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.
Department of Biochemistry, University of Cambridge, Building O, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK.
Frank R
FR
Wettey
Steve F C
SF
Hawkins
Abigail
A
Stewart
J Paul
JP
Luzio
Jonathan C
JC
Howard
Antony P
AP
Jackson
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