GPCRs, integrins, and receptor tyrosine kinases regulate actin polymerization, contraction, and adhesion turnover through Rho family small GTPases, thereby controlling cell migration, morphology, and tissue function.

Keywords: actin dynamics; cytoskeleton; GPCR; integrin; receptor tyrosine kinase; RhoA; Rac1; Cdc42; ROCK; PAK; Arp2/3; cell migration

1 Dynamic Remodeling Basis of the Actin Cytoskeleton

1.1 Dynamic Conversion Between G-actin and F-actin

Actin exists mainly in two states: globular actin (G-actin) and filamentous actin (F-actin). ATP-bound G-actin can be incorporated into actin filaments, and ATP is gradually hydrolyzed after polymerization, making older filament segments more susceptible to severing and depolymerization. Actin filaments are polarized. The barbed end usually has a faster monomer addition rate, while the pointed end has relatively weaker polymerization capacity. Barbed-end extension, filament severing, pointed-end depolymerization, and monomer recycling together maintain rapid turnover of the actin network. Increased F-actin content does not necessarily indicate faster polymerization; it may also result from impaired depolymerization, enhanced filament stability, or redistribution of actin structures. Therefore, actin dynamics cannot be judged only by static fluorescence intensity.

1.2 Formation and Turnover of Actin Networks

Dynamic remodeling of actin networks mainly includes three connected processes: nucleation, elongation, and severing/recycling.

(1) Nucleation and branch formation

The Arp2/3 complex can form new branches on the side of existing actin filaments, building dense networks in lamellipodia, phagocytic cups, and immune synapses. Formin proteins mainly promote the formation of longer linear actin filaments and participate in the construction of stress fibers, filopodia, and cytokinetic rings.

(2) Actin filament elongation

Profilin binds G-actin and promotes its delivery to the barbed end of actin filaments. Ena/VASP can maintain filament-end elongation, while capping proteins terminate continued polymerization at some filament ends, thereby regulating network density and filament length.

(3) Severing and monomer recycling

Cofilin preferentially binds older ADP-containing actin filaments, promoting filament twisting, severing, and depolymerization, while also generating new polymerizable filament ends. LIM kinase-mediated Ser3 phosphorylation inhibits cofilin activity, and slingshot phosphatase can restore its severing function by removing this phosphate group.

1.3 Major Actin Structures and Their Functions

2 Upstream Signal Inputs Mediated by GPCRs, Integrins, and RTKs

2.1 GPCR-Mediated Chemotactic and Contractile Signaling

G protein-coupled receptors (GPCRs) can sense chemokines, lipid mediators, hormones, and neurotransmitters, and regulate actin structures through different G protein subtypes. Gα12/13 can activate RhoGEFs and promote RhoA-ROCK-mediated contraction. Some Gαi-coupled receptors promote Rac1 activation through Gβγ, PI3Kγ, and Rac-related GEFs, forming PIP3 enrichment and branched actin networks at the cell leading edge. Gαq can regulate adhesion and myosin contraction through PLCβ, Ca²⁺, and protein kinase C. The specific effect depends on receptor type, cellular background, and stimulation duration. A single G protein subtype should not be fixed to one specific cytoskeletal phenotype.

2.2 Integrin-Mediated Adhesion and Mechanical Signaling

Integrins connect the extracellular matrix with the intracellular actin cytoskeleton. Chemokine or growth factor signaling can promote talin and kindlin binding to integrin cytoplasmic tails, shifting integrins into a high-affinity state. After integrins bind the matrix, FAK, Src, paxillin, and vinculin are further recruited to form focal adhesions, transmitting traction force generated by actin-myosin to the extracellular matrix. The FAK-Src complex can also control Rac1 and RhoA through p130Cas, Crk, DOCK, and other Rho regulatory proteins. Therefore, integrins are both mechanical connection points and signaling receptors that sense matrix composition, stiffness, and tension.

