SUMMARY The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.

As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679 -685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol ⑀ are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol ⑀ are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol ⑀ may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol ⑀. We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteinerich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol ⑀, but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site.In Saccharomyces cerevisiae, three DNA polymerases participate in chromosomal DNA replication, pol 1 ␣, pol ␦, and pol ⑀. pol ␣ is primarily involved in the initiation of DNA replication and priming of Okazaki fragments (2), whereas pol ␦ and pol ⑀ are required for completion of synthesis of both the leading and lagging strands. The precise reactions performed by pol ␦ and pol ⑀ on leading and lagging strands, however, have not yet been delineated. In an interesting contrast to yeast chromosomes, simian virus 40 DNA replication does not require pol ⑀. Instead, pol ␣ and pol ␦ are sufficient for viral DNA replication (3). Thus, there appears to be some plasticity in the eukaryotic replication fork.Pol ⑀ is a multi-subunit complex consisting of Pol2p, Dpb2p, Dpb3p, and Dpb4p (4). The Pol2p is the catalytic subunit, and it is encoded by the POL2 gene (5). The Pol2p is a class B polymerase, characterized by a series of conserved domains, called domains I-VI, containing the exonuclease subdomains and the DNA polymerase active site residues in the N-terminal half of the protein (Fig. 1A) (6, 7). Mutations M643I and P710S (the pol2-9 and pol2-18 alleles, respectively) within the polymerase domain in POL2 result in temperature sensitivity (8). The remaining half of POL2 consists of a long region that is conserved in pol ⑀ from all organisms but is not found in any other class B polymerase. An interesting feature of the extreme C terminus is a cysteine-rich stretch of amino acids containing two putative zinc fingers...

Summary Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate [1-3]. While the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property [4-7], it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development [8]. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. While previous studies suggested a role for the N/C volume ratio in MBT timing [1, 9-13], none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. While the DNA-to-cytoplasmic ratio has been implicated in MBT timing [1, 9-18], our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG 1-3 repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.Essential for faithful chromosome maintenance and replication, the ends of eukaryotic chromosomes, telomeres, are dynamic entities whose structures are regulated (26, 43). In the budding yeast Saccharomyces cerevisiae, each telomeric terminal DNA region consists of a tract of irregular TG 1-3 repeat sequences onto which sequence-specific DNA binding proteins and associated proteins assemble. Telomeres in dividing yeast cells are maintained between 250 to 350 bp through a dynamic balance of lengthening and shortening activities. Telomeric DNA shortens as a result of the inability of the general DNA replication machinery to fully replicate the ends of linear DNA molecules (the "end replication problem") and nuclease action (31, 51). Telomere lengthening is primarily mediated by telomerase, a cellular reverse transcriptase that catalyzes the de novo addition of telomeric DNA to chromosome ends, using a sequence within its intrinsic RNA subunit as a template (3).How telomere structure is modulated by the telomeric DNA tract length and how length-dependent structural changes regulate the lengthening and shortening activities at the telomere are central but unresolved questions. Available data support the proposal that long telomeres assume an as-yet-unknown structural state that is inhibitory for telomerase-mediated telomere elongation, while short telomeres can undergo a structural change that results in either recruitment or activation of telomerase (4, 46). Physical models for higher-order telomere structure have been proposed. A "fold-back" model places the end of the telomere inward toward the subtelomeric region, preventing action by telomerase (11,33,45). In a related model, suggested by the clustering of telomeres at the nuclear periphery (17), telomere-telomere interactions restrict accessibility to telomerase.

The size and shape of the nucleus are tightly regulated, indicating the physiological significance of proper nuclear morphology, yet the mechanisms and functions of nuclear size and shape regulation remain poorly understood. Correlations between altered nuclear morphology and certain disease states have long been observed, most notably many cancers are diagnosed and staged based on graded increases in nuclear size. Here we review recent studies investigating the mechanisms regulating nuclear size and shape, how mitotic events influence nuclear morphology, and the role of nuclear size and shape in subnuclear chromatin organization and cancer progression.

It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) ⑀ (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29 -39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol ⑀ holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol ⑀. The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol ⑀, but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay.

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, which places variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components scale to accommodate cell size differences. Nuclear transport has emerged as a conserved means of scaling nuclear size. A meiotic spindle scaling factor has been identified as the microtubule-severing protein katanin, which is differentially regulated by phosphorylation in two different-sized frog species. Anaphase mechanisms and levels of chromatin compaction both act to coordinate cell size with spindle and chromosome dimensions to ensure accurate genome distribution during cell division. Scaling relationships and mechanisms for many membrane-bound compartments remain largely unknown and are complicated by their heterogeneity and dynamic nature. This review summarizes cell and organelle size relationships and the experimental approaches that have elucidated mechanisms of intracellular scaling.

Changes in nuclear size have long been used by cytopathologists as an important parameter to diagnose, stage, and prognose many cancers. Mechanisms underlying these changes and functional links between nuclear size and malignancy are largely unknown. Understanding mechanisms of nuclear size regulation and the physiological significance of proper nuclear size control will inform the interplay between altered nuclear size and oncogenesis. In this chapter we review what is known about molecular mechanisms of nuclear size control based on research in model experimental systems including yeast, Xenopus, Tetrahymena, Drosophila, plants, mice, and mammalian cell culture. We discuss how nuclear size is influenced by DNA ploidy, nuclear structural components, cytoplasmic factors and nucleocytoplasmic transport, the cytoskeleton, and the extracellular matrix. Based on these mechanistic insights, we speculate about how nuclear size might impact cell physiology and whether altered nuclear size could contribute to cancer development and progression. We end with some outstanding questions about mechanisms and functions of nuclear size regulation.