Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis

During animal cell chemotaxis, signalling at the plasma membrane induces actin polymerisation to drive forward cell movement. Since the cellular pool of actin is limited, efficient protrusion formation also requires the coordinated disassembly of pre-existing actin filaments. To search for proteins that can monitor filamentous and globular actin levels to maintain the balance of polymerisation and disassembly, we followed changes in the proteome induced by RNA interference (RNAi)mediated alterations in actin signalling. This unbiased approach revealed an increase in the levels of an inactive, phosphorylated form of the actin-severing protein cofilin in cells unable to generate actin-based lamellipodia. Conversely, an increase in F-actin levels induced the dephosphorylation and activation of cofilin via activation of the Ssh phosphatase. Similarly, in the context of acute phosphoinositide 3-kinase (PI3K) signalling, dynamic changes in cofilin phosphorylation were found to depend on the Ssh phosphatase and on changes in lamellipodial Factin. These results indicate that changes in the extent of cofilin phosphorylation are regulated by Ssh in response to changes in the levels and/or organisation of F-actin. Together with the recent finding that Ssh phosphatase activity is augmented by F-actin binding, these results identify Ssh-dependent regulation of phosphorylated cofilin levels as an important feedback control mechanism that maintains actin filament homeostasis during actin signalling

Functional proteomic and genomic analysis of cytoskeletal organisation in Drosophila.

Phosphoinositide 3-kinase (PI3K) plays an important role in cellular signalling by generating phospholipid second messengers at the plasma membrane. A large repertoire of signalling and actin-binding proteins, which consistently regulate the dynamic assembly and spatial organisation of actin filaments, binds phospholipid second messengers, through their pleckstrin homology (PH) domains, and regulates changes in actin cytoskeleton dynamics and organisation in response to external stimuli. Thus, the actin cytoskeleton, which functions in the generation and maintenance of cell morphology and polarity, regulation of endocytosis and intracellular trafficking, contractility, motility and cell division, is considered as an integral part of the cell signal transduction system. PI3K-dependent actin cytoskeleton reorganisation has been the subject of intensive studies, as alteration in the cytoskeleton and thus in cell morphology and migration appear to be common signatures of malignancy where PI3K activation is significantly involved. PI3K- dependent regulation of actin cytoskeleton dynamics is proposed to be achieved by cross-talk with the Rho-family small GTPases, major regulators of actin cytoskeleton organisation. However, the molecular mechanisms behind PI3K-dependent actin reorganisation and their interaction with small GTPases in not yet clearly defined. The aim of this project was to investigate the role of the PI3K signalling in controlling actin cytoskeleton, and to explore possible common targets of PI3K and Rho-family small GTPase signalling pathways, as well as to search for new targets downstream of PI3K. Initially, the role of PI3K in the regulation of the actin cytoskeleton in Drosophila cells was defined. Furthermore, a "loss-of-function approach" based on RNA interference for genes involved in PI3K and small GTPase signalling was combined with quantitative differential protein expression analysis and mass spectrometry. The differentially expressed proteins, many of which were cytoskeleton proteins, metabolic and redox enzymes, were linked to signalling pathways and associated with the morphological phenotype of each knockdown. Finally, the research was focused on studying the regulation of phosphorylation of cofilin, an actin depolymerising protein. It has been established that cofilin phosphorylation and activity is not directly regulated by upstream signalling events, but by changes in the levels of filamentous actin itself, with slingshot, the cofilin phosphatase, being a key regulator in sensing the dynamic changes in F-actin levels. Thus, cofilin phosphorylation is a homeostatic sensor of actin polymerisation, which self-limits protrusive response to external stimuli

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

Insulin increases GLUT4 at the muscle cell surface, and this process requires actin remodeling. We show that a dynamic cycle of actin polymerization and severing is induced by insulin, governed by Arp2/3 and dephosphorylation of cofilin, respectively. The cycle is self-perpetuating and is essential for GLUT4 translocation

B cell receptor-induced Ca2+ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes

B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PL