2.3 RTK-Mediated Growth Factor Signaling

After activation of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor, platelet-derived growth factor receptor, and vascular endothelial growth factor receptor, PI3K, PLCγ, Grb2, and other adaptor proteins can be recruited. PIP3 generated by PI3K locally recruits Rac1- and Cdc42-related GEFs at the plasma membrane, promoting leading-edge actin polymerization. PLCγ-mediated phospholipid hydrolysis and Ca²⁺ signaling can regulate myosin contraction, adhesion turnover, and membrane trafficking. The RAS-RAF-MEK-ERK pathway connects immediate cytoskeletal remodeling with longer-term protein expression, cell migration, and proliferation responses.

2.4 Coordinated Integration of Different Receptor Signals

Effective migration usually requires the joint participation of chemotactic receptors, integrins, and growth factor receptors. GPCRs and RTKs provide directional and leading-edge polymerization signals. Integrins establish matrix binding and transmit traction force. FAK, Src, PI3K, and Rho family small GTPases integrate inputs from different receptors. Leading-edge extension, adhesion formation, cell body contraction, and rear detachment can be converted into sustained displacement only when coordinated in time and space.

3 Activation and Signal Regulation of Rho Family Small GTPases

3.1 Molecular Switch Cycle of Rho Family Small GTPases

Rho family small GTPases switch between a GDP-bound inactive state and a GTP-bound active state. Guanine nucleotide exchange factors (GEFs) promote GDP release and GTP binding, GTPase-activating proteins (GAPs) accelerate GTP hydrolysis, and guanine nucleotide dissociation inhibitors (GDIs) restrict membrane localization of some Rho proteins. Receptor signals mainly determine the activation intensity and duration of Rho proteins in specific membrane regions by controlling the localization and activity of GEFs, GAPs, and GDIs.

3.2 Functional Division Among RhoA, Rac1, and Cdc42

(1) RhoA

RhoA promotes myosin II contraction, stress fiber formation, focal adhesion maturation, and rear retraction through ROCK and mDia, and also participates in cortical tension and cytokinesis regulation.

(2) Rac1

Rac1 promotes lamellipodia formation through the WAVE regulatory complex and Arp2/3, controlling cell spreading, leading-edge adhesion, and directional migration.

(3) Cdc42

Cdc42 promotes filopodia formation through WASP, N-WASP, and formins, and participates in cell polarity, centrosome positioning, and directed vesicle trafficking.

Figure 1. Schematic of GPCR-, RTK-, and Integrin-Mediated Regulation of Actin Structures through Rho Family GTPases

3.3 Spatiotemporal Coordination of Rho GTPases

Migrating cells usually show local Rac1 and Cdc42 activation at the leading edge to maintain membrane protrusion and directional sensing. RhoA promotes contraction in the cell body and rear, and can also be transiently activated at the leading edge to participate in new adhesion formation. These three GTPases show both cooperation and mutual inhibition. Their spatial distribution changes with matrix stiffness, adhesion strength, and migration mode. Therefore, Rac1 and Cdc42 should not be absolutely restricted to the leading edge, and RhoA should not be interpreted only as a rear contraction signal.

4 Effector Mechanisms of ROCK, PAK, and Actin Regulatory Proteins

4.1 RhoA-ROCK-Mediated Contraction and Tension Regulation

After activated RhoA binds ROCK1 or ROCK2, it can directly promote myosin light chain phosphorylation and inhibit myosin light chain phosphatase, thereby enhancing non-muscle myosin II activity, stress fiber contraction, and focal adhesion maturation. ROCK can also activate LIM kinase and inhibit cofilin-mediated actin filament severing. Moderate activation helps maintain contractile structures, while persistently excessive activation may reduce actin turnover and restrict cell migration.

4.2 Rac1/Cdc42-PAK-Mediated Cytoskeletal Rearrangement

The PAK family is an important effector of Rac1 and Cdc42. It can regulate LIM kinase, myosin light chain kinase, focal adhesion proteins, and microtubule-related molecules, coordinating leading-edge polymerization, adhesion disassembly, and cell polarity. PAK can stabilize local actin networks through the LIMK-cofilin axis and can also regulate cellular tension by altering myosin light chain phosphorylation. Its specific role depends on PAK subtype, cell type, and subcellular localization.

