Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex

Area of Science:

• Cell Biology
• Developmental Biology
• Molecular Biology

Background:

• Asymmetric cell division (ACD) is vital for stem cell function, enabling self-renewal and differentiation.
• In Drosophila neuroblasts (NBs), ACD relies on mitotic spindle orientation guided by cortical polarity cues.
• The Partner of Inscuteable (Pins) and Mushroom body defect (Mud) complex is key for spindle orientation in NBs, but its assembly mechanism is not fully understood.

Purpose of the Study:

• To elucidate the molecular mechanics governing the assembly of the cortical Pins/Mud complex in Drosophila NBs.
• To identify novel components involved in the regulation of spindle orientation during ACD.
• To investigate the role of liquid-liquid phase separation in the assembly of apical protein complexes.

Main Methods:

• Co-immunoprecipitation and in vitro binding assays to identify Mud-binding proteins.
• Analysis of protein localization and function in Drosophila neuroblasts using genetic manipulation.
• In vitro liquid-liquid phase separation assays to study complex formation and properties.
• Domain mapping and mutational analyses to determine the roles of specific protein regions.

Main Results:

• Hu li tai shao (Hts) was identified as a direct Mud-binding protein, interacting via its head domain.
• Hts is apically localized in mitotic NBs and is essential for Mud-dependent spindle orientation.
• Pins and Mud undergo liquid-liquid phase separation, a process modulated by Hts.
• Hts binding alters the phase separation properties of the Pins/Mud complex, involving multivalent interactions and protein disorder.

Conclusions:

• Hts is a novel component of the spindle positioning machinery in Drosophila neuroblasts.
• Liquid-liquid phase separation of protein complexes may regulate ordered assembly at the apical domain to ensure proper signaling output during ACD.
• The findings provide insights into the differential assembly mechanics of competing apical protein complexes.