Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane

Abstract

Changes in nuclear pore complex (NPC) structure are studied following treatments modifying the cisternal calcium levels located between the two lipid bilayers that together form the nuclear envelope. Since the NPC forms the only known passageway across the nuclear envelope, it plays a central role in nucleocytoplasmic transport. Understanding the origin of conformational changes that may affect this trafficking or modify cargo interactions with the NPC is, therefore, necessary to completely understand the function of these complex molecules. In previous studies on the cytoplasmic side of the nuclear envelope, a central mass was observed in the pore of the NPC and its location was shown to be sensitive to the cisternal calcium levels. Here we report atomic force microscopy (AFM) measurements on the nuclear side of the envelope, which also reveal a cisternal calcium dependence in the conformational state of the NPC. These measurements, made at the single nuclear pore level, reveal a displacement of the central mass towards the nuclear side of the membrane following treatments with adenophostin A, a specific agonist of calcium channels (inositol 1,4,5-trisphosphate (IP(3)) receptors) located in the nuclear envelope. We further demonstrate that these conformational changes are observed in nuclear pores lacking the basket structure while samples prepared in the presence of protease inhibitors retain baskets and block AFM measurements of the channel. While these measurements are unable to distinguish whether the central mass is cargo or an integral component of the NPC, its dose-dependent displacement with cisternal calcium levels does suggest links to transport or to changes in cargo interactions with the NPC. Taken together with previous measurements done on the cytoplasmic side of the nuclear envelope, these studies argue against a piston-like displacement of the central mass and instead suggest a more complicated mechanism. One possibility involves a concerted collapse of the NPC rings towards one another following cisternal calcium release, thus leading to the apparent emergence of the central mass from each side of the NPC.

Brownian Dynamics Simulation of Nucleocytoplasmic Transport: A Coarse-Grained Model for the Functional State of the Nuclear Pore Complex

“…The nuclear pore complex (NPC; see Figure 1) is the exclusive gateway of material transport across the nuclear envelope (NE) [1], [2], [3]. This selective gateway plays a critical role in regulating transcription and protecting the genetic material of eukaryotic cells; consequently, its structure is highly conserved from yeast to vertebrates.…”

Changes in Nucleoporin Domain Topology in Response to Chemical Effectors

“…15 Atomic force microscopy (AFM) studies further showed that depletion of nuclear Ca 2+ stores or variations in extranuclear calcium concentrations led to conformational changes within the NPC. Such changes included the appearance of a central plug on the cytoplasmic and nuclear face of the NPC, [16][17][18][19] or alterations in the arrangement of the nuclear basket by ∼ 20-30 nm. 20 ATP can influence NPC conformation as well, and in this context AFM studies revealed that addition of ATP causes dramatic conformational changes on the cytoplasmic surface of the NPC.…”

Calcium regulation of nucleocytoplasmic transport

“…Dunn et al found a shift of central granule up to 9 nm towards the cytoplasmic side and conformational changes on nuclear side at a decreased nuclear cisternal calcium concentration (Moore-Nichols et al, 2002). Activation of IP 3 receptors (Stehno-Bittel et al, 1995a) and rynodine receptors (Erickson et al, 2004) induced calcium to be released from the cisternal spaces, and subsequently caused conformational changes in NPC with an apparent outward displacement of the central mass on both sides (Mooren et al, 2004). Several research groups also speculated that the FG Nups in the NPC might play a dominant role in the NPC conformational change.…”