Abstract
ABSTRACTDrosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin-binding protein 2 (CTTNBP2) and Cortactin-binding protein 2 N-terminal-like (CTTNBP2NL). Here, we have named this gene Nausicaa (naus) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence microscopy, electron microscopy, RNAi depletion and genetics. We found that Naus co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using permeabilization activated reduction in fluorescence and fluorescence recovery after photobleaching, we find that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, we discovered that Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. We demonstrate that these alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. We present the hypothesis that Naus functions to slow Cortactin's disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability.
Highlights
Cell migration is critical to a number of physiological processes including wound healing and immune function, development, neurogenesis and vascularization
The three proteins share a highly conserved proline-rich patch located near their C-termini that has been shown to facilitate the interaction between Cortactin and Cortactin-binding protein 2 (CTTNBP2) in COS cells (Fig. S1) (Chen et al, 2012)
Using cultured and primary Drosophila cells we demonstrate that Naus, through its interaction with Cortactin, regulates actin-branch dynamics, lamellipodial protrusion and the morphology of neurons
Summary
Cell migration is critical to a number of physiological processes including wound healing and immune function, development, neurogenesis and vascularization. One protein that defines the lamellipodia is the actinrelated protein 2/3 (Arp2/3) complex which generates new branches from the sides of pre-existing filaments resulting in a highly branched actin network (Machesky et al, 1999; Mullins et al, 1997; Suraneni et al, 2012; Svitkina and Borisy, 1999). It is the addition of actin subunits (G-actin), spread across the entire expanse of the lamellipodium that leads to protrusion of this organelle. While type I NFPs generally bind and activate Arp2/3 via a shared VCA (verprolin homology, central, acidic) region, Cortactin and HS1 use an N-terminal acidic region (NtA) (Goley and Welch, 2006; Weaver et al, 2001)
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