Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis

Sigrid E. Myrdal, Katherine C. Johnson, Peter Steyger

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Understanding the cellular mechanism(s) by which the oto- and nephrotoxic aminoglycoside antibiotics penetrate cells, and the precise intracellular distribution of these molecules, will enable identification of aminoglycoside-sensitive targets, and potential uptake blockers. Clones of two kidney cell lines, OK and MDCK, were treated with the aminoglycoside gentamicin linked to the fluorophore Texas Red (GTTR). As in earlier reports, endosomal accumulation was observed in live cells, or cells fixed with formaldehyde only. However, delipidation of fixed cells revealed GTTR fluorescence in cytoplasmic and nuclear compartments. Immunolabeling of both GTTR and unconjugated gentamicin corresponded to the cytoplasmic distribution of GTTR fluorescence. Intra-nuclear GTTR binding co-localized with labeled RNA in the nucleoli and trans-nuclear tubules. Cytoplasmic and nuclear distribution of GTTR was quenched by phosphatidylinositol-bisphosphate (PIP2), a known ligand for gentamicin. Cytoplasmic and nuclear GTTR binding increased over time (at 37°C, or on ice to inhibit endocytosis), and was serially competed off by increasing concentrations of unconjugated gentamicin, i.e., GTTR binding is saturable. In contrast, little or no reduction of endocytotic GTTR uptake was observed when cells were co-incubated with up to 4 mg/mL unconjugated gentamicin. Thus, cytoplasmic and nuclear GTTR uptake is time-dependent, weakly temperature-dependent and saturable, suggesting that it occurs via an endosome-independent mechanism, implicating ion channels, transporters or pores in the plasma membrane as bioregulatory routes for gentamicin entry into cells.

Original languageEnglish (US)
Pages (from-to)156-169
Number of pages14
JournalHearing Research
Volume204
Issue number1-2
DOIs
StatePublished - Jun 2005

Fingerprint

Endocytosis
Gentamicins
Aminoglycosides
Fluorescence
Texas red
Endosomes
Ice
Phosphatidylinositols
Ion Channels
Formaldehyde
Clone Cells

Keywords

  • Aminoglycoside
  • Cytoplasmic
  • Drug uptake
  • Gentamicin
  • Non-endocytotic

ASJC Scopus subject areas

  • Sensory Systems

Cite this

Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis. / Myrdal, Sigrid E.; Johnson, Katherine C.; Steyger, Peter.

In: Hearing Research, Vol. 204, No. 1-2, 06.2005, p. 156-169.

Research output: Contribution to journalArticle

Myrdal, Sigrid E. ; Johnson, Katherine C. ; Steyger, Peter. / Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis. In: Hearing Research. 2005 ; Vol. 204, No. 1-2. pp. 156-169.
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