Figure 5. APC-dependent RNAs associate with heterogeneous clusters at the tips of protrusions.

(A-C) The indicated endogenous RNAs were detected by in situ hybridization. Signal intensities of observed spots are shown in the associated surface plot profiles, which also indicate the size of each image in microns. In internal regions all detected RNAs exist as single molecules. At the tips of protrusions, they exist in clusters of multiple RNAs. (D) In situ hybridization images and surface plot profiles of endogenous Ddr2 and Net1 RNAs detected in the same cell. Peripheral clusters can contain distinct RNA species. (E) In situ hybridization images and surface plot profiles of endogenous Pkp4 RNA and polyA RNA detected in the same cell. Peripheral clusters are characterized by a visible accumulation of polyA RNA. (Note that only enlarged views of individual protrusions are shown in panels A-E). (F) Whole cell masks of cells processed for FISH were used to derive a 2 μm-wide peripheral edge mask. (G) Whole-cell FISH images of the indicated endogenous RNAs (for additional examples see Figure 5—figure supplement 1). Scale bars: 15 μm. (H) For each RNA, signal intensity histograms of all detected particles found within the 2μm-wide peripheral edge area, were used to group particles into single RNAs or RNA clusters (see Figure 5—figure supplement 1 ). Table lists number of particles in each category for the indicated RNAs. p-values based on Fisher’s exact test against Arpc3 RNA. (I) Percent of overlap of the indicated RNA clusters with polyA clusters. n = number of particles observed in ca. 25 cells.

Figure 5—figure supplement 1. Intensity histograms of endogenous APC-dependent or control RNAs.

(A) Whole cell masks of cells processed for FISH were used to derive a 2 μm-wide peripheral edge mask. (B) FISH images of the indicated endogenous APC-dependent RNAs or non-localized RNAs (Arpc3 and P4hb). For each RNA, the associated graphs present a frequency distribution histogram of the signal intensities (in arbitrary units) of all detected particles found within the 2μm-wide peripheral edge area, as shown in (A). Intensities > 400 were all grouped in one bin. Number of analyzed particles: 150–1500, depending on the RNA, imaged in >25 cells. The existence of one major peak indicates that the majority of analyzed particles reflect single RNA molecules. Particles with higher intensities (>400) reflect homotypic RNA clusters. (See Figure 5H). Note that peripheral clusters of APC-dependent RNAs are not observed in all cells. The amount of each RNA in clusters is a small proportion of the total RNA amount.

Figure 5—figure supplement 2. Amount of APC-dependent RNAs per cell.

Graph shows the average number of the indicated RNAs detected per cell, based on FISH images such as those shown in Figure 5—figure supplement 1. n > 25 cells; error bars: standard error.

Figure 5—figure supplement 3. Peripheral cluster formation by MS2-reporter RNAs.

Localized reporter RNAs, which carry the 3’UTR of Rab13 or Net1 RNAs, as well as 24 copies of MS2-binding sites, were expressed in cells expressing GFP-tagged MS2 coat protein. Snapshots of live cell images show that the RNAs exist as single particles in internal regions and as clusters at the tips of protrusions. Scale bars: 5 μm.