Integrin-mediated Focal Adhesion Research Articles

Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation.

Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

T cells use focal adhesions to pull themselves through confined environments.

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical compositions. Their migration has classically been defined as amoeboid under the assumption that it is integrin independent. Here, we show that activated primary Th1 T cells require both confinement and extracellular matrix proteins to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal cell focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cells preferentially follow tracks of other T cells, suggesting that these adhesions modify the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated focal adhesions play a key role in T cell motility.

Abstract 5874: Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways

Abstract The acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) restricts its clinical utility. Clinically, acquired resistance has been highly associated with epithelial-mesenchymal transition (EMT) traits. In this study, we characterized shared phenotypic plasticity features of TKI-resistant cells and aimed to find its key driver in three different cell lines harboring mutant EGFR. In vitro resistance cell lines were generated by dose-escalation exposure to osimertinib, a 3rd generation TKI, for more than 2 months. We employed canonical markers (E-cadherin, vimentin, pan-cytokeratin, and N-cadherin) along with additional markers (EpCAM and fibronectin) to define the EMT status of resistant cells. Moreover, cytoskeleton architecture (F-actin and Vimentin) was utilized to clarify the types of cell motility. Transcriptomics analysis illustrated enriched pathways at focal adhesion and adherens junction. Loss of adherens junction-related catenin (α-E-catenin, β-catenin, γ-catenin, p120 catenin) and aberrant activation of focal adhesion-related protein (p-FAK Y397 and Y576/57, p-Src Y416) confirmed the transcriptomics analysis of reduced contact inhibition and cell polarity. The resulting migration mode of resistant cells highly resembles ameboid movement. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. In conclusion, resistant cells display ameboid-like movement resulting from loss of contact inhibition and aberrant activation of focal adhesion. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. YTHDF3 serves as an RNA epitranscriptomic regulator that destabilizes CDH1 mRNA, thereby disrupting adherens junction formation and reducing integrin-mediated focal adhesion stabilization. Citation Format: Chun Ma, Jerry Chieh-Yu Chen, Mong-Lien Wang. Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5874.

Dermal stiffness governs the topography of the epidermis and the underlying basement membrane in young and old human skin.

The epidermis is a stratified epithelium that forms the outer layer of the skin. It is composed primarily of keratinocytes and is constantly renewed by the proliferation of stem cells and their progeny that undergo terminal differentiation as they leave the basal layer and migrate to the skin surface. Basal keratinocytes rest on a basement membrane composed of an extracellular matrix that controls their fate via integrin-mediated focal adhesions and hemidesmosomes which are critical elements of the epidermal barrier and promote its regenerative capabilities. The distribution of basal cells with optimal activity provides the basement membrane with its characteristic undulating shape; this configuration disappears with age, leading to epidermal weakness. In this study, we present an in-depth imaging analysis of basal keratinocyte anchorage in samples of human skin from participants across the age spectrum. Our findings reveal that skin aging is associated with the depletion of hemidesmosomes that provide crucial support for stem cell maintenance; their depletion correlates with the loss of the characteristic basement membrane structure. Atomic force microscopy studies of skin and invitro experiments revealed that the increase in tissue stiffness observed with aging triggers mechanical signals that alter the basement membrane structure and reduce the extent of basal keratinocyte anchorage, forcing them to differentiate. Genomic analysis revealed that epidermal aging was associated with mechanical induction of the transcription factor Krüppel-like factor 4. The altered mechanical properties of tissue being a new hallmark of aging, our work opens new avenues for the development of skin rejuvenation strategies.

PIEZO1 channels as regulators of integrin-mediated focal adhesions in cardiac fibroblasts

PIEZO1 channels as regulators of integrin-mediated focal adhesions in cardiac fibroblasts

Abstract 16799: Galectin-1 Alleviates Aortic Valve Calcification via Modulating Integrin-Mediated Cytoskeletal Reorganization and Focal Adhesion

