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Dendrimer-assisted patch-clamp sizing of nuclear pores
J O Bustamante1, E R Michelette, J P Geibel
1The Nuclear Physiology Laboratory, Universidade Tiradentes, Praia Aruana, Brazil. jobustamante@macafeemail.com
Pflugers Archiv : European Journal of Physiology
|April 28, 2000
Summary
This study reveals the nuclear pore complex channel diameter in cardiomyocytes is 8-9 nm using patch-clamp and dendrimers, offering new insights into macromolecular translocation.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Biology
Background:
- Macromolecular translocation (MMT) across the nuclear envelope (NE) is crucial for cellular function and occurs via the nuclear pore complex (NPC).
- The diameter of the NPC aqueous/electrolytic channel (NPCC) is a key determinant of MMT, with previous estimates around 10 nm using electron microscopy and gold particles.
- Previous methods using colloidal gold particles for NPCC diameter assessment were found to interfere with NPC gating.
Purpose of the Study:
- To develop and validate a novel patch-clamp method for accurately measuring the NPCC diameter in live cardiomyocyte nuclei.
- To identify inert probes suitable for assessing NPCC diameter without altering NPC gating dynamics.
- To determine the NPCC diameter in adult cardiomyocytes under physiological conditions.
Main Methods:
- Utilized patch-clamp electrophysiology combined with fluorescence microscopy on adult cardiomyocyte nuclei.
- Verified NPC-mediated macromolecular translocation competency using B-phycoerythrin (BPE) conjugated to a nuclear localization signal (NLS) and observed enhanced green fluorescence protein (EGFP) expression.
- Employed fluorescently labeled Starburst dendrimers as inert probes for patch-clamp measurements after demonstrating colloidal gold particles altered NPCC gating.
Main Results:
- Confirmed cardiomyocyte nuclei competency for NPC-mediated MMT and subsequent protein expression.
- Demonstrated that colloidal gold particles significantly alter NPCC gating, rendering them unsuitable for accurate diameter measurements.
- Calibrated dendrimer measurements using patch-clamp and fluorescence microscopy indicated a cardiomyocyte NPCC diameter ranging from 8 to 9 nm.
Conclusions:
- Patch-clamp combined with fluorescently labeled dendrimers provides a reliable method for assessing NPCC diameter in live cells.
- The NPCC diameter in adult cardiomyocytes is approximately 8-9 nm, refining previous estimates.
- This research establishes a new approach for investigating dynamic NPC transport under physiological conditions.