Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes

Science. 2002 Aug 30;297(5586):1521-5. doi: 10.1126/science.1074222.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • B-Lymphocytes / metabolism*
  • B-Lymphocytes / ultrastructure
  • Cell Line
  • Chickens
  • Clathrin / biosynthesis
  • Clathrin / genetics*
  • Clathrin / physiology
  • Clathrin Heavy Chains
  • Down-Regulation
  • Doxycycline / pharmacology
  • Endocytosis / physiology
  • Gene Expression Regulation* / drug effects
  • Lysosomes / physiology
  • Membrane Proteins / physiology
  • Molecular Sequence Data
  • Signal Transduction

Substances

  • Clathrin
  • Membrane Proteins
  • Clathrin Heavy Chains
  • Doxycycline

Associated data

  • GENBANK/AJ427965
  • GENBANK/AJ429072
  • GENBANK/AJ429073
  • GENBANK/AJ429074
  • GENBANK/AJ429075
  • GENBANK/AJ429076