Background Galectin-1, a beta-glycoside binding protein, has been reported to involve a variety of cell functions including proliferation, cell migration and focal adhesion. The role of cell-matrix interaction in calcific aortic valve disease (CAVD) is not fully investigated and whether galectin-1 is involved in this process remains unclear. We hypothesized that galectin-1 mitigates the fibrocalcific response and mechanosensitivity of valve interstitial cells (AVICs) by modulating integrin-mediated cytoskeletal reorganization and focal adhesion. Methods and Results We enrolled patients with CAVD and age- and sex-matched controls to detect serum galectin-1 and found that circulating galectin-1 was lower in CAVD. The expression of galectin-1 decreased in fibrocalcific valves and AVICs cultured in osteogenic medium (OM), as shown by western blot, qPCR, immunohistochemistry (IHC) and immunofluorescence. Silencing galectin-1 by short-interfering RNA exacerbated the fibrocalcific change of AVICs in vitro while recombinant galectin-1 (rGal-1) prevented these effects. AVICs cultured in OM also presented enhanced integrin signaling, cytoskeletal reorganization and focal adhesion. Anti-integrin blocking experiments and co-immunoprecipitation assays demonstrated that the galectin-1 directly interacted with integrin αvβ1 to regulate AVICs adhesion to a fibronectin matrix. Western blot, qPCR and Immunofluorescence assays confirmed that actin cytoskeletal reorganization was induced by αvβ1 activation through Rac1-WRC-Arp2/3 signaling axis. Western blot, alkaline phosphatase (ALP) as well as Alizarin red staining demonstrated that silencing galectin-1 enhanced AVICs sensitivity to piezo1 activation, which is dependent on αvβ1 binding and actin polymerization. In vivo, aortic valve disease was constructed by direct wire injury (DWI), and we showed that overexpression of galectin-1 by adeno-associated virus significantly decelerated the progression of aortic valve lesion induced by DWI in mice. Conclusion Galectin-1 interacts with integrin αvβ1 to modulate cytoskeletal reorganization and AVICs adhesion to fibronectin, thus mitigating the fibrocalcific response of AVICs and aortic valve calcification.

Joining forces: crosstalk between mechanosensitive PIEZO1 ion channels and integrin-mediated focal adhesions.

Both integrin-mediated focal adhesions (FAs) and mechanosensitive ion channels such as PIEZO1 are critical in mechanotransduction processes that influence cell differentiation, development, and cancer. Ample evidence now exists for regulatory crosstalk between FAs and PIEZO1 channels with the molecular mechanisms underlying this process remaining unclear. However, an emerging picture is developing based on spatial crosstalk between FAs and PIEZO1 revealing a synergistic model involving the cytoskeleton, extracellular matrix (ECM) and calcium-dependent signaling. Already cell type, cell contractility, integrin subtypes and ECM composition have been shown to regulate this crosstalk, implying a highly fine-tuned relationship between these two major mechanosensing systems. In this review, we summarize the latest advances in this area, highlight the physiological implications of this crosstalk and identify gaps in our knowledge that will improve our understanding of cellular mechanosensing.

Curved adhesions mediate cell attachment to soft matrix fibres in three dimensions

Integrin-mediated focal adhesions are the primary architectures that transmit forces between the extracellular matrix (ECM) and the actin cytoskeleton. Although focal adhesions are abundant on rigid and flat substrates that support high mechanical tensions, they are sparse in soft three-dimensional (3D) environments. Here we report curvature-dependent integrin-mediated adhesions called curved adhesions. Their formation is regulated by the membrane curvatures imposed by the topography of ECM protein fibres. Curved adhesions are mediated by integrin ɑvβ5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin β5 and a curvature-sensing protein, FCHo2. We find that curved adhesions are prevalent in physiological conditions, and disruption of curved adhesions inhibits the migration of some cancer cell lines in 3D fibre matrices. These findings provide a mechanism for cell anchorage to natural protein fibres and suggest that curved adhesions may serve as a potential therapeutic target.

Development of an integrin αv-based universal marker, capable of both prediction and direction of stem cell fate.

