Actin filaments, in the form of microfilaments, are one of three major components of the cytoskeleton. In addition, actin forms thin filaments, which are part of the contractile apparatus in muscle cells. Focal adhesions are large protein complexes that link the actin cytoskeleton to the extracellular matrix (ECM). Examples of proteins localized to these structures can be seen in Figure 1. Actin filaments and focal adhesions provide an important structural framework and signal transduction system that plays essential roles in cell morphology and polarity, organization of organelles, motility, mitosis, cytokinesis, and cell signalling (Pollard TD et al. (2009),Alberts B et al, 2002). Dynamic remodeling of the actin network provides a mode of regulating cellular morphology, organization and motility in response to various chemical and mechanical signals (Mitchison TJ et al. (1996)). Dysfunction of proteins in the actin and focal adhesion proteomes have been linked to several severe diseases, including muscular disorders and cancers.
In the Subcellular Section, 363 genes (2% of all protein-coding human genes) have been shown to encode proteins that localize to actin filaments or focal adhesion sites (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the core actin proteins reveals enrichment of terms describing biological processes related to actin binding, cytoskeletal organization, and cell signalling. Roughly 83% (n=300) of the proteins that localize to actin filaments also localize to at least one additional cellular compartment. Two of the most common additional localizations observed with actin filaments are cytoplasm and plasma membrane, which is where actin monomers and actin binding proteins (ABPs) polymerize.
Figure 1. Examples of proteins localized to the actin filaments and focal adhesions. SEPTIN9 is a highly conserved actin binding protein necessary for cell cycle progression and cytokinesis (shown in A-431 cells). CNN3 is an actin-binding protein that is involved in regulation of smooth muscle contraction (shown in U-2 OS cells). FGD4 is an actin binding protein that regulates cell shape (shown in A-431 cells). PXN is a member of the focal adhesion complex that binds actin filaments (shown in U-2 OS cells). TNS1 is another member of the focal adhesion complex that binds actin filaments (shown in U-2 OS cells). ZYX is also a member of the focal adhesion complex that binds actin filaments and may be involved in extracellular signal transduction (shown in A-431 cells).
Figure 2. 2% of all human protein-coding genes encode proteins localized to actin filaments or focal adhesions. Each bar is clickable and gives a search result of proteins that belong to the selected category.
The structure of actin filaments
Actin is a highly conserved family of proteins that are abundant in eukaryotic cells. In humans, there are three major types of actin; α-actin is found in contractile structures in muscle cells, while β-actin and γ-actin are prominent in different structures in non-muscle cells. Actin proteins have a characteristic globular structure with an ATP-binding site and ATP hydrolytic activity. Monomers of actin (G-actin) can polymerize into long filaments (F-actin) with a helical structure of around 7 nm in diameter. Due to the fact that all subunits of F-actin are oriented in the same direction, the filaments have a polarized structure with a pointed (-) end and a barbed (+) end. Individual actin filaments can branch as well as bundle together forming an elaborate network, together with associated proteins, regulatory factors and motor proteins, known as the actin cytoskeleton.
The actin cytoskeleton is generally more prominent near the plasma membrane, where focal adhesions provide important mechanical links to the ECM. Focal adhesions are large multi-protein structures with a highly dynamic composition. An important constituent are transmembrane proteins called integrins, with an intracellular part that binds to the cytoskeleton via adaptor proteins and an extracellular part that binds to various components of the ECM.
Actin, together with myosin-II motor proteins, also play an important role during cytokinesis (Pollard TD et al. (2019)). During mitosis, actin and myosin-II are assembled into a contractile ring, positioned around the equator of the cell. Constriction of this ring results in the formation of a cleavage furrow, which continues to ingress until a cytokinetic bridge is formed. Table 1 provides a list of genes that may be used as markers for actin filaments and focal adhesions and Table 2 provides a list of highly expressed genes encoding proteins that localize to these structures.
Table 1. Selection of proteins suitable as markers for the actin filaments and focal adhesions .
Table 2. Highly expressed single localizing actin and focal adhesion proteins across different cell lines.
Actin polymerization and depolymerization are highly dynamic processes that are regulated by a large number of actin binding proteins (ABPs) (dos Remedios CG et al. (2003); Campellone KG et al. (2010); Rottner K et al. (2017)) (Actin Filament Assembly). The first step of polymerization is nucleation, which involves formation of short polymers stabilized by nucleating factors, such as FMN1 and LMOD2. Actin polymerization can also occur by branching from existing filaments with the help of the ARP2/3 complex (including ARPC1B). Nucleation is followed by an elongation phase, stimulated by elongation factors like FMN1, in which the filaments grows by further addition of actin monomers, preferentially to the barbed (+) end. As the monomers age in the filament, the bound ATP is hydrolyzed to ADP, which promotes dissociation. A steady-state dynamic equilibrium is reached when polymerization and depolymerization occurs at the same rate, but can rapidly be shifted by regulatory factors. Many of these regulatory factors are driven, at least in part, by the activity of specific members of the Rho family of small GTPases.
