Actin filamentsThe microfilament network in the cell, composed primarily of actin and the transmembrane focal adhesions, provide an important structural framework as well as a a powerful signal transduction system through which cells interact with the environment (Charras G et al, 2014). Examples of proteins localized to these structures can be seen in Figure 1. Actin filaments and focal adhesions play essential roles in many complex cellular processes including mitosis, motility, cellular polarity and embryogenesis (Alberts B et al, 2002). Dynamic remodeling of the actin network provides a mode of controlling dynamic cellular morphology, organelle 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. Of all human proteins, 357 (approximately 2%) have been experimentally shown to localize to actin filaments in the Cell Atlas (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the core actin proteins shows highly enriched terms for biological processes related to actin binding, cytoskeletal organization, and cell signalling. Roughly 82% (n=291) of proteins observed in actin filaments localize to at least one additional cellular compartment. Two of the most common localizations observed with actin filaments are cytoplasm and plasma membrane, which is where actin monomers and actin binding proteins (ABPs) perform actin polymerization.
Figure 1. Examples of proteins localized to the actin filaments and focal adhesions. SEPT9 is a highly conserved actin binding protein necessary for cell cycle progression and cytokinesis (shown in A-431 cells). CSGALNACT1 is localized to the actin filaments in three cell lines (U-2 OS, A549, and SiHa) (shown in SiHa 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 a member of the focal adhesion complex that binds actin filaments (shown in U-2 OS cells). ZYX is 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 the 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 filamentsSubstructures Basic structure Filamentous actin (F-actin) consists of long polar microfilaments roughly 7 nm in diameter that forms a double helix structure with a pointed (-) end and a barbed (+) end made up of monomers of globular actin (G-actin). The structure of these monomers was first observed via crystallization in 2001 (Graceffa P et al, 2003). Actin filaments are linked together by ACTN1 and VCL, forming larger fibrous bundles. Myosin (TPM1) motors on these bundles can be used to exert large contractile forces for dynamically reshaping the cell (HUXLEY AF et al, 1954; HUXLEY H et al, 1954). F-actin is linked to the transmembrane component of focal adhesions, ITGB1, via a protein complex consisting of TLN2, PTK2, PXN, and ENAH (Cvrčková F. 2013). Single localizing actin filament and focal adhesion proteins are of great interest when seeking to understand cellular morphology, migration, and dynamics. Table 1 provides a list of antibodies that appear to be highly consistent across many cell types and that may be used as markers for studying the actin and focal adhesion network. Table 1. Selection of proteins suitable as markers for the actin filaments, focal adhesions or their substructures.
Actin filament formation Molecules of globular actin (G-actin) in the cytoplasm are activated through binding with ATP, which is assisted by the ABP PFN1. Once activated, G-actin can join with other G-actin molecules to form a new actin filament through a process called nucleation where the filament initially grows from both (+) and (-) ends (Cytoskeleton Dynamics, Actin Filament Assembly). Though spontaneous actin nucleation in the cytoplasm is possible, it is often assisted by various ABPs which stabilize the interaction of actin monomers allowing them to bind more easily (dos Remedios CG et al, 2003). One of the main nucleators of actin is FMN1, a member of the formin family of proteins which is anchored to the plasma membrane and is activated by members of the Rho GTPase (ARHGAP4) protein family, forming a ring shape which holds actin monomers in place at the (+) end of the actin filament allowing it to elongate and stabilize actin filaments (Watanabe N et al, 1997; Ando Y et al, 2007). Actin treadmilling Once added to the filament, G-actin monomers slowly convert their bound ATP to ADP through hydrolysis over the period of days in solution and hours in filaments (Korn ED et al, 1987). Eventually, monomers near the (-) end of the filament dissociate and are recycled to be re-incorporated in the (+) end of the filament again in an action known as "treadmilling". Under steady-state conditions, this process is relatively slow, however assisted by ABPs such as cofilin (CFL2), actin is broken off the (-) end of the filament and broken down into G-actin monomers much more quickly. This is often seen at the leading edge of the cell where actin is quickly remodeled to aid in cellular motility before binding to and stabilizing nascent focal adhesions. In fact, this treadmilling action can happen at a rate of 200 monomers/second (Selve N et al, 1986). Actin branching Another way through which actin filament formation occurs is by branching from existing filaments. Branches are formed on existing F-actin by the binding with the ARP2/3 (ARPC1B) protein family. This protein complex is activated through interaction with WAS which is first activated through interactions with CDC42 (Rouiller I et al, 2008). The WAS-ARP2/3 complex then binds to existing actin filaments and begins nucleation of actin filaments through further interactions with ATP-G-actin. This branching event happens at a highly conserved angle of 78 degrees. Though actin filaments are typically found near the cell periphery, 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 PGM1 in A-431, U-2 OS and U-251 cells.
