br Cell motility is accompanied by
Cell motility is accompanied by the dramatic remodeling of the cellular cortex, driven by the extension of plasma membrane protru-sions at the migrating leading edge and membrane retraction at the trailing cell edge [7,8]. The forces that enable such remodeling are
generated by the cortical actomyosin cytoskeleton. Indeed, the forma-tion of the membrane protrusions is known to be driven by the Stearamide polymerization motor, whereas membrane retraction is mediated by the contractile activity of the non-muscle myosin II (NM II) motor [9,10]. The eﬃcient transduction of pushing and pulling forces from the ac-tomyosin cytoskeleton to the plasma membrane requires physical in-teractions between the membrane and the underlying actin filaments [11,12]. Therefore, the molecular complexes that mediate such inter-actions could serve as essential regulators of cell motility . Al-though several mechanisms are known to connect plasma membrane to the actin cytoskeleton, the most common involves the spectrin-ankyrin-adducin membrane skeleton [14–16]. A core component of this mem-brane skeleton is a polymeric lattice composed of spectrin oligomers that lines the inner side of the plasma membrane. This spectrin lattice is linked to both transmembrane proteins and actin filaments, thereby servings as an essential interphase between the membrane and the actin
Corresponding author at: Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC22, Cleveland, OH 44195, United States of America.
E-mail address: [email protected] (A.I. Ivanov).
S. Lechuga et al.
Adducins are important components of the spectrin-based mem-brane skeleton. They bind both spectrin oligomers and actin filaments, thereby promoting and stabilizing association of these cellular struc-tures [17,18]. Furthermore, adducins could interact with the actin cy-toskeleton in spectrin-independent fashion, by capping and bundling actin filaments [18–21]. The adducin family is composed of three clo-sely related members, α-adducin (ADD1), β-adducin (ADD2) and γ-adducin (ADD3) [22,23]. ADD1 and ADD3 are ubiquitously expressed in diﬀerent mammalian tissues, whereas expression of ADD2 is limited to the brain and the hematopoietic system [24,25]. All three adducins have a similar molecular organization, which is characterized by a globular head domain at the N-terminus, a neck domain, and a tail domain at the C-terminus of the molecule [20,24,25]. The neck and head domains possess oligomerization sites that facilitate formation of adducin heterodimers and heterotetramers [22,23]. The C-terminal tail mediates adducin interactions with main binding partners such as actin filaments, spectrins and transmembrane proteins [22,23]. It also con-tains a highly-conserved 22-residue MARCKS-related motif with phos-phorylation sites for protein kinase A (PKA) and protein kinase C (PKC) [20,22,23]. Phosphorylation of these MARCKS motifs was shown to disrupt interactions between adducins and both actin filaments and spectrin oligomers [20,26–28].
Due to their involvement in the assembly of the cortical actin cy-toskeleton and the spectrin-based membrane skeleton, adducins have multiple functional roles in diﬀerent eukaryotic cells. For example, in epithelial cells, ADD1 and ADD3 control the formation of the lateral plasma membrane domain and regulate the dynamics of intercellular junctions [29–33]. In neural tissue, adducins regulate synaptic plasti-city and synaptic contacts [34,35] and play a role in neuronal mor-phology .
Surprisingly, little is known about the roles and mechanisms of adducin-dependent regulation of cell motility. Several previous studies that focused on the roles of ADD1 in the migration of normal epithelial cells and cancer cells yielded controversial results with some studies describing ADD1 as a positive regulator [27,37], while other suggesting it as a negative regulator of cell motility [38,39]. The reason for this discrepancy is not clear. Importantly, the involvement of ADD3 in cell migration and invasion have not been previously investigated. Since expression and activity of adducins is altered in diﬀerent tumors  it is essential to understand how these membrane skeleton proteins reg-ulate migration and invasion of cancer cells and contribute to tumor metastasis.
The overall goal of this study was to understand the roles of ad-ducins in the regulation of non-small cell lung cancer (NSCLC) cell motility in vitro. Clinical evidence suggests altered adducin expression and activity in lung cancer cells. A recent study demonstrated that oncogenic transcription factor ZNF322A upregulated ADD1 expression in a subset of NSCLC patients and connected this event to tumor growth and metastasis . Another study documented hepatocyte growth factor-dependent phosphorylation of ADD1 and ADD3 in small cell lung cancer cells, which may promote lung cancer cell invasion . In-terestingly, accumulation of an alternative spliced, long isoform of ADD3 has been reported in NSCLC, although the functional significance of such tumor-related alternative splicing remains elusive . Finally, loss of ADD1 was shown to impair the establishment of the basolateral plasma membrane in normal lung epithelial cells , which may af-fect cell surface expression of adhesion proteins and chemotactic re-ceptors. In the present study, we found that adducins serve as negative regulators of NSCLC cell motility, acting via diﬀerent mechanisms that involve modulation of cell-matrix adhesion and cellular level of cad-herin-11.