Cytoskeletal Activity

The RHO family is part of the RAS superfamily of guanine nucleotide-binding proteins regulating cellular processes in eukaryotes, including morphogenesis, polarity, movement, cell division, gene expression and cytoskeleton reorganization, and is linked to many diseases (19).

The Rho family of proteins acts as molecular switches in cells, transitioning between inactive GDP-bound and active GTP-bound states. GEFs speed up GDP/GTP exchange, while GAPs enhance GTP hydrolysis.

Activated Rho GTPases, including RhoA, Rac1 and Cdc42, regulate the actin cytoskeleton and associated actin-based processes, including actin organization and motility, by binding and activating actin nucleators like WASP/WAVE proteins and Diaphanous-related formins (DRFs). The WASP/WAVE proteins, for example, stimulate actin polymerization, enabling actin-based motility through the Arp2/3 complex, resulting in the creation of new actin filaments that extend from the existing ones (20, 21).

Cell movement is essential to physiological processes, including embryo development, immune defense, angiogenesis, tissue repair and regeneration, and pathological events, including cancer progression and metastasis (3).

Actin is one of the three main types of cytoskeletal polymers that control cells' shape, structure and movement (2). Actin is primarily responsible for generating force within the cell to power processes associated with cell movement (4). The actin cytoskeleton generates protrusive forces by coordinating the polymerization of multiple actin filaments and contractile forces by sliding actin filaments along actin-based motor protein myosin II. Protrusive and contractile forces combine to enable whole-cell migration (5).

Actin functions by binding to adenosine-5′-triphosphate (ATP) and transitions from monomeric globular actin (G-actin) to polymeric filamentous actin (F-actin), regulated by actin-binding proteins (ABPs), including profilin and cofilin (6). Multiple factors, particularly Rho GTPases like Cdc42, Rac and Rho, regulate actin reorganization, the primary mechanism enabling motility-related functions (7).

Calpains are a 15-member class of calcium-dependent intracellular cysteine proteases. They are localized to the cytosol and mitochondria. Calpain activation and catalytic activity are tightly regulated by cytosolic calcium (Ca2+) and its endogenous inhibitor, calpastatin (22, 23).

Calpain regulates many cellular functions, including cell signaling, cytoskeletal changes, cell differentiation, apoptosis and necrosis by targeting proteins, including cytoskeletal and plasma membrane-associated proteins such as α-fodrin, neurofilaments, ion channels and growth factor receptors (24).

Dysregulation of calpain activation and activity has been implicated in many diseases involving the brain, eyes, heart, vascular system, lungs, pancreas, kidneys and skeletal muscle, highlighting its critical role in maintaining cellular function and integrity (23).

The actin cytoskeleton is essential for many cellular functions. Hence, any abnormalities in its regulation or functioning can cause or contribute to diseases, including cancer, neurological disorders, cardiovascular diseases and Wiskott-Aldrich syndrome (6).

The cytoskeleton is a dynamic and adaptive network consisting of protein fibers and other molecules that serve as a support system for cells in the body (1). It coordinates the activity of cytoplasmic proteins to organize the contents of the cells, connect the cell to the external environment and produce the forces required for cell movement and shape change. Abnormal changes in the cytoskeleton can contribute to the onset and progression of diseases, including cancer (2).

PAKs (p21-activated kinases) are a family of serine/threonine protein kinases consisting of PAK1–6. They are effectors of GTPases Rac and Cdc42 that are essential to cytoskeletal dynamics, cell survival and proliferation.

PAK activation by Rac and Cdc42 leads to the phosphorylation of substrates that regulate cytoskeletal dynamics, including LIM kinase, p41-ARC, filamin A, Op18/stathmin and TCoB.

The LIM kinase is the most researched substrate of Rac and Cdc42 (13). It phosphorylates the actin-regulatory protein cofilin to inhibit its ability to depolymerize actin filaments, thus promoting filament accumulation and stability by preventing depolymerization (14).

Cdc42 is a member of the Rho GTPase family of intracellular molecular switches regulating multiple signaling pathways involved in actomyosin organization, cell-to-cell adhesion, cell cycle regulation, cell proliferation and actin-based morphogenesis. It also plays a critical role in regulating cell polarity. The formation and maintenance of cell polarity are essential for cellular processes, including differentiation, chemotaxis, morphogenesis, cell movement and cell division (10).

The Cdc42 acts as a molecular switch that is open in the GTP-bound state and closed in the GDP-bound state. This cycling between an inactive GDP-bound state and an active GTP-bound state is regulated by intracellular molecules guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GEFs initiate the exchange of GDP for GTP to activate Cdc42, while GAP promotes its GTPase activity, leading to the transition of Cdc42 to its inactive state, and GDIs inhibit reactivation (11).

When active, Cdc42 recruits and regulates various downstream effectors proteins that regulate the actin cytoskeleton and cell polarity, including N-WASP and p21-activated protein kinases (PAK) (12).

The actin-related protein 2/actin-related protein 3 (Arp2/3) complex is activated by WASP (Wiskott–Aldrich syndrome protein) family proteins (8). Upon activation, the ARP2/3 complex nucleates branched actin filaments on the sides of preexisting actin filaments, the first step in actin polymerization, which generates the protrusive forces required in cell movement (9, 4).

Phospholipase C (PLC) is activated through G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) (15).

This enzyme preferentially hydrolyzes phospholipid phosphatidylinositol 4,5-biphosphate (PIP2) into two-second messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) to regulate various cellular processes. These second messengers regulate cellular signaling by facilitating the mobilization of calcium (Ca²⁺) and activation of protein kinase C (PKC), other kinases and ion channels (16).

Ca²⁺ and PKC then modulate the activity of various actin-binding proteins, in turn, modulating actin polymerization, organization and other cytoskeleton-related processes (17, 18).