Understanding the RCC1-dependent spindle formation process in cell-like droplets generated using microfluidic devices

Abstract

Microtubules (MTs) are key components of the cytoskeleton and play a critical role in cell growth and division. During mitosis, the mitotic spindle, composed of dynamic MTs, acts as a structural framework that captures and segregates chromosomes into daughter cells. Although MTs begin assembling the spindle during prophase, the precise mechanisms initiating this process remain unclear. It is known that RCC1, the most upstream regulator of the Ran pathway, generates RanGTP to release spindle assembly factors that promote MT formation. However, the spatial and quantitative requirements for RCC1 in spindle assembly are still poorly understood. Given RCC1’s essential role in initiating this process, determining its minimum effective concentration and localization could provide important insights into how the spindle facilitates mitosis and where errors might arise. To investigate this, I used microfluidic devices to generate cell-like droplets encapsulating MTgenerating particles, allowing for precise titration of RCC1 concentrations. These devices were fabricated using the soft lithography method, where PDMS with the channel design was bonded to glass coverslips using plasma treatment. The encapsulated RCC1-coated particles facilitate the exchange of RanGDP for RanGTP, establishing a Ran gradient that drives MT formation and spindle assembly. Droplets were formed by injecting Xenopus egg extract as the aqueous phase and HFE-7500 oil as the oil phase. Xenopus egg extract is a cell-free system derived from frog eggs that preserves the biochemical machinery of cell division, making it an ideal model for studying MT dynamics in vitro. HFE-7500 is a fluorinated oil widely used in droplet microfluidics due to its chemical inertness, low solubility of aqueous components, and compatibility with surfactants that maintain droplet stability. My experiments revealed a power-law relationship between the volumetric flow rate ratio of the oil and egg extract and the resulting droplet sizes. Additionally, I observed that larger numbers of smaller RCC1-coated particles produced more complex MT network architectures around the self-localizing particles. To deepen our understanding of how RCC1 drives spindle formation, further experiments using varying concentrations of RanQ69L and RCC1 particles in microfluidic systems are needed.