Significant Proportions of Nuclear Transport Proteins with Reduced Intracellular Mobilities Resolved by Fluorescence Correlation Spectroscopy

Allison Paradise, Mikhail K. Levin, George Korza, John H. Carson

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin α, importin β, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin α, importin β, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.

Original languageEnglish (US)
Pages (from-to)50-65
Number of pages16
JournalJournal of Molecular Biology
Volume365
Issue number1
DOIs
StatePublished - Jan 5 2007

Keywords

  • NTF2
  • Ran
  • fluorescence correlation spectroscopy
  • importin
  • nuclear pore complex

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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