Cofilin-1 is a small protein (19kDa) ubiquitously present throughout the eukaryotic kingdom and it belongs to the ADF/cofilin family.The ADF/cofilin family in mammalian systems consists of three highly similar paralogs: cofilin-1 (Cfl1, non-muscle cofilin, n-cofilin), cofilin-2 (Cfl2, muscle cofilin, m-cofilin) and ADF (actin-depolymerising factor or destrin).
For sequence and structure see: http://www.uniprot.org/uniprot/P23528
During development, Cfl1 is the predominant isoform and it remains ubiquitously expressed in most adult tissues. ADF becomes post-natally upregulated mainly in epithelial and endothelial tissues, albeit usually at concentrations lower than Cfl1. In late embryogenesis and after birth, Cfl2 replaces Cfl1 in striated muscle and forms the only isoform expressed in differentiated skeletal muscle and the main one in cardiac muscle.
Cofilin plays a role in migrating and invading cells . Cell migration is needed for proper morphogenesis during development, for wound healing and for lymphocyte-mediated immune responses. It is also a determining feature of cancer malignancy as it is required for dissemination of tumour cells throughout the metastatic process.
Cofilin-1 appears crucial for cell migration events in morphogenesis, it is similar to the process of metastasis diffusion beginning form a preliminary tumour mass.
ADF/cofilins have been termed actin-dynamising proteins based on their capacity to enhance the turnover of actin filaments in vitro. At steady state, actin filaments (F-actin) preferentially grow at one end (called the fast growing or barbed end) by association of ATP-loaded monomeric actin molecules (ATP-G-actin, globular actin) whereas monomers dissociate at the other (slow growing or pointed end).
Incorporated monomers undergo hydrolysis of the bound ATP to ADP, with ADP/Pi-loaded actin as important intermediates. ADP-loaded actin is more prone to dissociation and recycles back to the monomer pool. Dissociated ADP-loaded actin monomers need to exchange their nucleotide before entering a new polymerisation cycle.
Modified from: Van Troys, Huyck et al. 2008
The polarised growing and shrinking, resulting in dynamic turnover of actin filaments (also called treadmilling), forms the fundament of protrusive forces in cells. It occurs in lamellipodia and filopodia in migrating cells and it is also the basis of propulsion of specific vesicles in the cytosol. In these cellular nano-scaled force machineries, actin filaments are organised in networks. The dynamic turnover of such an actin array can be enhanced either by an increase in the number of filament ends (resulting from de novo nucleation of filaments or from severing existing filaments) or by an increase in the extent or rate of monomer association at barbed filament ends and/or dissociation from pointed filament ends.
Each cofilin molecule is contacting two actin subunits in an actin filament by binding in a cleft between them. Cofilins have a higher affinity for ADP-loaded than for ATP-loaded actin; they decrease nucleotide exchange on ADP-loaded monomers and promote Pi release from ADP/Pi subunits in the filament. They accelerate spontaneous polymerisation of monomers (nucleation) and are hypothesised to increase the rate of actin subunit dissociation from the pointed end. Cofilin binding to F-actin induces a conformational twist in the actin filament structure that propagates over a long range from the actual cofilin-binding site, and this is suggested to underlie their fragmenting/severing activity.
How exactly ADF/cofilins dynamise the actin polymerisation process in vitro has been strongly debated for several decades and resulted in two leading models: either ADF/cofilins increase the dissociation rate of actin subunits from pointed ends (model 1) or they sever actin filaments (model 2). Whereas both models imply a direct role of ADF/cofilins in F-actin disassembly, only the latter supports a direct function in assembly through the rapid generation of new barbed ends. Barbed end-capping proteins promoting F-actin disassembly at the pointed end; actin-binding proteins (Arp2/3 complex) bind F-actin and promoting filament nucleation or barbed end elongation. These proteins are proposed to act in synergy with cofilin.
Modified from: Van Troys, Huyck et al. 2008
In model 2 there is a switch in cofilin acitivity depending on itd concentration. This means that in a cell region where a gradient of high to low cofilin activity is present, the activity of ADF/cofilin could shift from: (1) nucleating new filaments (that can initiate the formation of a branched network by the Arp2/3 complex) to (2) actin filament stabilisation and aging by cofilin decoration, and finally (3) filament severing, which creates barbed ends capable of elongation or will, in the presence of barbed end-capping proteins, results in net disassembly from pointed ends.
REGULATORY MECHANISMS OF ADF/COFILIN ACTIVITY
Multiple mechanisms have been identified that regulate cofilin, including cofilin inactivation via phosphorylation and by polyphosphoinositide interaction, the effects of pH and the synergistic or competitive interactions of cofilin with other ABPs
Cofilin-1 is inactivated by phosphorylation on Ser3. This posttranslational modification results in inhibition of G- and F-actin binding and of F-actin severing. Two families of ubiquitous kinases, with related catalytic domains, are responsible for the inactivation of cofilins by phosphorylation: the LIM kinases (LIMK) and testicular kinases (TESK). The phosphatases (PPases) of the Slingshot (SSH) family and the haloacid dehalogenase phosphatase chronophin (CIN) are the enzymes that reactivate phosphorylated cofilin.
