Live imaging system for visualizing nuclear pore complex (NPC) formation during interphase in mammalian cells

Abstract

Nuclear pore complexes (NPCs) are 'supramolecular complexes' on the nuclear envelope assembled from multiple copies of approximately 30 different proteins called nucleoporins (Nups) that provide aqueous channels for nucleocytoplasmic transport during interphase. Although the structural aspects of NPCs have been characterized in detail, NPC formation and its regulation, especially during interphase, are poorly understood. In this study, using the temperature-sensitive RCC1 mutant tsBN2, a baby hamster kidney 21 cell line, we found that a lack of RCC1 activity inhibited NPC formation during interphase, suggesting that RanGTP is required for NPC formation during interphase in mammalian cells. Utilizing the reversible RCC1 activity in tsBN2 cells, we established a live-cell system that allows for the inhibition or initiation of NPC formation by changes in temperature. Our system enables the examination of NPC formation during interphase in living cells. As a lack of RCC1 decreased some Nups containing unstructured phenylalanine-glycine repeats in the NPC structure, we propose that RCC1 is also involved in maintaining NPC integrity during interphase in mammalian cells.

Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

“…Since importins always release their cargoes when bound to RanGTP (Vasu and Forbes, 2001; Hetzer et al , 2002; Harel et al , 2003a; Tahara et al , 2008), the release of Pom121 from importin β must be a crucial step for both its NPC targeting and interphase NPC assembly. The requirement for RanGTP in this step agrees with several reports showing the importance of RanGTP in interphase NPC assembly (Ryan et al , 2003; D’Angelo et al , 2006; Iino et al , 2010; Maeshima et al , 2010). Because our present data demonstrate the importance of interactions between Pom121 and the INM for interphase NPC assembly (Figures 6, 4Bc, and 4Cc), we predict that Pom121 molecules enter the nucleus through the nuclear pores as an integral membrane protein bound to importin α/β.…”

Nuclear size, nuclear pore number and cell cycle

“…[3][4][5][6][7] To address this question, we established novel techniques to study nuclear volume and NPC formation, and investigated how they were regulated during interphase in dividing human cells. 8,9 Our results indicate that Cdks, especially Cdk1 and Cdk2, control NPC formation during interphase. 9 Cdk inhibition suppressed the generation of the "nascent pores," which are immature NPCs in the process of forming and interrupted expression and localization of some nucleoporins.…”

“…9 Recently, using baby hamster kidney tsBN2 cells, a temperature-sensitive mutant of RCC1, we established a system allowing the inhibition or initiation of interphase NPC formation by changing the temperature. 8 In this system, interphase NPC formation actively occurs at permissive temperature (32°C), but not at non-permissive temperature (39.7°C) cells. 9 Nups 62, 88, 93, 98, 107 and 133 were expressed at similar levels in roscovitine-treated and control cells.…”

Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase

“…By comparison, the process of NPC assembly in interphase in metazoan cells is poorly characterized and has only recently become the focus of research (D’Angelo et al, 2006; Doucet et al, 2010; Iino et al, 2010). Although assembly during both phases of the cell cycle is thought to produce indistinguishable pores, and some of the regulatory requirements such as activity of the small GTPase Ran are conserved between interphase and mitosis (Walther et al, 2003b; D’Angelo et al, 2006; Iino et al, 2010), the assembly takes place under fundamentally different conditions. At the end of mitosis, a large number of NPCs assemble simultaneously.…”