Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy

Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded in the double membrane nuclear envelope. They are the major gateways mediat ing transport of ions, small molecules, proteins, RNAs, and ribonucleoprote in particles in and out of the nucleus in interphase cells. Understanding s tructural changes at the level of individual pores will be a prerequisite t o eventually correlate the molecular architecture of the NPC with its disti nct functional states during nucleocytoplasmic transport. Toward this goal, we have employed time-lapse atomic force microscopy of native NPCs kept in buffer, and recorded calcium-mediated structural changes such as the openi ng (i.e. +Ca2+) and closing (i.e. -Ca2+) of individual nuclear baskets. Mos t likely, this structural change of the nuclear basket involves its distal ring which may act as an iris-like diaphragm. In order to directly correlat e distinct structural features with corresponding functional states and dyn amic aspects, we also addressed the question of whether the "central plug" or "transporter" actually represents a calcium-sensitive component of the N PC involved in mediating nucleocytoplasmic transport. Our data indicate tha t in the absence of ATP, cytoplasmic plugging/unplugging of the NPC is inse nsitive to calcium. (C) 1999 Academic Press.