Nuclear growth and import can be uncoupled.

What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/ß-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than Xenopus grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.

Nuclear growth and import can be uncoupled.

What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/ß-mediated nuclear import, we show that, while nuclear growth depends on nuclear import, nuclear growth and import can be uncoupled. Nuclei containing fragmented DNA grew slowly despite exhibiting normal import rates, suggesting nuclear import itself is insufficient to drive nuclear growth. Nuclei containing more DNA grew larger but imported more slowly. Altering chromatin modifications caused nuclei to grow less while still importing to the same extent or to grow larger without increasing nuclear import. Increasing heterochromatin in vivo in sea urchin embryos increased nuclear growth but not import. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, live imaging showed that nuclear growth preferentially occurred at sites of high chromatin density and lamin addition, whereas small nuclei lacking DNA exhibited less lamin incorporation. Our hypothesized model is that lamin incorporation and nuclear growth are driven by chromatin mechanical properties, which depend on and can be tuned by nuclear import.

Nuclear F-actin and Lamin A antagonistically modulate nuclear shape.

Nuclear shape influences cell migration, gene expression and cell cycle progression, and is altered in disease states like laminopathies and cancer. What factors and forces determine nuclear shape? We find that nuclei assembled in Xenopus egg extracts in the presence of dynamic F-actin exhibit a striking bilobed nuclear morphology with distinct membrane compositions in the two lobes and accumulation of F-actin at the inner nuclear envelope. The addition of Lamin A (encoded by lmna), which is absent from Xenopus eggs, results in rounder nuclei, suggesting that opposing nuclear F-actin and Lamin A forces contribute to the regulation of nuclear shape. Nuclear F-actin also promotes altered nuclear shape in Lamin A-knockdown HeLa cells and, in both systems, abnormal nuclear shape is driven by formins and not Arp2/3 or myosin. Although the underlying mechanisms might differ in Xenopus and HeLa cells, we propose that nuclear F-actin filaments nucleated by formins impart outward forces that lead to altered nuclear morphology unless Lamin A is present. Targeting nuclear actin dynamics might represent a novel approach to rescuing disease-associated defects in nuclear shape.

Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts.

The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract.

Nuclear Transport Factor 2 (NTF2) suppresses WM983B metastatic melanoma by modifying cell migration, metastasis, and gene expression.

While changes in nuclear structure and organization are frequently observed in cancer cells, relatively little is known about how nuclear architecture impacts cancer progression and pathology. To begin to address this question, we studied Nuclear Transport Factor 2 (NTF2) because its levels decrease during melanoma progression. We show that increasing NTF2 expression in WM983B metastatic melanoma cells reduces cell proliferation and motility while increasing apoptosis. We also demonstrate that increasing NTF2 expression in these cells significantly inhibits metastasis and prolongs survival of mice. NTF2 levels affect the expression and nuclear positioning of a number of genes associated with cell proliferation and migration, and increasing NTF2 expression leads to changes in nuclear size, nuclear lamin A levels, and chromatin organization. Thus, ectopic expression of NTF2 in WM983B metastatic melanoma abrogates phenotypes associated with advanced stage cancer both in vitro and in vivo, concomitantly altering nuclear and chromatin structure and generating a gene expression profile with characteristics of primary melanoma. We propose that NTF2 is a melanoma tumor suppressor and could be a novel therapeutic target to improve health outcomes of melanoma patients.

Organelle size scaling over embryonic development.

Cell division without growth results in progressive cell size reductions during early embryonic development. How do the sizes of intracellular structures and organelles scale with cell size and what are the functional implications of such scaling relationships? Model organisms, in particular Caenorhabditis elegans worms, Drosophila melanogaster flies, Xenopus laevis frogs, and Mus musculus mice, have provided insights into developmental size scaling of the nucleus, mitotic spindle, and chromosomes. Nuclear size is regulated by nucleocytoplasmic transport, nuclear envelope proteins, and the cytoskeleton. Regulators of microtubule dynamics and chromatin compaction modulate spindle and mitotic chromosome size scaling, respectively. Developmental scaling relationships for membrane-bound organelles, like the endoplasmic reticulum, Golgi, mitochondria, and lysosomes, have been less studied, although new imaging approaches promise to rectify this deficiency. While models that invoke limiting components and dynamic regulation of assembly and disassembly can account for some size scaling relationships in early embryos, it will be exciting to investigate the contribution of newer concepts in cell biology such as phase separation and interorganellar contacts. With a growing understanding of the underlying mechanisms of organelle size scaling, future studies promise to uncover the significance of proper scaling for cell function and embryonic development, as well as how aberrant scaling contributes to disease. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Early Embryonic Development > Fertilization to Gastrulation Comparative Development and Evolution > Model Systems.