How the sizes of the mitotic spindle and interphase nucleus are regulated within a cell remains largely unknown. This gap in knowledge prevents us from understanding the functional significance of organelle size control, particularly in the context of various cancers in which the scaling relationship between organelle and cell size has gone awry. Our long-term goal is to identify mechanisms of organelle size regulation in order to better understand how organelle size and morphology impact cell function. The objective of this proposal is to elucidate the molecular basis of organelle size control. Specifically, we will address the question of how physical constraints imposed by cell-size impact the size, shape, and function of both the mitotic spindle and interphase nucleus. Our central hypothesis is that scaling of nuclear and spindle size with cell size is mediated through a limiting component mechanism. To test this hypothesis, we have developed an innovative experimental platform that utilizes microfluidic-based technology to encapsulate cell-free extracts, allowing us to address previously intractable questions regarding organelle scaling. The rationale for completion of this research is to provide information that can be used to develop more accurate and predictive models of organelle assembly and function, which in turn may lead to new strategies for treatment of cancers and other conditions linked to improper nuclear and spindle function.
Aim 1 : To determine how cytoplasmic volume regulates nuclear scaling. In this aim we will utilize microfluidics and Xenopus extracts to assemble nuclei in cytoplasmic droplets of defined size, shape, and composition to determine whether changes in cytoplasmic volume are sufficient to account for in vivo nuclear scaling.
Aim 2 : To identify molecular effectors of mitoti spindle and interphase nuclear scaling using microfluidic encapsulation. In this aim, we will employ microfluidic emulsion/droplet-generating devices to characterize the molecular mechanisms of the scaling relationship between cytoplasmic volume and spindle/nuclear size. Using an unbiased biochemical screen in combination with candidate molecule approaches, we expect to identify components, i.e. scaling factors, whose relative amounts determine spindle/nuclear size.
Aim 3 : To develop microfluidic droplet manipulation techniques to enable dynamic control over cytoplasm volume and content in four dimensions (geometry and time).
This aim will develop microfluidic techniques by which droplet volume or composition may be changed at specified time points to induce and observe dynamic changes in organelle size. Completion of the work proposed in these aims is expected to (i) produce a fundamental advance in our basic understanding of the mechanisms that control the size of the mitotic spindle and nucleus and (ii) demonstrate the tremendous utility and potential of combining microfluidics with an already powerful biological model system, cell-free extracts derived from Xenopus eggs and embryos. This is significant because it will fundamentally advance our knowledge of how the size of the mitotic spindle and nucleus are regulated, providing targets for new therapeutic approaches.
Nuclear and spindle morphology are often deregulated in cancer cells, many cancers are diagnosed and staged based on graded increases in nuclear size, and aberrant organelle size might actually be required for cell homeostasis and viability. Therapeutic manipulation of organelle size could provide a means to selectively target and kill only affected cells, so our proposal to elucidate factors and mechanisms of scaling will shed light on novel approaches and targets for the diagnosis and treatment of cancer.
