Aminoglycoside antibiotics traffic to the Golgi complex in LLC-PK1 cells.
Aminoglycoside antibiotics are known to be internalized via endocytosis and have been associated with subcellular organelle dysfunction; however, the route of intracellular trafficking and their distribution remain largely unknown. To address these questions, a Texas Red conjugate of gentamicin (TRG) was synthesized for dual-labeling experiments with the endoplasmic reticulum, Golgi, and lysosomal markers DiOC6-3, C6-NBD-ceramide, and fluorescent dextrans, respectively. Confocal images were overlaid to determine areas of colocalization. Initial characterization studies of the fluorescent gentamicin analogue revealed that both internalization and accumulation were inhibited by excess unlabeled gentamicin. Furthermore, the fluorescent gentamicin label was colocalized with unlabeled gentamicin, using immunologic techniques. LLC-PK1 cells were exposed to the fluorescent gentamicin in media containing 1 mg/ml labeled gentamicin for 8 h and then either fixed or chased with gentamicin-free media for an additional 16 or 40 h (24 to 48 h total). Studies with fluorescent dextrans revealed rapid intracellular colocalization within the endosomal and lysosomal systems. Neither endoplasmic reticulum nor mitochondrial colocalization could be detected. However, Golgi colocalization was revealed using both confocal and electron microscopic techniques at 8 h of TRG incubation, and continued to be present for an additional 40 h. Protein synthetic rates were quantified and revealed decreased synthesis at the 24-h chase mark. These results suggest that TRG can serve as a fluorescent tracer for aminoglycoside trafficking within cells. The fluorescent marker remained associated with vesicular structures at all times and colocalized with the Golgi apparatus. It is postulated that this early association of gentamicin with the Golgi complex may be an avenue for delivery of aminoglycosides to other intracellular compartments.
Sandoval
R
R
Department of Medicine, Roudebush Veterans Administration Medical Center, Indiana University Medical Center, Indianapolis 46202-5116, USA.
Leiser
J
J
Molitoris
B A
BA
D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics*; D002518:Ceramides / Q000032:analysis; D004721:Endoplasmic Reticulum; D011992:Endosomes / Q000648:ultrastructure; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000032:analysis; D005839:Gentamicins / Q000493:pharmacokinetics*; D006056:Golgi Apparatus / Q000378:metabolism*; D018374:LLC-PK1 Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000648:ultrastructure; D008715:Methionine / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D011506:Proteins / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014966:Xanthenes / Q000032:analysis
D000900:Anti-Bacterial Agents; D002518:Ceramides; D005456:Fluorescent Dyes; D005839:Gentamicins; D011506:Proteins; D014966:Xanthenes; D015100:3,3'-Diaminobenzidine; C034657:Texas red; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D008715:Methionine; D009327:4-Chloro-7-nitrobenzofurazan