4.3 WASP/WAVE-Arp2/3-Mediated Branched Polymerization

WASP and N-WASP are mainly regulated by Cdc42, membrane phospholipids, and binding proteins, while the WAVE regulatory complex is mainly located downstream of Rac1. Activated WASP or WAVE can relieve self-inhibition or complex inhibition and recruit Arp2/3 to form new branches on the side of existing actin filaments. This is an important basis for the formation of lamellipodia, phagocytic cups, immune synapses, and some endocytic structures.

4.4 Filament Turnover Mediated by Formin, Profilin, and Cofilin

Formin can continuously promote linear filament elongation at the barbed end of actin filaments and cooperate with profilin to improve delivery efficiency of ATP-actin. Cofilin generates new filament ends and recyclable monomers after severing aged filament segments. These two mechanisms are not simply an opposition between polymerization and depolymerization, but jointly maintain actin structure renewal, enabling cells to rapidly change migration direction while maintaining tension and morphology.

5 Regulation of Cell Adhesion, Migration, and Morphology by Actin Dynamics

5.1 Formation and Turnover of Cell Adhesion

After cells contact the matrix, integrins cluster and form initial adhesion complexes. Rac1-driven leading-edge polymerization facilitates new adhesion formation, while traction force generated by RhoA-ROCK-myosin II promotes expansion and maturation of some adhesion structures. Under force, talin can expose new vinculin-binding sites, strengthening the connection between focal adhesions and actin bundles. Adhesion that is too weak causes insufficient traction force, while adhesion that is too strong restricts rear detachment; both can reduce migration efficiency.

5.2 Cell Migration Process

Cell migration consists of a continuous sequence of leading-edge protrusion, new adhesion formation, forward movement of the cell body, and rear retraction. Rac1, Cdc42, and Arp2/3 drive membrane extension toward the migration direction. Integrins establish new matrix binding at the leading edge. Actin-myosin contraction pulls the cell body forward, and rear adhesion disassembly plus RhoA-ROCK-related contraction completes tail detachment. Impairment of any step may appear as reduced migration speed, but the underlying molecular causes are not the same.

5.3 Cell Polarity and Morphology Maintenance

Asymmetric distribution of the actin cytoskeleton determines the front-rear axis, apical-basal axis, and local membrane tension. Cdc42 participates in localization of polarity complexes, Rac1 promotes leading-edge expansion, and RhoA regulates cortical tension and contractile structures. These three GTPases cooperate to control epithelial cell polarity, neurite formation, immune cell deformation, and endothelial barrier stability.

5.4 Phagocytosis, Endocytosis, and Secretion

During phagocytosis, local actin polymerization drives the plasma membrane to wrap particles and form phagocytic cups. Endocytosis and vesicle trafficking require transient cortical actin rearrangement. Secretory granule exocytosis depends on opening pathways in the actin network and regulating granule movement toward the plasma membrane. Excessive actin stabilization or broad depolymerization can both interfere with membrane deformation, vesicle positioning, and secretion efficiency.

6 Abnormal Actin Signaling in Immune Diseases, Developmental Defects, and Tumors

6.1 Immune Dysfunction and Immune Diseases

Immune cell chemotaxis, immune synapses, phagocytosis, and degranulation all depend on actin dynamics. WAS deficiency can weaken Arp2/3-mediated branched polymerization, affecting lymphocyte migration, antigen recognition, and immune synapse formation. Abnormalities in DOCK8, RAC2, and other cytoskeletal regulatory genes can also impair chemotaxis, cell survival, and killing function. In chronic inflammation, sustained enhancement of chemokine receptor, integrin, or Rho-ROCK signaling can promote immune cell entry into tissues. Therefore, related pathways may cause immunodeficiency when function is insufficient and may drive inflammatory injury when overactivated.

