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Updated: Jul 20, 2026
08:53
Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells
Published on: May 16, 2017
Ultrastructural nuclear import assay.
Hualin Zhong1, Helen Shio, Nabeel R Yaseen
1Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10021, USA.
Methods (San Diego, Calif.)
|August 29, 2006
Summary
This study uses electron microscopy to show that the transcription factor PU.1 requires energy for nuclear import but not a specific carrier. PU.1 moves to the nucleus via Ran-dependent binding to the nuclear pore complex component NUP153.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Nuclear transport mechanisms have been studied for decades using electron microscopy (EM).
- Early studies employed microinjected gold-conjugated proteins, evolving to immuno-EM with gold-conjugated antibodies and recombinant proteins to map nuclear pore complex (NPC) interactions.
Purpose of the Study:
- To investigate the nuclear import mechanism of the transcription factor PU.1 using an ultrastructural nuclear import assay.
- To characterize the energy dependence and carrier requirements of PU.1 nuclear import.
Main Methods:
- Preparation of recombinant, gold-conjugated PU.1 protein.
- Performing in vitro nuclear import assays with permeabilized cells or isolated nuclear envelopes.
- Utilizing transmission electron microscopy (TEM) for ultrastructural analysis and data interpretation.
Main Results:
- Nuclear import of PU.1 is energy-dependent but carrier-independent.
- Gold-conjugated PU.1 translocated to the nuclear side of the NPC and into the nucleus when energy was supplied.
- Biochemical assays suggested Ran-dependent binding of PU.1 to NUP153 at the NPC nuclear face.
Conclusions:
- The study details methods for ultrastructural nuclear import assays.
- Findings elucidate a novel Ran-dependent mechanism for PU.1 nuclear import involving NUP153 interaction.
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