Selective blocking of clathrin-mediated endocytosis by RNA interference: epsin as target protein.
Epsin is an essential accessory protein exclusively implicated in clathrin-mediated endocytosis and therefore an ideal target to study involvement of this entry route in the uptake of bioligands. The technique of RNA interference (RNAi) was exploited to generate a cell line constitutively silencing epsin expression in a sequence-specific manner In these Caco-2(eps-) cells, quantitative reverse transcription PCR (RT-PCR) revealed a severe depletion of the epsin messenger RNA (mRNA) level in cells, reaching a factor > 10(6). The reduction at the mRNA level in the Caco-2(eps-) cells was paralleled by a decrease of 75% at the protein level. In order to evaluate transfection effects at the functional level, uptake of transferrin and epidermal growth factor (EGF) in transfected Caco-2(eps-) and control cells was evaluated. In control cells, respectively, approximately 72% of transferrin and approximately 66% of EGF were internalized, whereas in Caco-2(eps-) cells only approximately 25% of transferrin and approximately 34% of EGF was taken up, confirming that in the transfected cells, endocytosis via coated pits was prominently compromised. The reduced uptake was not the result of an inhibition of transferrin recycling. The effects of direct treatment with chlorpromazine on Caco-2 cells, also monitored from the degree of transferrin internalization, were compared with those elicited by RNAi.
Vanden Broeck
Davy
D
University of Antwerp, Antwerp, Belgium.
De Wolf
Marc J S
MJ
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D018938:Caco-2 Cells; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014168:Transferrin; C114154:epsin; D004815:Epidermal Growth Factor; D002746:Chlorpromazine