6.2 Developmental Defects and Abnormal Tissue Morphogenesis

Cell migration, tissue folding, neurite extension, and cytokinesis during embryonic development all require precise actin regulation. Variants in ACTB, ACTG1, FLNA, DIAPH, and other cytoskeleton-related genes can alter cell morphology, mechanical stability, and migration capacity, and are associated with neurodevelopmental abnormalities, craniofacial defects, hearing impairment, and tissue developmental abnormalities. Disease severity depends not only on actin content, but also on mutation site, protein interactions, and tissue-specific expression.

6.3 Tumor Invasion and Metastasis

Tumor cells can promote membrane protrusion, invadopodia formation, cell contraction, and matrix traversal by enhancing RhoA, Rac1, Cdc42, and their downstream ROCK, PAK, Arp2/3, and formin signaling. Rac1-Arp2/3-related networks favor mesenchymal-like migration, while the high-contractility state mediated by RhoA-ROCK can support rounded or amoeboid migration. These two modes can switch according to matrix pore size, adhesion strength, and therapeutic pressure. Actin remodeling also affects receptor trafficking, cytokinesis, and cell survival. However, detection of Rho proteins or cytoskeletal protein expression alone is insufficient to prove that they drive metastasis; activation status, spatial localization, and functional intervention results should also be combined.

7 Experimental Research and Result Evaluation of Actin Dynamics

7.1 Detection of Actin Structure and Turnover

(1) Fluorescent phalloidin staining

This method is suitable for observing F-actin, stress fibers, cortical actin, and cell protrusions, but fixation, permeabilization, cell thickness, and imaging parameters can affect signal intensity.

(2) F-actin/G-actin separation

This method can evaluate the overall actin polymerization status of cells, but cannot display structural changes in different subcellular regions.

(3) Live-cell imaging

Fluorescent fusion actin, LifeAct, and other live-cell probes can be used to observe real-time actin turnover, but excessive probe expression may interfere with normal dynamics.

7.2 Detection of Rho Family Small GTPase Activity

RhoA activity can be detected by enriching GTP-bound RhoA using Rhotekin-RBD. Rac1 and Cdc42 activity can be detected by enriching the corresponding activated proteins using PAK-PBD. Pull-down assays are suitable for comparing overall activity among samples, but cannot show intracellular activation locations. FRET biosensors can observe local dynamic changes, but probe expression needs to be controlled, and photobleaching and cell morphology changes need to be corrected. Total protein levels of RhoA, Rac1, and Cdc42 cannot replace detection of the GTP-bound state.

7.3 Downstream Signal Detection

After receptor stimulation, phosphorylation changes in FAK, Src, PAK, LIMK, cofilin, and myosin light chain can be detected, and pathway dependence can be verified using ROCK, PAK, Arp2/3, or myosin II inhibitors. Phosphorylated proteins should be detected together with total proteins. Increased cofilin Ser3 phosphorylation usually indicates that actin filament severing activity is inhibited, and should not be interpreted as enhanced cofilin function.

7.4 Cell Behavior and Result Interpretation

Scratch assays are suitable for observing collective migration, but are affected by cell proliferation and death. Transwell assays can evaluate directional migration or invasion, but cannot distinguish abnormalities in leading-edge polymerization, adhesion, or contraction. Single-cell tracking, focal adhesion imaging, traction force microscopy, and three-dimensional matrix migration can further analyze speed, directionality, adhesion lifetime, and mechanical output.

8 Products Related to Actin Dynamics Research

Actin dynamics convert GPCR, integrin, and RTK inputs into cell protrusion, adhesion, contraction, and morphological changes. Related research should jointly evaluate Rho GTPase activity, actin structure, adhesion signaling, and cell behavior, and should avoid judging specific regulatory mechanisms only based on F-actin fluorescence intensity, total Rho protein level, or a single migration assay.

For more related articles, please see below:

[1] G Protein–Coupled Receptors (GPCRs): Structure, Signaling, and Drug Targets

[2] Ras-Raf-MEK-ERK Signaling