Inscuteable regulates the Pins-Mud spindle orientation pathway.

Area of Science:

• Cell Biology
• Developmental Biology
• Molecular Biology

Background:

• Asymmetric cell division requires precise mitotic spindle alignment with the cell polarity axis.
• The Inscuteable (Insc) protein is a key mediator linking cell polarity proteins like Bazooka (Baz) to spindle orienting proteins such as Partner of Inscuteable (Pins).

Purpose of the Study:

• To investigate the mechanism by which the Insc-Pins complex regulates downstream spindle orientation pathways.
• To elucidate the specific roles of Inscuteable in mediating the interactions between Pins and its downstream effectors, Mushroom body defect (Mud) and Discs large (Dlg).

Main Methods:

• Investigated the composition and interactions within the Insc-Pins complex using biochemical assays.
• Performed in vitro competition experiments to determine binding preferences of Insc, Mud, and Dlg to Pins.
• Assessed the functional requirements of Gαi, Dlg, and Mud for Insc-Pins complex activity.

Main Results:

• The Insc-Pins complex requires Gαi for partial activity and specifically recruits Discs large (Dlg), but not Mushroom body defect (Mud).
• In vitro experiments demonstrated that Insc and Mud compete for binding to Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins.
• Inscuteable preferentially inhibits the Mud-dependent pathway of spindle orientation, while permitting the Dlg-dependent pathway.

Conclusions:

• Inscuteable acts as a regulator of complex assembly, not a passive linker, influencing spindle orientation.
• Insc's preferential inhibition of the Mud pathway suggests a mechanism to ensure spindle attachment to the cortex via Dlg before activating pulling forces mediated by Mud, Dynein/Dynactin.