Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts.
The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.
Lange
Yvonne
Y
Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612, USA. ylange@rush.edu
Ye
Jin
J
Rigney
Mike
M
Steck
Theodore L
TL
D000666:Amphotericin B / Q000494:pharmacology; D000736:Androstenes / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000924:Anticholesteremic Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000633:toxicity; D004351:Drug Resistance / Q000502:physiology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides; D009542:Niemann-Pick Diseases / Q000378:metabolism* / Q000473:pathology; D015739:Nocodazole / Q000494:pharmacology
D000736:Androstenes; D000900:Anti-Bacterial Agents; D000924:Anticholesteremic Agents; D000970:Antineoplastic Agents; D003505:Cyclodextrins; D018942:Macrolides; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D000666:Amphotericin B; C040929:bafilomycin A1; D002784:Cholesterol; D015739:Nocodazole