A Cell-Free Assay Using <em>Xenopus laevis </em>Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

University of Wyoming

Cell size varies greatly among different cell types and organisms, especially during early development when cell division is rapid with little overall growth. A fundamental question is how organelle size is regulated relative to cell size. The nucleus exhibits exquisite size scaling during development and between species, and nuclear size is often altered in cancer cells. Recent studies have elucidated mechanisms of nuclear size regulation in a variety of experimental systems, opening the door to future research on how nuclear size impacts upon cell and nuclear function and subnuclear organization. In this review we discuss studies that have clarified nuclear size control mechanisms and how these results have or will contribute to our understanding of the functional significance of nuclear size.

Nuclear size regulation: from single cells to development and disease.

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.

Sizing and shaping the nucleus: mechanisms and significance.

Dramatic changes in cell and nuclear size occur during development and differentiation, and aberrant nuclear size is associated with many disease states. However, the mechanisms that regulate nuclear size are largely unknown. A robust system for investigating nuclear size is early Xenopus laevis development, during which reductions in nuclear size occur without changes in DNA content. To identify cellular factors that regulate nuclear size during development, we developed a novel nuclear resizing assay wherein nuclei assembled in Xenopus egg extract become smaller in the presence of cytoplasmic interphase extract isolated from post-gastrula Xenopus embryos. We show that nuclear shrinkage depends on conventional protein kinase C (cPKC). Increased nuclear cPKC localization and activity and decreased nuclear association of lamins mediate nuclear size reductions during development, and manipulating cPKC activity in vivo during interphase alters nuclear size in the embryo. We propose a model of steady-state nuclear size regulation whereby nuclear expansion is balanced by an active cPKC-dependent mechanism that reduces nuclear size.

cPKC regulates interphase nuclear size during Xenopus development.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells.

Nuclear size is generally maintained within a defined range in a given cell type. Changes in cell size that occur during cell growth, development, and differentiation are accompanied by dynamic nuclear size adjustments in order to establish appropriate nuclear-to-cytoplasmic volume relationships. It has long been recognized that aberrations in nuclear size are associated with certain disease states, most notably cancer. Nuclear size and morphology must impact nuclear and cellular functions. Understanding these functional implications requires an understanding of the mechanisms that control nuclear size. In this review, we first provide a general overview of the diverse cellular structures and activities that contribute to nuclear size control, including structural components of the nucleus, effects of DNA amount and chromatin compaction, signaling, and transport pathways that impinge on the nucleus, extranuclear structures, and cell cycle state. We then detail some of the key mechanistic findings about nuclear size regulation that have been gleaned from a variety of model organisms. Lastly, we review studies that have implicated nuclear size in the regulation of cell and nuclear function and speculate on the potential functional significance of nuclear size in chromatin organization, gene expression, nuclear mechanics, and disease. With many fundamental cell biological questions remaining to be answered, the field of nuclear size regulation is still wide open.

Lisa J. Edens

Department of Molecular Biology

University of Wyoming

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Lisa J. Edens

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Biology

University of Wyoming

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Department of Molecular Biology