Fig 1.
RPE fractionation method and EM images of isolated RPE fractions.
A) In addition to melanosomes, the crude melanosomal fraction contains a large quantity of other membranous structures. B) In the crude lysosomal fraction, only few melanosomes were observed among non-pigmented vesicles. C) The purified melanosomal fraction contains mainly round or ellipsoidal melanosomes. Scale bar 2 μm, 6000x magnification.
Fig 2.
Na+K+ATPase (112 kDa), HSP60 (60 kDa), V-ATPase (96 kDa) and Rab27a (27 kDa) expression in the porcine RPE melanosomal fractions and in the porcine RPE crude lysosomal fraction.
The mitochondrial marker HSP60 is absent in the purified melanosomal fraction whereas the V-ATPase as well as the Na+K+ATPase are expressed in all fractions. Rab27a is enriched in the purified melanosomes.
Fig 3.
Purified melanosomes display intact membrane structure.
Staining of isolated porcine RPE melanosomes with Vybrant™DiO (A-C), a fluorescent membrane dye (green) showed that melanosomal membrane remains intact after isolation process. (D-E) represent brightfield images. Scale bar 5 μm.
Fig 4.
Melanosomes retain their biological activity after isolation.
(A) In ATP hydrolysis assay, purified melanosomes were incubated with ATP and concentration of generated free phosphate (Pi) was measured. Assay was carried out with or without V-ATPase inhibitor bafilomycin A1 (BAF). Values are shown as mean +/- S.E.M (n = 3). (B) Statistically significant difference in Pi formation with BAF is shown as 95% Confidence Interval (indicated by dashed lines, *P < 0.001, unpaired t-test).
Fig 5.
Size analysis of isolated melanosomes.
(A) At lower melanosomal concentration 0.01 μg/μl, one population with an average diameter of 934.5 nm was observed whereas at higher melanosomal concentration (B) 0.1 μg/μl, two populations with average diameters 1533 and 5577 nm were observed.