arbitrary units). Here we find a deviation from the pattern in A and B: only LN1 by itself causes density increase, while in the presence of agrin this increase is largely blocked. E, the mean Z corresponding to the average separation between aggregates. Significant deviations from random separation (p < .01) are marked with *. Unlike the AChR distributions shown in Figure 2D, α-DG aggregate distributions are found closer together than random under all experimental conditions. This cooperativity is enhanced by LN1 alone or in combination with agrin.
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Figure 5. Analysis of AChR and α-DG label correlations for the stimulus protocols. The stimulus protocols for all panels are shown at the bottom: C control (no stimulus), A agrin, L laminin-1, AL agrin and laminin. Red bars, raw data correlation. Green bars, high frequency correlation (see Figure 7, Materials and Methods). Blue bars, above threshold correlation. The latter two metrics (green and blue) are the most relevant to the question of molecular aggregation, and show little if any correlation under control conditions. All metrics show progressive and significant increases as the stimulus protocol is changed to agrin, LN1, and agrin with LN1.
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Figure 6. Aggregate size distributions following the various stimulation protocols. Each left-right pair represents the frequency histograms of aggregate size for AChRs (left) and α-DG (right). A, controls (no stimulus). B, agrin. C, laminin. D, agrin and laminin. All abscissas are aggregate size in microns.
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Figure 7. Quantitative analysis of fluorescent micrographs. A, fluorescent micrograph of rhodamine labeled AChRs at the meridian of the cell, showing six aggregates labeled 1–6. For each aggregate there is an associated nearest neighbor distance – the lesser of the distances to its two flanking neighbors. Aggregates #1 and #2 happen to have the same nearest neighbor distance, indicated by the white arc. B, the same cell as in A overlain to illustrate sector analysis. The software identifies the cell perimeter and represents it as a series of 256 sectors, starting at the left and proceeding counter-clockwise. In B every fourth sector is marked in white to illustrate its size and position. C, plots of sequential sector analysis. Raw Data is simply a plot of sector intensity vs. position in degrees, where the arrows indicate the corresponding aggregates identified in A. The data are processed to produce the High Frequency components, which can be thought of as the original data with the Low Frequency (gradually changing) components removed. A threshold is applied to the high frequency components (dashed line) to identify the Above Threshold aggregates, where again the arrows indicate aggregates corresponding to A.
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