Distribution and Co-localization of endosome markers in cells

Abstract

Clathrin mediated endocytosis is one pathway for internalization of extracellular nano materials into cells [1, 2]. In this pathway, proteins attached to receptors and the internalized materials are encapsulated in clathrin coated membrane vesicles that subsequently fuse with or transform into intracellular compartments (early and late endosomes) as their contents are being directed to the lysosomes for degradation. The following proteins are commonly used to mark the pathway at various stages: Rab5 (early endosome), Rab7 (late endosome), and LAMP-1 (lysosome). In this work, we studied the distribution and co-localization of these marker proteins in two cell lines (C2C12 and A549) to determine whether these markers are unique for specific endosome types or whether they can co-exist with other markers. We estimate the densities and sizes of the endosomes containing the three markers, as well as the number of marker antibodies attached to each endosome. We determine that the markers are not unique to one endosome type but that the extent of co-localization is different for the two cell types. In fact, we find endosomes that contain all three markers simultaneously. Our results suggest that the use of these proteins as specific markers for specific endosome types should be reevaluated. This was the first successful use of triple image cross correlation spectroscopy to qualitatively and quantitatively study the extent of interaction among three different species in cells and also the first experimental study of three-way interactions of clathrin mediated endocytic markers.

Internalization of Phospholipid-Coated Gold Nanoparticles

“…The pink and purple spots in all four images reflect the co-localization of red with blue markers. Consistent with previous work, we observed that there are a few purple and pink spots in all images to indicate co-localization of Rab5 with Rab7, Rab11 and LAMP-1, and of Rab7 with LAMP-1 [50]. The association of PCAuNPs and Rab5 would be shown by a yellow color and the association of PCAuNPs with Rab7 and LAMP-1 would be shown by a cyan.…”

“…Table 5 shows the averages of the auto correlation amplitude, average fitted laser beam width, and average intensity from a data set of 20-30 images of A549 cells exposed to phospholipid-coated gold nanoparticles for 1, 2, 4, and 24 h. The images show that there is a gradual increase in intensity and size of PCAuNP compartments over the 24 h. Table 5 shows the averages of the auto correlation amplitude, average fitted laser beam width, and average intensity from a data set of 20-30 images of A549 cells exposed to phospholipid-coated gold nanoparticles for 1, 2, 4, and 24 h. We observed an increasing trend of the auto correlation amplitude (g(0,0)) with time. The fit for the laser beam width ranges between 0.38-0.48 microns; this is consistent with the images shown in Figure 7 as larger size clusters were observed after 24 h. In previous work, we characterized the sizes of clathrin-mediated endocytic markers to be approximately 0.40 microns [50]. Thus, simply from this observation, one can assume phospholipid-coated gold nanoparticles may be contained in endocytic compartments.…”

“…Specifically, the fraction of PCAuNP-containing compartments that also contain two markers range from 8-40% in C2C12 cells and from 24−39% in A549 cells. These numbers are consistent with the fact that between 50 and 80% of these markers co-exist on the same endosome compartments [50].…”

“…In previous work, we observed that there was significant co-localization of Rab5 and Rab7 markers, of Rab5 and LAMP-1 markers, and of Rab7 and LAMP-1 markers, demonstrating that these markers are not uniquely defining their respective compartments [50]. We therefore sought to determine the fractions of PCAuNPs that associate with compartments with more than one marker.…”

“…We can conclude that in both cell lines, these gold nanoparticles are found in the endocytic compartments. It is important to note that, because the markers for the various compartments are not unique [50], some of these compartments will have more than one marker associated with them. This point will be discussed further later.…”