Integrin-mediated focal adhesion (FA) and subsequent cytoskeletal reorganization influence cell morphology, migration, and ultimately cell fate. Previous studies have used various patterned surfaces with defined macroscopic cell shapes or nanoscopic FA distributions to explore how different substrates affect the fate of human bone marrow mesenchymal stem cells (BMSCs). However, there is currently no straightforward relationship between BMSC cell fates induced by patterned surfaces and FA distribution substrates. In this study, we conducted single-cell image analysis of integrin αv-mediated FA and cell morphological features of BMSCs during biochemically induced differentiation. This enabled the identification of distinct FA features that can discriminate between osteogenic and adipogenic differentiation, demonstrating that integrin αv-mediated focal adhesion (FA) can be used as a non-invasive biomarker for real time observation. Based on these results, we developed an organized microscale fibronectin (FN) patterned surface where the fate of BMSC could be precisely manipulated by these FA features. Notably, even in the absence of any biochemical inducers, such as those contained in the differentiation medium, BMSCs cultured on these FN patterned surfaces exhibited upregulation of differentiation markers comparable to BMSCs cultured using conventional differentiation methods. Therefore, the present study reveals the application of these FA features as universal markers not only for predicting differentiation status, but also for regulating cell fate by precisely controlling the FA features with a new cell culture platform. STATEMENT OF SIGNIFICANCE: Although the effects of material physiochemical properties on cell morphology and subsequent cell fate decisions have been extensively studied, a simple yet intuitive correlation between cellular features and differentiation remains unavailable. We present a single cell image-based strategy for predicting and directing stem cell fate. By using a specific integrin isoform, integrin αv, we identified distinct geometric features that can be used as a universal marker for discriminating between osteogenic and adipogenic differentiation in real-time. From these data, new cell culture platform capable of regulating cell fate by precisely controlling FA features and cell area can be developed.

Electroconductive and mechano-competent PUCL@CNT nanohybrid scaffolds guiding neuronal specification of neural stem/progenitor cells

Diseased or injured nervous systems lead to lifelong disability and even death due to their limited self-repair and regenerative capability. For successful neuronal regeneration and functional recovery, it is critical to selectively guide neural stem/progenitor cells (NSPCs) to differentiate into neurons and oligodendrocytes rather than astrocytes. Biomaterial scaffolds with specific properties such as electroconductivity can boost this event and guide the specific differentiation of NSPCs. Here, we prepared electroconductive and mechano-competent scaffolds made of shape-memory polyurethane‐polycaprolactone (PUCL) nanofibers and carbon nanotubes (CNTs) (namely, PUCL@CNT). The CNT-decorated PUCL platforms provided excellent shape-formable and shape-memorable properties for surgical compatibility as well as electroconductivity (∼ 8 S/m) and nanoscale-topography (tens-of-nm), which could promote the initial cell adhesion via the integrin-mediated focal adhesion signaling pathway, and promote neuron and oligodendrocyte differentiation. The potential mechanism of this lineage-specific differentiation of NSPCs upon the PUCL@CNT was evaluated by transcriptome sequencing analysis and pharmacological intervention tests, revealing the involvement of the intracellular calcium levels and the FAK-AKT-β-catenin pathway. When implanted in a rat corticectomy model, PUCL@CNT could induce a larger number of endogenous neural stem cells (both Nestin+ and β-catenin+) and neurons (Tuj1+) near the injured region (vs. PUCL and PBS), demonstrating the in vivo therapeutic role of PUCL@CNT. Taken together, the CNT surface-decoration was effective in driving NSPCs toward neuronal specification and their maturation, and the PUCL@CNT scaffold is potentially useful for neural tissue regeneration that can benefit via the neuronal activation of endogenous NSPCs.

Probing mechanical forces in curvature-sensitive cell adhesions

Integrin-mediated focal adhesions facilitate mammalian cell adhesion to the extracellular matrix (ECM), a topologically complex network of protein fibers. While extensive studies on cellular adhesion to the ECM have been done, outstanding questions remain regarding the relationship between three-dimensional (3D) ECM geometry and cellular adhesion, namely how the physical properties of the 3D ECM affect cellular mechanotransduction. To probe this relationship, we used a quartz nanostructure platform to induce different curvature geometries in cells and found that cells exhibit curvature-induced adhesions. Since the formation of focal adhesions is tied to mechanical cues in the environment, we then explored whether mechanical forces are involved in these curvature-sensitive adhesions. We utilized two genetically encoded, FRET-based talin tension biosensors, one sensitive to forces in the range of 3-5 pN and the other sensitive to forces in the range of 9-11 pN. Using ratiometric imaging, we then compared these adhesions with focal adhesions and found that while these curvature-sensitive adhesions bear mechanical forces, they experience lower tension compared to focal adhesions. In this study, we aim to further explore the intra- and extracellular forces involved in these curvature-sensitive adhesions.

