Intracellular late endosomal tubules (LET's), discovered during studies on Niemann Pick C disease, were found to be an essential component of the late endocytic pathway that includes vesicular late endosomes and lysosomes. The NPC I gene, deciphered, in 1997 elucidated the structure of the NPC I membrane spanning protein which was shown, with immunocytochemistry, to reside in late endosomes in cells after internalization of LDL cholesterol. Further studies with a fluorescent NPC I ?GFP construct showed that NPC I ?GFP is a valuable membrane marker for late endosomes by illuminating the dynamic tubular component this compartment in living cells. These studies showed also, that cholesterol blocked the mobility of LETs leading to formation of immobile, elongated tubules in mutant cells and decreased communication between the late endosomal and lysosomal organelles. CT60 CHO cells, an NPC I null mutant, were primarily studied with the NPC I ?GFP construct since they are more easily transfected than human fibroblasts. However, in present quantitative study of several mutant human fibroblasts we use an adenoviral construct of WT?NPCI?GFP (Ad?NPCI?GFP) advantageous for infecting human fibroblasts with high efficiency. Studies on normal and NPC human fibroblasts transduced with Ad?NPCI?GFP were done to show that its trafficking is identical to that reported previously for NPC?GFP. Normal fibroblasts contain dynamic LET's whereas cholesterol laden NPC I and NPC2 fibroblasts initially contain immobile LET's labeled with Ad?NPCI?GFP. Depletion of cholesterol from mutant cells after extended expression of WT Ad?NPC?GFP or, as shown previously in nontransduced cells, by incubation in lipid depleted serum, re?establishes late endosomal tubular trafficking and egress of accumulated lysosomal lipids. Thus the cellular phenotype of the NPC lesion includes cholesterol laden immobilized late endosomal tubules and lysosomes and dysfunctional communication between those organelles. Structure of Late Endosomal Tubules (LET). NPC1-GFP traffics between late endosomal vesicles and lysosomes via an extensive system of branching tubules with rapid rates of movement ranging between 1-5mm/s. Late endocytic tubules (LETS) move by a dynamic process involving tubulation and fission, followed by rapid anterograde and retrograde migration along microtubules. LET membranes are rich in unesterified cholesterol as shown by filipin cytochemistry. Electron microscopic analysis shows that LET's range dramatically in diameter (500-60nm) in size dependent on their content of luminal bilayered lamellae that appear to emanate from the internal luminal leaflet of the tubule bounding membrane. Further 3D EM analysis shows concentric lamellae accumulate in these tubules. A cholesterol-loaded endocytic compartment (CLC) containing mostly elongated membranous tubules with luminal lamellae (visualized with electron microscopy) has been isolated by density gradient centrifugation, from normal and mutant human diploid fibroblasts fed high levels of low density lipoprotein (LDL). We intend to characterize the cholesterol/phospholipid content of the CLC fraction. Understanding the structure, lipid and protein content of these anommolous LET tubules, containing luminal bilayers, can provide clues to the role of cholesterol and lipid transport proteins such as the NPC1 protein. Dysfunction of the late endosomal tubular component of the endocytic pathway is a phenotype of Niemann Pick C disease. We have found that cholesterol overload is responsible for the stasis of this membrane flow pathway. Our studies are designed to discover ways to maintain the integrity of membrane transport in this arm of the endocytic pathway and thus maintain normal trafficking of intracellular lipids.
|Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K et al. (2009) Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors. Virol J 6:61|