Actin filaments are typically found near the cell periphery, but the actin network may appear very different depending on the characteristics of a given cell type (Figure 3).
Figure 3. Examples of the morphology of actin filaments in different cell lines, represented by immunofluorescent staining of protein SEPTIN9 in A-431, U-2 OS and U-251 cells.
The function of actin filaments
It is well known that actin filaments and focal adhesions are the main regulators of cellular morphology and motility (Mitchison TJ et al. (1996); Pollard TD et al. (2009); Bird RP. (1987)). Myosin (TPM1) motors on these bundles can be used to exert large contractile forces for dynamically reshaping the cell as well as creating locomotion (HUXLEY AF et al. (1954); HUXLEY H et al. (1954)). The latter generally involves formation of an actin-dependent prostrution, like filopodia and lamellipodia (Svitkina T. (2018)). Actin filaments are also involved in endocytosis and provide important avenues for transport of cargo, especially organelles, throughout the cell. Again, motor proteins of the myosin family play an important role in the latter. Another important function of actin filaments is in cytokinesis, where a contractile ring of actin and myosin II is used to create a cleavage furrow and pinch the cell into two daughter cells.
Gene Ontology (GO) analysis of proteins localized to actin filaments and focal adhesions show enrichment of terms describing biological processes and molecular functions in line with known functions of the actin cytoskeleton (Figure 5). The most highly enriched terms for Biological Process are integrin-mediated signalling, regulation of cell shape and cytokinesis. The most highly enriched terms for Molecular Function are binding to different components of the actin cytoskeleton, microfilament motor activity and cell adhesion mediator activity.
Figure 5a Gene Ontology-based enrichment analysis for the actin filament proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 5b Gene Ontology-based enrichment analysis for the actin filament proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Actin filament proteins with multiple locations
Among proteins that localize to actin filaments and focal adhesions in the Subcellular Section, 83% (n=300) are also detected in other cellular compartments (Figure 6). The network plot shows that the most common locations shared with the actin cytoskeleton are plasma membrane, nucleoplasm and cytosol. Compared to all other proteins in the Subcellular Section, actin and focal adhesion associated proteins are significantly more likely to also localize to the plasma membrane (Figure 6, blue, see Figure 7 for example). This may reflect the role of the actin cytoskeleton in transducing mechanical forces and signals across the plasma membrane, and in controlling the shape of the cell cortex. The cytosol, in turn, is where non-polymerized globular actin and actin associated proteins localize. Co-localization of actin filaments with the nucleoplasm could reflect the existence of nuclear actin (Kelpsch DJ et al. (2018)), however these co-localizing proteins are underrepresented. Examples of multilocalizing proteins within the actin filament proteome can be seen in Figure 7.
Figure 6. Interactive network plot of actin filament and focal adhesion proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to actin filaments or focal adhesions and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the actin filaments and focal adhesion proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.
Figure 7. Examples of multilocalizing proteins in the actin filament and focal adhesion proteome. The first two examples show common or overrepresented combinations for multilocalizing proteins in the actin filament and focal adhesion proteome while the last shows an example of the underrepresented overlap between this proteome and vesicles. PDLIM7 is likely an adapter protein that is involved in the assembly of actin filaments and focal adhesions (shown in U-251 MG cells). LIMA1 is another member of the LIM family of proteins and can be found at the actin filaments, focal adhesion sites, plasma membrane and cytoplasm. It inhibits actin filament depolymerization and stabilizes filaments via crosslinking of filament bundles (shown in U-2 OS cells). WASHC3 is a vesicle (endosome)-associated protein that is involved in the regulation of actin polymerization through interactions with ARP 2/3 (shown in U-2 OS cells).
Expression levels of actin filaments proteins in tissue
Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that genes encoding proteins that localize to actin filaments and focal adhesion sites are more likely to be expressed in many tissues, but less likely to be detected in all tissues, compared to all genes presented in the Subcellular Section. Thus, these genes tend to show a somewhat more restricted pattern of tissue expression.
Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for actin filaments-associated protein-coding genes compared to all genes in the Subcellular Section. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Relevant links and publications
Uhlen M et al., A proposal for validation of antibodies. Nat Methods. (2016)
Alberts B et al, 2002. Molecular Biology of the Cell. 4th edition. The Self-Assembly and Dynamic Structure of Cytoskeletal Filaments. New York: Garland Science.
TheFunsuman. Actin Filament Assembly. YouTube. Accessed November 25, 2016. http://www.youtube.com/watch?v=n-b7Zz-sfBk.