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 s tructure and function. The NPCC diameter was previously determined to be co ngruent to 10 nm with electron microscopy (EM) using the translocation of c olloidal gold particles. Here we present patch-clamp and fluorescence micro scopy data from adult cardiomyocyte nuclei that demonstrate the use of patc h-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoer ythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) dem onstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei express ed enhanced green fluorescence pro-rein (EGFP) after 5-10 h of incubation w ith the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-c lamp showed that colloidal gold particles were not useful probes; they modi fied NPCC gating. As a re suit of this finding, we searched for an inert cl ass of par tides that could be used without irreversibly affecting NPCC gat ing and found that fluorescently labeled Starburst dendrimers, a distinct c lass of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diamet er varies between 8 and 9 nm. These studies open a new direction in the inv estigation of live, continuous NPC dynamics under physiological conditions.