Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.
We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation. This variant cell line did not undergo apoptosis in the absence of clathrin and was used to examine the effects of clathrin depletion on membrane-trafficking pathways. Receptor-mediated and fluid-phase endocytosis were both substantially inhibited, and transferrin-receptor recycling was modestly inhibited. Surprisingly, clathrin removal did not affect the morphology or biochemical composition of lysosomes.
Wettey
Frank R
FR
Department of Biochemistry, University of Cambridge, Building O, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK.
Hawkins
Steve F C
SF
Stewart
Abigail
A
Luzio
J Paul
JP
Howard
Jonathan C
JC
Jackson
Antony P
AP
D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002645:Chickens; D002966:Clathrin / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D033922:Clathrin Heavy Chains; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D008247:Lysosomes / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D015398:Signal Transduction
D002966:Clathrin; D008565:Membrane Proteins; D033922:Clathrin Heavy Chains; D004318:Doxycycline