The microfilament network in the cell, composed primarily of
actin and the transmembrane focal adhesions,
provide 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. This system allows the cell to interact with the extracellular matrix and is necessary for many complex cellular processes including mitosis, motility, cellular polarity
(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 in proteins in the actin and focal adhesion proteomes have been linked
to several severe diseases including muscular disorders and cancers.Focal adhesion sites: 133
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)
(Focal Adhesion Assembly, www.mechanobio.info/topics/mechanosignaling/cell-matrix-adhesion/focal-adhesion/focal-adhesion-assembly).
The structure of these
monomers was first observed via crystallization in 2001
(Graceffa P et al, 2003). Actin filaments are linked together by
VCL, forming larger fibrous
(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
(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
Table 1. Selection of proteins suitable as markers for the actin filaments, focal adhesions or their substructures.
||Chondroitin sulfate N-acetylgalactosaminyltransferase 1
||FYVE, RhoGEF and PH domain containing 4
||WD repeat domain 93
||Focal adhesion sites
||N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2
||Focal adhesion sites
||Focal adhesion sites
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 with ATP, G-actin can join with other G-actin molecules to form a new actin filament through a
process called nucleation where the filament grows from both (+) and (-) ends initially
(Cytoskeleton Dynamics, www.mechanobio.info/topics/cytoskeleton-dynamics; Actin Filament Assembly, www.youtube.com/watch?v=n-b7Zz-sfBk).
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).
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-encorporated 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).
Another way actin nucleation occurs is through branching from existing filaments. Branches are formed on existing F-actin by the binding with the ARP 2/3
(ARPC1B) protein family. This protein complex is activated through interaction with
which is first activated through interactions with
(Rouiller I et al, 2008). The WAS-ARP 2/3 complex then binds to existing actin
filaments and begins actin nucleation through further interactions with ATP-G-actin. This branching event happens at a highly conserved angle of 78o.
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.
The function of the 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; 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 4). In cellular motility, actin is present at the leading edge of the cell and
forms several structures that assist cellular motility. 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 4.a 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 4.b 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 this 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 then 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, namely
(DePina AS et al, 1999).
In muscle cells, certain myosin proteins have been shown to 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 its structural role in the cell, actin 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
(Wang W et al, 2008). Later, actin is responsible for forming the cleavage furrow, and contractile ring, that eventually aids in 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.
||Metastasis suppressor 1-like
||Sperm antigen with calponin homology and coiled-coil domains 1-like
Actin filament proteins with multiple locations
Of all actin filament and focal adhesion associated proteins localized by the Cell Atlas,
82% (n=277) are also detected in other compartments in the cell (Figure 5).
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 5, blue, see Figure 6 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. These mapped actin and focal adhesion proteins also appear in the
cytosol significantly more frequently than expected as seen in Figure 5, likely indicative of non-polymerized globular actin and actin associated proteins freely diffusing
in the cytosol. Although several proteins are found both in the nucleoplasm and actin filaments, there are significantly fewer than expected given the high proportion of proteins
observed in the nucleoplasm.
Figure 5. 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 6. 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).
CCDC53 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
The transcriptome analysis (Figure 7) shows that actin filament and focal adhesion proteins are significantly more likely to be tissue
enhanced as compared to other proteins in the Cell Atlas.
Figure 7. Bar plot showing the distribution of expression categories, based on the gene expression in tissues, for actin filaments-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks statistically significant deviation(s) (p≤0.05) from all other organelles 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
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. www.mechanobio.info/topics/cytoskeleton-dynamics.
DePina AS et al, 1999. Vesicle transport: the role of actin filaments and myosin motors. Microsc Res Tech.
PubMed: 10523788 DOI: 10.1002/(SICI)1097-0029(19991015)47:2<93::AID-JEMT2>3.0.CO;2-P
dos Remedios CG et al, 2003. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev.
PubMed: 12663865 DOI: 10.1152/physrev.00026.2002
Driscoll MK et al, 2015. Quantifying Modes of 3D Cell Migration. Trends Cell Biol.
PubMed: 26603943 DOI: 10.1016/j.tcb.2015.09.010
Focal Adhesion Assembly. MBInfo. Accessed November 25, 2016. 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.
PubMed: 12813032 DOI: 10.1074/jbc.M303689200
Heng YW et al, 2010. Actin cytoskeleton dynamics and the cell division cycle. Int J Biochem Cell Biol.
PubMed: 20412868 DOI: 10.1016/j.biocel.2010.04.007
HUXLEY AF et al, 1954. Structural changes in muscle during contraction; interference microscopy of living muscle fibres. Nature.
HUXLEY H et al, 1954. Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature.
Korn ED et al, 1987. Actin polymerization and ATP hydrolysis. Science.
Mitchison TJ et al, 1996. Actin-based cell motility and cell locomotion. Cell.
Nishihama R et al, 2011. New insights into the phylogenetic distribution and evolutionary origins of the septins. Biol Chem.
PubMed: 21824002 DOI: 10.1515/BC.2011.086
Rouiller I et al, 2008. The structural basis of actin filament branching by the Arp2/3 complex. J Cell Biol.
PubMed: 18316411 DOI: 10.1083/jcb.200709092
Russell SE et al, 2005. Do septins have a role in cancer? Br J Cancer.
PubMed: 16136025 DOI: 10.1038/sj.bjc.6602753
Selve N et al, 1986. Rate of treadmilling of actin filaments in vitro. J Mol Biol.
Sparrow JC et al, 2003. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord.
Stevenson RP et al, 2012. Actin-bundling proteins in cancer progression at a glance. J Cell Sci.
PubMed: 22492983 DOI: 10.1242/jcs.093799
TheFunsuman. Actin Filament Assembly. YouTube. Accessed November 25, 2016. www.youtube.com/watch?v=n-b7Zz-sfBk.
Théry M et al, 2006. Cell shape and cell division. Curr Opin Cell Biol.
PubMed: 17046223 DOI: 10.1016/j.ceb.2006.10.001
Wang W et al, 2008. Centrosome separation driven by actin-microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin. J Cell Sci.
PubMed: 18388321 DOI: 10.1242/jcs.018176
Watanabe N et al, 1997. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J.
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Wilson K et al, 2013. Mechanisms of leading edge protrusion in interstitial migration. Nat Commun.
PubMed: 24305616 DOI: 10.1038/ncomms3896
Wolfenson H et al, 2009. The heel and toe of the cell's foot: a multifaceted approach for understanding the structure and dynamics of focal adhesions. Cell Motil Cytoskeleton.
PubMed: 19598236 DOI: 10.1002/cm.20410
Zhao JH et al, 1998. Regulation of the cell cycle by focal adhesion kinase. J Cell Biol.