Dendrimer-assisted patch-clamp sizing of nuclear pores.
Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.
Bustamante
J O
JO
The Nuclear Physiology Laboratory, Universidade Tiradentes, Praia Aruana, Brazil. jobustamante@macafeemail.com
Michelette
E R
ER
Geibel
J P
JP
Hanover
J A
JA
McDonnell
T J
TJ
Dean
D A
DA
D000818:Animals; D001692:Biological Transport; D001693:Biological Transport, Active; D017979:Gold Colloid / Q000493:pharmacokinetics; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D007473:Ion Channels / Q000378:metabolism; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D010539:Permeability; D011108:Polymers / Q000493:pharmacokinetics*
D017979:Gold Colloid; D007202:Indicators and Reagents; D007473:Ion Channels; D008164:Luminescent Proteins; D011108:Polymers; D049452:Green Fluorescent Proteins