Perforated MDCK cells support intracellular transport.
We have developed a method for perforating the plasma membrane of MDCK cells while retaining cellular functions. A nitrocellulose acetate filter was applied to the apical side of cells, grown on a glass coverslip, and allowed to dry. Segments of the apical plasma membrane adhered to the filter and were detached from the cell layer by shearing when the filter was peeled off. This allowed macromolecules such as antibodies and enzymes to diffuse into the cells. The cells were otherwise intact as judged by light and electron microscopy. The perforated cells maintained their capacity to support vesicular transport of proteins and lipids. Vesicular stomatitis virus infected cells readily incorporated [35S]methionine into G protein following permeabilization. This G protein was core-glycosylated during assembly in the endoplasmic reticulum, and was further transported to the trans Golgi with high efficiency. Experiments using lipid probes demonstrated that newly synthesized fluorescent sphingolipids were transported from the Golgi complex to the basolateral cell surface in perforated cells. Our results show that perforated cells provide a convenient and efficient alternative to cell-free assays for studying the molecular mechanism of intracellular transport.
Simons
K
K
European Molecular Biology Laboratory, Heidelberg, FRG.
Virta
H
H
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D007668:Kidney; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins