Molecular principles of spindle orientation complex organization and function

University Of New Mexico, Albuquerque NM

Investigators

Abstract

Development of multicellular organisms is a complex process that rests fundamentally on the ability to generate many diverse cell types that ultimately result in adult tissues with unique functions. The creation of cell diversity is tasked to stem cells, whose divisions can produce distinct, non-identical daughter cells capable of assuming different fates. This so-called asymmetric cell division (ACD) process is essential to proper development and viability. Understanding the cellular and molecular mechanisms involved in ACD, therefore, has broad and significant biological importance, particularly considering the conservation of this process across all multicellular life. The goal of this project is to understand how conserved protein complexes that control specific aspects of asymmetric stem cell divisions are regulated to ensure proper development. Although many of the specific components that will be investigated are known, important knowledge gaps remain with respect to how they function at the molecular level. The project will focus particularly on how specific protein complexes assemble and ultimately achieve a cooperative function within the cell. Disruption of these events can lead to defects in tissue development that impact the viability and function of adult organisms. These studies will utilize the common fruit fly as a model genetic organism, whose genes important for ACD are highly conserved to humans. The Broader Impact activities include the intrinsic merit of the research as all multicellular life involves ACD. The work will also provide training for undergraduate and graduate students, with opportunities provided to underrepresented groups to inspire their involvement in a more diverse scientific community. Asymmetric cell division (ACD) is an evolutionarily conserved process that allows progenitor stem cells to produce non-identical daughter cells that ultimately diversify cell fates during development. Proper ACD requires coordinated function between cortical polarity and spindle positioning complexes, and defects that uncouple these disrupt tissue development and homeostasis. The goal of this project is to understand how the conserved Pins/Mud spindle orientation complex organizes at the cell cortex and functions despite competing interactions with cell polarity factors. We provide preliminary data demonstrating that the Pins/Mud complex phase separates into dense liquid droplets in vitro. This is facilitated through a newly-identified interaction between Mud and the actin-binding protein Hts, which we also show to be polarized in Drosophila neural stem cells in vivo. We hypothesize that Hts/Pins/Mud complexes form clustered biological condensates shielded from competing interactions to ensure spindle orientation precision in asymmetrically dividing stem cells. To test this hypothesis, we will use loss-of-function genetic approaches to first determine how Hts polarity is established in neural stem cells and how it impacts localization of core polarity and spindle orientation components. Next, we will use quantitative fluorescence imaging approaches to determine the physical properties of phase separated Pins/Mud droplets in the absence and presence of Hts. Finally, we will use genome editing and high-resolution microscopy to directly ascertain the role of phase separation in the assembly and function of Pins/Mud complexes in vivo. Results from this project will provide insights into the biophysical properties and cellular function of an evolutionarily conserved spindle positioning complex and resolve long-standing knowledge gaps in the molecular underpinnings of ACD. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.