Research

Mechanisms of Steady-State Nuclear Size Regulation in Xenopus (NIH R15GM106318; 9/1/13 - 8/31/17)

PI: Levy

Cell size varies greatly among different cell types and organisms, especially during early development when cells divide rapidly without overall embryonic growth. A fundamental question is how organelle size is appropriately regulated relative to cell size, a phenomenon we refer to as scaling. The nucleus is one organelle that exhibits exquisite size scaling both during development and between species. Importantly, the normal relationship between nuclear and cell size is often abrogated in cancers and other disease states. Many cancers are diagnosed and staged based on graded increases in nuclear size, yet mechanisms that regulate nuclear size are largely unknown and may directly contribute to cancer progression. How steady-state nuclear size is determined is poorly understood. This knowledge gap prevents us from understanding how nuclear size impacts chromatin organization, gene expression, and cell function, particularly in the context of oncogenesis.

Our long-term goal is to elucidate mechanisms of nuclear size regulation to understand how nuclear size impacts cell and nuclear function and sub-nuclear organization. The objective of this proposal is to identify the molecular mechanisms that regulate nuclear expansion and shrinking and to demonstrate how these mechanisms control nuclear size in vivo in the intact embryo. Our central hypothesis is that steady-state nuclear size is determined by balanced nuclear growth and shrinking activities. Previous work demonstrated that a nuclear import mechanism contributes to nuclear scaling between two different size Xenopus frog species, identified some of the key proteins involved, and showed that a similar mechanism contributes to reductions in nuclear size during early frog development. Using these results as a starting point, we will test our central hypothesis by pursuing the following three specific aims.

Aim 1: Identify mechanisms that regulate nuclear expansion. Nuclei reconstituted in egg extracts from two different size Xenopus species exhibit differential nuclear growth rates. Through biochemical characterization of these extracts and microscopy, we will identify how nuclear import cargos contribute to interspecies differences in nuclear expansion.
Aim 2: Identify mechanisms that regulate nuclear shrinking. Early stage Xenopus embryos contain larger cells and nuclei than later stage embryos. We find that large nuclei isolated from early stage embryos become smaller when incubated in cell extract from later stage embryos. We will utilize live time-lapse microscopy to characterize the dynamics of this novel activity and biochemical approaches to identify factors responsible for nuclear shrinking.
Aim 3: Demonstrate the in vivo activities of nuclear scaling factors. In vitro Xenopus extract approaches have already identified several factors that control nuclear size and others will be identified in Aims 1 and 2 of this proposal. To determine if mechanisms of nuclear size control are similar in vivo, we will manipulate these nuclear scaling activities in intact Xenopus embryos by mRNA and inhibitor microinjection and use live cell microscopy to examine effects on nuclear size and dynamics. These experiments will test our central hypothesis in an in vivo context to determine if a balance of nuclear growth and shrinking activities sets steady-state nuclear size in the embryo.

The proposed study will transform our understanding of nuclear size regulation because it combines biochemical, genetic, and experimental manipulations in the highly tractable Xenopus system. The expected outcome is elucidation of nuclear size control mechanisms, which will shed light on size regulation of other organelles. This project will also lay the foundation to develop R01 funding to approach the novel hypothesis that nuclear size regulates chromatin structure, gene expression, and cellular function. Ultimately, our studies will have implications beyond nuclear size control. It has long been noted that organellar scaling is essential to cellular balance, yet it has been difficult to clarify the mechanisms that maintain size ratios in a cell. This research will thus significantly impact our understanding of how scaling is regulated during biogenesis and growth, as well as provide insight on cancer development and progression.