The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics

Abstract

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro . Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼μM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4–6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1–3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4–6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization

“…Complete loss-of-function mutations, on the other hand, severely disrupt F-actin organization during cellularization and gastrulation (9,10). In vitro studies have demonstrated the mechanistic importance of FLII in focal adhesions, where it associates with the actin cytoskeleton (6,7,29,40). Taken together, these data point to a critical role for FLII in cell adhesion dynamics and associated cytoskeletal rearrangements.…”

The C-terminal tail extension of myosin 16 acts as a molten globule, including intrinsically disordered regions, and interacts with the N-terminal ankyrin

“…For this reason, we performed a binding assay using steady-state fluorescence anisotropy measurements to test the interaction between Myo16Tails with the N-terminal Myo16Ank region. Anisotropy is a powerful method to investigate the size, shape, dynamics, conformation, and interactions of proteins ( 62 ) and was used successfully to monitor protein binding ( 63 , 64 , 65 , 66 , 67 ). The steady-state anisotropy of fluorescently labeled Alexa568–Myo16Ank (1.2 μM) increased by the addition of an increasing concentration of Myo16Tail as expected for binding interaction.…”