Highly Adhesive Amyloid-Polyphenol Hydrogels for Cell Scaffolding.

Rationally designing microstructures of soft hydrogels for specific biological functionalization is a challenge in tissue engineering applications. A novel and affordable soft hydrogel scaffold is constructed here by incorporating polyphenol modules with lysozyme amyloid fibrils (Lys AFs) via non-covalent self-assembly. Embedded polyphenols not only trigger hydrogel formation but also determine gel behavior by regulating the polyphenol gallol density and complex ratio. The feasibility of using a polyphenol-Lys AF hydrogel as a biocompatible cell scaffold, which is conducive to cell proliferation and spreading, is also shown. Notably, introducing polyphenols imparts the corresponding hydrogels a superior cell bioadhesive efficiency without further biofunctional decoration and thus may be successfully employed in both healthy and cancer cell lines. Confocal laser scanning microscopy also reveals that the highly expressed integrin-mediated focal adhesions form due to stimulation of the polyphenol-AF composite hydrogel, direct cell adhesion, proliferation, and spreading. Overall, this work constitutes a significant step forward in creating highly adhesive tissue culture platforms for in vitro culture of different cell types and may greatly expand prospects for future biomaterial design and development.

α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

Cell-matrix adhesions are mainly provided by integrin-mediated focal adhesions (FAs). We previously found that Shp2 is essential for FA maturation by promoting ROCK2 activation at FAs. In this study, we further delineated the role of α-actinin-4 in the FA recruitment and activation of Shp2. We used the conditional immortalized mouse podocytes to examine the role of α-actinin-4 in the regulation of Shp2 and ROCK2 signaling. After the induction of podocyte differentiation, Shp2 and ROCK2 were strongly activated, concomitant with the formation of matured FAs, stress fibers, and interdigitating intracellular junctions in a ROCK-dependent manner. Gene knockout of α-actinin-4 abolished the Shp2 activation and subsequently reduced matured FAs in podocytes. We also demonstrated that gene knockout of ROCK2 impaired the generation of contractility and interdigitating intercellular junctions. Our results reveal the role of α-actinin-4 in the recruitment of Shp2 at FAs to potentiate ROCK2 activation for the maintenance of cellular contractility and cytoskeletal architecture in the cultured podocytes.

Ihog proteins contribute to integrin-mediated focal adhesions.

Integrin expression forms focal adhesions, but how this process is physiologically regulated is unclear. Ihog proteins are evolutionarily conserved, playing roles in Hedgehog signaling and serving as trans-homophilic adhesion molecules to mediate cell-cell interactions. Whether these proteins are also engaged in other cell adhesion processes remains unknown. Here, we report that Drosophila Ihog proteins function in the integrin-mediated adhesions. Removal of Ihog proteins causes blister and spheroidal muscle in wings and embryos, respectively. We demonstrate that Ihog proteins interact with integrin via the extracellular portion and that their removal perturbs integrin distribution. Finally, we show that Boc, a mammalian Ihog protein, rescues the embryonic defects caused by removing its Drosophila homologs. We thus propose that Ihog proteins contribute to integrin-mediated focal adhesions.

Endotoxins Induced ECM-Receptor Interaction Pathway Signal Effect on the Function of MUC2 in Caco2/HT29 Co-Culture Cells.

Endotoxins are toxic substances that widely exist in the environment and can enter the intestine with food and other substances. Intestinal epithelial cells are protected by a mucus layer that contains MUC2 as its main structural component. However, a detailed understanding of the mechanisms involved in the function of the mucus barrier in endotoxin penetration is lacking. Here, we established the most suitable proportion of Caco-2/HT-29 co-culture cells as a powerful tool to evaluate the intestinal mucus layer. Our findings significantly advance current knowledge as focal adhesion and ECM-receptor interaction were identified as the two most significantly implicated pathways in MUC2 small interfering RNA (siRNA)-transfected Caco-2/HT-29 co-culture cells after 24 h of LPS stimulation. When the mucus layer was not intact, LPS was found to damage the tight junctions of Caco-2/HT29 co-cultured cells. Furthermore, LPS was demonstrated to inhibit the integrin-mediated focal adhesion structure and damage the matrix network structure of the extracellular and actin microfilament skeletons. Ultimately, LPS inhibited the interactive communication between the extracellular matrix and the cytoskeleton for 24 h in the siMUC2 group compared with the LPS(+) and LPS(-) groups. Overall, we recognized the potential of MUC2 as a tool for barrier function in several intestinal bacterial diseases.

