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Science 30 August 2002:
Vol. 297. no. 5586, pp. 1521 - 1525
DOI: 10.1126/science.1074222
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Research Articles

Controlled Elimination of Clathrin Heavy-Chain Expression in DT40 Lymphocytes

Frank R. Wettey,12 Steve F. C. Hawkins,1 Abigail Stewart,3 J. Paul Luzio,3 Jonathan C. Howard,2 Antony P. Jackson1*

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.

1 Department of Biochemistry, University of Cambridge, Building O, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK.
2 Institute for Genetics, University of Cologne, Zülpicher Strasse 47, D-50674 Cologne, Germany.
3 Department of Clinical Biochemistry and Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.
*   To whom correspondence should be addressed. E-mail: a.p.jackson{at}bioc.cam.ac.uk

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