Nuclear volume and the number of nuclear pore complexes (NPCs) on the nucleus almost double during interphase in dividing cells. How these events are coordinated with the cell cycle is poorly understood, particularly in mammalian cells. We report here, based on newly developed techniques for visualizing NPC formation, that cyclin-dependent kinases (Cdks), especially Cdk1 and Cdk2, promote interphase NPC formation in human dividing cells. Cdks seem to drive an early step of NPC formation because Cdk inhibition suppressed generation of 'nascent pores', which we argue are immature NPCs under the formation process. Consistent with this, Cdk inhibition disturbed proper expression and localization of some nucleoporins, including Elys/Mel-28, which triggers postmitotic NPC assembly. Strikingly, Cdk suppression did not notably affect nuclear growth, suggesting that interphase NPC formation and nuclear growth have distinct regulation mechanisms.

The nuclear basket (NB) scaffold, a fibrillar structure anchored to the nuclear pore complex (NPC), is regarded as constructed of polypeptides of the coiled-coil dominated protein TPR to which other proteins can bind without contributing to the NB’s structural integrity. Here we report vertebrate protein ZC3HC1 as a novel inherent constituent of the NB, common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. Formerly described as a protein of other functions, we instead present the NB component ZC3HC1 as a protein required for enabling distinct amounts of TPR to occur NB-appended, with such ZC3HC1-dependency applying to about half the total amount of TPR at the NEs of different somatic cell types. Furthermore, pointing to an NB structure more complex than previously anticipated, we discuss how ZC3HC1 and the ZC3HC1-dependent TPR polypeptides could enlarge the NB’s functional repertoire.

In eukaryotic cells, the nucleus is a complex and sophisticated organelle containing genomic DNA and supports essential cellular activities. Its surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It has been observed that the nuclear volume and the number of NPCs almost doubles during interphase in dividing cells, but the coordination of these events with the cell cycle was poorly understood, particularly in mammalian cells. Recently, we demonstrated that cyclin-dependent protein kinases (Cdks) control interphase NPC formation in dividing human cells. Cdks drive the very early step of NPC formation because Cdk inhibition suppressed the generation of "nascent pores," which are considered to be immature NPCs, and disturbed expression and localization of some nucleoporins. Cdk inhibition did not affect nuclear volume, suggesting that these two processes have distinct regulatory mechanisms in the cell cycle. The details of our experimental systems and finding are discussed in more depth. With new findings recently reported, we also discuss possible molecular mechanisms of interphase NPC formation.

The proteins ZC3HC1 and TPR are structural components of the nuclear basket (NB), a fibrillar structure attached to the nucleoplasmic side of the nuclear pore complex (NPC). ZC3HC1 initially binds to the NB in a TPR-dependent manner and can subsequently recruit additional TPR polypeptides to this structure. Here, we examined the molecular properties of ZC3HC1 that enable its initial binding to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 that comprises two similarly built modules, both essential for binding the NB-resident TPR. We show that such a bimodular construction is evolutionarily conserved, which we further investigated in Dictyostelium discoideum and Saccharomyces cerevisiae. Presenting ScPml39p as the ZC3HC1 homolog in budding yeast, we show that the bimodular NuBaID of Pml39p is essential for binding to the yeast NB and its TPR homologs ScMlp1p and ScMlp2p, and we further demonstrate that Pml39p enables linkage between subpopulations of Mlp1p. We eventually delineate the common NuBaID of the human, amoebic, and yeast homolog as the defining structural entity of a unique protein not found in all but likely present in most taxa of the eukaryotic realm.

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.

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Proteins ZC3HC1 and TPR are construction elements of the nuclear pore complex (NPC)-attached nuclear basket (NB). NB-location of ZC3HC1 depends on TPR already occurring NPC-anchored, whereas additional TPR polypeptides are appended to the NB by ZC3HC1. The current study examined the molecular properties of ZC3HC1 that enable it to bind to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 comprising two similarly built modules, both essential for the binding to the NB's NPC-anchored HsTPR. Furthermore, we describe such a bimodular construction as evolutionarily conserved and exemplify the kinship of HsZC3HC1 by the NB- and DdTPR-interacting homolog of Dictyostelium discoideum and by characterizing protein Pml39 as the ZC3HC1 homolog in Saccharomyces cerevisiae. Among several properties shared by the different species' homologs, we unveil the integrity of the bimodular NuBaID of ScPml39p as being essential for binding to the yeast's NBs and its TPR homologs ScMlp1p and ScMlp2p, and we further present Pml39p as enabling interlinkage of Mlp1p subpopulations. In addition to phyla-specific features, we delineate the three species' common NuBaID as the characterizing structural entity of a one-of-a-kind protein found not in all but likely most taxa of the eukaryotic realm.