Cell division in tissues enables macrophage infiltration.

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Microtubules tune mechanosensitive cell responses.

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.

ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions, cell migration, and invasion.

The Rho family of GTPases are inactivated in a cell context–dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, one of the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 and overexpressing its two mutually independent upstream regulators, SEPT2 and SEPT9, all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion–associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the up-regulated Integrin Beta 1. These results provide novel insights into cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.

LncRNA ITGB8-AS1 functions as a ceRNA to promote colorectal cancer growth and migration through integrin-mediated focal adhesion signaling

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and progression of colorectal cancer (CRC). However, functions of most lncRNAs in CRC and their molecular mechanisms remain uncharacterized. Here we found that lncRNA ITGB8-AS1 was highly expressed in CRC. Knockdown of ITGB8-AS1 suppressed cell proliferation, colony formation, and tumor growth in CRC, suggesting oncogenic roles of ITGB8-AS1. Transcriptomic analysis followed by KEGG analysis revealed that focal adhesion signaling was the most significantly enriched pathway for genes positively regulated by ITGB8-AS1. Consistently, knockdown of ITGB8-AS1 attenuated the phosphorylation of SRC, ERK, and p38 MAPK. Mechanistically, ITGB8-AS1 could sponge miR-33b-5p and let-7c-5p/let-7d-5p to regulate the expression of integrin family genes ITGA3 and ITGB3, respectively, in the cytosol of cells. Targeting ITGB8-AS1 using antisense oligonucleotide (ASO) markedly reduced cell proliferation and tumor growth in CRC, indicating the therapeutic potential of ITGB8-AS1 in CRC. Furthermore, ITGB8-AS1 was easily detected in plasma of CRC patients, which was positively correlated with differentiation and TNM stage, as well as plasma levels of ITGA3 and ITGB3. In conclusion, ITGB8-AS1 functions as a competing endogenous RNA (ceRNA) to regulate cell proliferation and tumor growth of CRC via regulating focal adhesion signaling. Targeting ITGB8-AS1 is effective in suppressing CRC cell growth and tumor growth. Elevated plasma levels of ITGB8-AS1 were detected in advanced-stage CRC. Thus, ITGB8-AS1 could serve as a potential therapeutic target and circulating biomarker in CRC.

Regulation of Myogenic Differentiation by Topologically Microgrooved Surfaces for Skeletal Muscle Tissue Engineering.

Inspired by the natural topological structure of skeletal muscle tissue, the topological surface construction of bionic scaffolds for skeletal muscle repair has attracted great interest. Many previous studies have focused on the effects of the topological structure on myoblasts. However, these studies used only specific repeating sizes and shapes to achieve the myoblast alignment and myotube formation; moreover, the regulatory effects of the size of a topological structure on myogenic differentiation are often neglected, leading to a lack of guidance for the design of scaffolds for skeletal muscle tissue engineering. In this study, we fabricated a series of microgroove topographies with various widths and depths via a combination of soft lithography and melt-casting and studied their effects on the behaviors of skeletal muscle cells, especially myogenic differentiation, in detail. Microgrooved poly(lactic-co-glycolic acid) substrates were found to effectively regulate the proliferation, myogenic differentiation, and myotube formation of C2C12 cells, and the degree of myogenic differentiation was significantly dependent on signals in response to the size of the microgroove structure. Compared with their depth, the width of the microgroove structures can more strongly affect the myogenic differentiation of C2C12 cells, and the degree of myoblast differentiation was enhanced with increasing groove width. Microgroove structures with relatively large groove widths and small groove depths promoted the myogenic differentiation of C2C12 cells. In addition, the integrin-mediated focal adhesion kinase signaling pathway and MAPK signaling pathway were activated in cells in response to the external topological structure, and the size of the topological structure of the material surface effectively regulated the degree of the cellular response to the external topological structure. These results can guide the design of scaffolds for skeletal muscle tissue engineering and the construction of effective bionic scaffold surfaces for skeletal muscle regeneration.