Three aspects need to be considered in trying to understand the relative contributions of the cofilin kinases and PPases: first, their expression levels and tissue distributions, second, their activation pathways and activity levels and, finally, their subcellular localisation via potential scaffolding factors and alterations therein upon specific cell stimulation.
LIMKs are activated by phosphorylation on a threonine residue in the catalytic domain by various kinases that are themselves activated downstream of small Rho GTPases. LIMKs have been shown to be essential regulators of actin cytoskeletal reorganisation downstream of these GTPases and implicated in Rac-dependent lamellipodia formation and Rho-dependent stress fibre and focal adhesion formation. Rho signal to both LIMK1 and 2 via the Rho kinases ROCK I and II. PAK1 and 4, downstream of Rac activation, also activate LIMK1, but not LIMK2.
The only PPase known to dephosphorylate and inactivate LIMK 1 and 2 is a PPase that also dephosphorylates cofilin, namely SSH1L, suggesting a positive feedback loop via simultaneous cofilin activation and LIMK inhibition. The pathways affecting TESK activity are integrin mediated and adhesion dependent. TESK1 activity is inhibited by sequestration by α-parvin, 14-3-3β, and Sprouty-4.
Membrane polyphosphoinositides (PPI), in particular phosphatidylinositol-3,4,5-trisphosphate (PIP3) and phosphatidylinositol-4,5-bisphosphate (PIP2), as well as the enzymes producing or hydrolysing these lipids upon cell stimulation, are well-recognised signalling molecules controlling the dynamic turnover of the actin cytoskeleton and focal adhesions, and ultimately cell migration.
The actin-binding capacity of Cofilin is lost upon interaction with PIP2. This inhibition is due to competitive binding since F-actin and PIP2 target overlapping binding sites on cofilin. Actin turnover is needed at the cell periphery to form the membrane protrusions required for migration or at vesicles for their intracellular motility. In many cell types cofilin, but not P-cofilin, is present at the membrane, strongly suggesting that PIP2 acts to sequester unphosphorylated cofilin. Stimulus-induced PIP2 hydrolysis (by the hydrolysing enzyme phospholipase C, PLC) and subsequent cofilin release would allow mobilisation of relatively high local concentrations of active cofilin near the membrane, exactly where its activity is needed. Coflin is released in its active ofrm and subsequently translocates to the actin filaments close to the membrane to generate new barbed ends.
ROLE OF COFILIN-1 IN CELL MOTILITY
ADF/cofilins are implicated in several cellular processes including neuronal outgrowth, T-cell activation, phagocytosis, endocytosis, receptor recycling, regulation of ion channels, and maybe, via the formation of actin–cofilin rods, in cellular ATP-energy management.
Cell migration is needed for proper morphogenesis during development, for wound healing and for lymphocyte-mediated immune responses. It is also a determining feature of cancer malignancy as it is required for dissemination of tumour cells throughout the metastatic process. A common and early requirement for different forms of cell motility is the formation of membrane protrusions (lamellipodia, filopodia, pseudopods and also growth cones) via pushing forces mainly generated by actin polymerisation against the membrane.
Manipulation of cofilin activity has been shown to affect formation of protrusions (growth cones, lamellipodia) and cell migration. The different biochemical activities of cofilin are probably important for obtaining either local assembly or local disassembly of actin filaments in cells. These two cofilin-mediated outcomes are not necessarily mutually exclusive and may depend on the local presence of other ABPs.
In mammary carcinoma cells, the activation of caged cofilin leads to the generation of new actin filament barbed ends and to actin filament assembly. Local photo-release resulted in local lamellipodia formation. This supports the fact that active cofilin can initiate actin filament assembly in cells albeit in combination with other ABPs. A model has been proposed by which new barbed ends (or nuclei), generated by cofilin, subsequently elongate and serve as basis for the Arp2/3 complex to initiate a dendritic array and lamellipodia formation.
Cofilin is from this perspective an early effector of the formation of lamellipodia since its local activation sets the site where new lamellipodia appear as well as the direction that a migrating cell takes.
To result in filament disassembly, however, filament barbed ends that are locally generated by cofilin-mediated severing likely need to be barbed-end capped with high efficiency. This can lead to local depolymerisation of filaments from their pointed ends and an increase in actin monomers available for new polymerisation or assembly.
The localisation of cofilin in lamellipodia is also indicative of its function in this cellular structure. In rapidly moving keratinocytes, ADF/cofilins were shown to localise to the middle and rear of the lamellipodium (which is the small treadmilling zone of the protrusion).