Abstract
During brain development, axon outgrowth and its subsequent pathfinding are reliant on a highly motile growth cone located at the tip of the axon. Actin polymerization that is regulated by actin-depolymerizing factors homology (ADF-H) domain-containing family drives the formation of lamellipodia and filopodia at the leading edge of growth cones for axon guidance. However, the precise localization and function of ADF-H domain-containing proteins involved in axon extension and retraction remain unclear. We have previously shown that transcripts and proteins of coactosin-like protein 1 (COTL1), an ADF-H domain-containing protein, are observed in neurites and axons in chick embryos. Coactosin overexpression analysis revealed that this protein was localized to axonal growth cones and involved in axon extension in the midbrain. We further examined the specific distribution of coactosin and cofilin within the growth cone using superresolution microscopy, structured illumination microscopy, which overcomes the optical diffraction limitation and is suitable to the analysis of cellular dynamic movements. We found that coactosin was tightly associated with F-actin bundles at the growth cones and that coactosin overexpression promoted the expansion of lamellipodia and extension of growth cones. Coactosin knockdown in oculomotor neurons resulted in an increase in the levels of the inactive, phosphorylated form of cofilin and dysregulation of actin polymerization and axonal elongation, which suggests that coactosin promoted axonal growth in a cofilin-dependent manner. Indeed, the application of a dominant-negative form of LIMK1, a downstream effector of GTPases, reversed the effect of coactosin knockdown on axonal growth by enhancing cofilin activity. Combined, our results indicate that coactosin functions promote the assembly of protrusive actin filament arrays at the leading edge for growth cone motility.
Highlights
IntroductionNeuronal growth cones are identified by the presence of large lamellipodia with sharp filopodia projecting from the growing edge of axons
During embryonic brain development, neuronal growth cones are identified by the presence of large lamellipodia with sharp filopodia projecting from the growing edge of axons
Actin-depolymerizing factor homology (ADF-H) domaincontaining proteins are essential for actin treadmilling and polymerization/depolymerization processes in which actin monomers are removed from the pointed end and added to the barbed end of actin filaments (Lappalainen et al, 1998; Yang et al, 1998; Poukkula et al, 2011)
Summary
Neuronal growth cones are identified by the presence of large lamellipodia with sharp filopodia projecting from the growing edge of axons. Coactosin Remodels F-Actin in Axons actin-binding proteins are found in the growth cone (Ishikawa and Kohama, 2007; Nozumi et al, 2009), which suggests that these molecules are involved in axon formation and development. ADF/cofilin, an extensively characterized F-actin severing protein, more strongly binds to ADP-actin than do ATP- and ADP-Pi actin filaments and accelerates depolymerization by removing actin monomers from the pointed ends of actin filaments (Yang et al, 1998; Bamburg, 1999, Kiuchi et al, 2007). Coactosin prevents cofilin-mediated depolymerization, thereby promoting lamellipodia formation (de Hostos et al, 1993; Röhrig et al, 1995; Provost et al, 2001; Hou et al, 2013; Kim et al, 2014). Despite the increasing number of biochemical and structural studies relating to ADF-H family proteins, their roles in regulating actin dynamics in cellular structures are not yet incompletely understood
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