Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane.
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.
Mooren
Olivia L
OL
Department of Chemistry, University of Kansas, Malott Hall, Lawrence, KS 66045, USA. omooren@ku.edu
Erickson
Elizabeth S
ES
Moore-Nichols
David
D
Dunn
Robert C
RC
D021581:Active Transport, Cell Nucleus; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D020013:Calcium Signaling / Q000502:physiology*; D005260:Female; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008968:Molecular Conformation; D009685:Nuclear Envelope / Q000502:physiology*; D022022:Nuclear Pore / Q000737:chemistry* / Q000502:physiology*; D009865:Oocytes / Q000737:chemistry / Q000648:ultrastructure; D014982:Xenopus laevis