Figure 4. 3D-view of focal adhesions in U-2 OS, visualized by immunofluorescent staining of ZYX. The morphology of focal adhesions in human induced stem cells can be seen in the Allen Cell Explorer. The function of actin filamentsIt is well known that actin filaments and focal adhesions are the main regulators of cellular morphology and motility (Mitchison TJ et al, 1996; Driscoll MK et al, 2015). In the Cell Atlas, proteins localized to the actin and focal adhesion proteomes show enrichment for these well known biological processes and molecular functions (Figure 5). In cellular motility, actin is present at the leading edge of the cell and forms several structures that assist cellular movement. During migration, cells extend filopodia, long thin actin rich protrusions ahead of the leading edge of the cell where the lamellipodia, an actin sheet, pushes the membrane of the cell forward (Wilson K et al, 2013; Alblazi KM et al, 2015).
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. Along the leading edge of the cell, nascent focal adhesions are created by bindings between receptors on the cell surface and the extracellular matrix. A fraction of these adhesion seed points are stabilized by joining to the actin network via talin (TLN2) and begin to elongate in the direction of retrograde actin flow (away from the leading edge) (Wolfenson H et al, 2009). Once fully mature, the focal adhesions provide a crucial signal transduction link between actin fibers and the extracellular matrix. Actin filaments also provide important avenues for transport of cargo throughout the cell. Cargo inside vesicles is pulled along the actin filaments via motor proteins belonging to the myosin superfamily, such as TPM1 (DePina AS et al, 1999). In muscle cells, certain myosin proteins form filaments which are positioned next to actin filaments and oriented along the major axis of the cell. When the muscle contracts, these motors walk along the adjacent actin filament, pulling the myosin filament, exerting mechanical force and contracting the cell (HUXLEY AF et al, 1954; HUXLEY H et al, 1954). Disorders in genes coding for actin and focal-adhesion-associated proteins often cause diseases in muscular tissue where dynamic cellular contractions are crucial (HUXLEY AF et al, 1954; HUXLEY H et al, 1954; Sparrow JC et al, 2003; Costa CF et al, 2004)). As part of their structural role in cells, actin filaments and focal adhesions play a key role in cell cycle progression and cellular division (Théry M et al, 2006). Particularly, it has been shown that during early cell cycle phases (G1, S) focal adhesions promote cell cycle progression (Zhao JH et al, 1998; Heng YW et al, 2010). During mitosis, focal adhesions are degraded and centrosome separation driven by the actin network and corticle actin (CTTN) (Wang W et al, 2008). Later, actin is responsible for forming the cleavage furrow, and the contractile ring, that eventually mediates cytokinesis (Heng YW et al, 2010). Due to their essential role, many proteins from the actin associated proteome are highly expressed and conserved throughout evolution (Table 2). Septins for example are found in nearly all eukaryotic cells from humans to fungi and algae and appear to play a critical role in tumor formation (Nishihama R et al, 2011; Russell SE et al, 2005). Other members of the actin proteome have been linked to cancer progression and metastisis (Alblazi KM et al, 2015; Boettner B et al, 2002). And therapeutics targeting members of the focal adhesion and actin network provide a promising means of managing cancer invasiveness (Stevenson RP et al, 2012). Table 2. Highly expressed single localizing actin and focal adhesion proteins across different cell lines.
Actin filament proteins with multiple locationsOf all actin filament and focal adhesion associated proteins localized by the Cell Atlas, 82% (n=291) are also detected in other compartments in the cell (Figure 6). The network plot shows that the most common locations shared with the actin cytoskeleton are plasmamembrane, nucleoplasm and cytosol. Compared to all other proteins in the Cell Atlas, 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). Signal transduction from extracellular focal adhesions and cellular motility through focal adhesion sites and actin treadmilling are processes occurring at and for some proteins even across the plasma membrane, making this a logical association. The cytosol, in turn, is where non-polymerized globular actin and actin associated proteins localize. Although several proteins are found both in the nucleoplasm and actin filaments, co-localization between actin filaments and nucleoplasm is underrepresented, with respect to the large proportion of proteins observed in the nucleoplasm.
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). 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 tissueTranscriptome 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 Cell Atlas. 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 Cell Atlas. 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 Thul PJ et al, 2017. A subcellular map of the human proteome. Science. 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. Cytoskeleton Dynamics. MBInfo. Accessed November 25, 2016. http://www.mechanobio.info/topics/cytoskeleton-dynamics. DePina AS et al, 1999. Vesicle transport: the role of actin filaments and myosin motors. Microsc Res Tech. Focal Adhesion Assembly. MBInfo. Accessed November 25, 2016. http://www.mechanobio.info/topics/mechanosignaling/cell-matrix-adhesion/focal-adhesion/focal-adhesion-assembly. Graceffa P et al, 2003. Crystal structure of monomeric actin in the ATP state. Structural basis of nucleotide-dependent actin dynamics. J Biol Chem. TheFunsuman. Actin Filament Assembly. YouTube. Accessed November 25, 2016. http://www.youtube.com/watch?v=n-b7Zz-sfBk. Théry M et al, 2006. Cell shape and cell division. Curr Opin Cell Biol. |
The Project
The Human Protein Atlas
The Human Protein Atlas project is funded
MENU
