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Actin

Motor protein involved in muscle contraction

Top 10 Actin related articles

Actin
Ribbon diagram of G-actin. ADP bound to actin's active site (multi color sticks near center of figure) as well as a complexed calcium dication (green sphere) are highlighted.[1]
Identifiers
SymbolActin
PfamPF00022
InterProIPR004000
PROSITEPDOC00340
SCOP22btf / SCOPe / SUPFAM

Actin is a family of globular multi-functional proteins that form microfilaments. It is found in essentially all eukaryotic cells, where it may be present at a concentration of over 100 μM; its mass is roughly 42-kDa, with a diameter of 4 to 7 nm.

An actin protein is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. It can be present as either a free monomer called G-actin (globular) or as part of a linear polymer microfilament called F-actin (filamentous), both of which are essential for such important cellular functions as the mobility and contraction of cells during cell division.

Actin participates in many important cellular processes, including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes.[2] In vertebrates, three main groups of actin isoforms, alpha, beta, and gamma have been identified. The alpha actins, found in muscle tissues, are a major constituent of the contractile apparatus. The beta and gamma actins coexist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility. It is believed that the diverse range of structures formed by actin enabling it to fulfill such a large range of functions is regulated through the binding of tropomyosin along the filaments.[3]

A cell's ability to dynamically form microfilaments provides the scaffolding that allows it to rapidly remodel itself in response to its environment or to the organism's internal signals, for example, to increase cell membrane absorption or increase cell adhesion in order to form cell tissue. Other enzymes or organelles such as cilia can be anchored to this scaffolding in order to control the deformation of the external cell membrane, which allows endocytosis and cytokinesis. It can also produce movement either by itself or with the help of molecular motors. Actin therefore contributes to processes such as the intracellular transport of vesicles and organelles as well as muscular contraction and cellular migration. It therefore plays an important role in embryogenesis, the healing of wounds, and the invasivity of cancer cells. The evolutionary origin of actin can be traced to prokaryotic cells, which have equivalent proteins.[4] Actin homologs from prokaryotes and archaea polymerize into different helical or linear filaments consisting of one or multiple strands. However the in-strand contacts and nucleotide binding sites are preserved in prokaryotes and in archaea.[5] Lastly, actin plays an important role in the control of gene expression.

A large number of illnesses and diseases are caused by mutations in alleles of the genes that regulate the production of actin or of its associated proteins. The production of actin is also key to the process of infection by some pathogenic microorganisms. Mutations in the different genes that regulate actin production in humans can cause muscular diseases, variations in the size and function of the heart as well as deafness. The make-up of the cytoskeleton is also related to the pathogenicity of intracellular bacteria and viruses, particularly in the processes related to evading the actions of the immune system.[6]

Actin Intro articles: 42

Discovery and early investigation

Actin was first observed experimentally in 1887 by W.D. Halliburton, who extracted a protein from muscle that 'coagulated' preparations of myosin that he called "myosin-ferment".[7] However, Halliburton was unable to further refine his findings, and the discovery of actin is credited instead to Brunó Ferenc Straub, a young biochemist working in Albert Szent-Györgyi's laboratory at the Institute of Medical Chemistry at the University of Szeged, Hungary.

Following up on the discovery of Ilona Banga & Szent-Györgyi in 1941 that the coagulation only occurs in some mysosin extractions and was reversed upon the addition of ATP,[8] Straub identified and purified actin from those myosin preparations that did coagulate. Building on Banga's original extraction method, he developed a novel technique for extracting muscle protein that allowed him to isolate substantial amounts of relatively pure actin, published in 1942.[9] Straub's method is essentially the same as that used in laboratories today. Since Straub's protein was necessary to activate the coagulation of myosin, it was dubbed actin.[8][10] Realizing that Banga's coagulating myosin preparations contained actin as well, Szent-Györgyi called the mixture of both proteins actomyosin.[11]

The hostilities of World War II meant Szent-Gyorgyi was unable to publish his lab's work in Western scientific journals. Actin therefore only became well known in the West in 1945, when their paper was published as a supplement to the Acta Physiologica Scandinavica.[12] Straub continued to work on actin, and in 1950 reported that actin contains bound ATP[13] and that, during polymerization of the protein into microfilaments, the nucleotide is hydrolyzed to ADP and inorganic phosphate (which remain bound to the microfilament). Straub suggested that the transformation of ATP-bound actin to ADP-bound actin played a role in muscular contraction. In fact, this is true only in smooth muscle, and was not supported through experimentation until 2001.[13][14]

The amino acid sequencing of actin was completed by M. Elzinga and co-workers in 1973.[15] The crystal structure of G-actin was solved in 1990 by Kabsch and colleagues.[16] In the same year, a model for F-actin was proposed by Holmes and colleagues following experiments using co-crystallization with different proteins.[17] The procedure of co-crystallization with different proteins was used repeatedly during the following years, until in 2001 the isolated protein was crystallized along with ADP. However, there is still no high-resolution X-ray structure of F-actin. The crystallization of F-actin was possible due to the use of a rhodamine conjugate that impedes polymerization by blocking the amino acid cys-374.[1] Christine Oriol-Audit died in the same year that actin was first crystallized but she was the researcher that in 1977 first crystallized actin in the absence of Actin Binding Proteins (ABPs). However, the resulting crystals were too small for the available technology of the time.[18]

Although no high-resolution model of actin's filamentous form currently exists, in 2008 Sawaya's team were able to produce a more exact model of its structure based on multiple crystals of actin dimers that bind in different places.[19] This model has subsequently been further refined by Sawaya and Lorenz. Other approaches such as the use of cryo-electron microscopy and synchrotron radiation have recently allowed increasing resolution and better understanding of the nature of the interactions and conformational changes implicated in the formation of actin filaments.[20][21][22]

Actin Discovery and early investigation articles: 29

Structure

Actin's amino acid sequence is one of the most highly conserved of the proteins as it has changed little over the course of evolution, differing by no more than 20% in species as diverse as algae and humans.[23] It is therefore considered to have an optimised structure.[4] It has two distinguishing features: it is an enzyme that slowly hydrolizes ATP, the "universal energy currency" of biological processes. However, the ATP is required in order to maintain its structural integrity. Its efficient structure is formed by an almost unique folding process. In addition, it is able to carry out more interactions than any other protein, which allows it to perform a wider variety of functions than other proteins at almost every level of cellular life.[4] Myosin is an example of a protein that bonds with actin. Another example is villin, which can weave actin into bundles or cut the filaments depending on the concentration of calcium cations in the surrounding medium.[24]

Actin is one of the most abundant proteins in eukaryotes, where it is found throughout the cytoplasm.[24] In fact, in muscle fibres it comprises 20% of total cellular protein by weight and between 1% and 5% in other cells. However, there is not only one type of actin; the genes that code for actin are defined as a gene family (a family that in plants contains more than 60 elements, including genes and pseudogenes and in humans more than 30 elements).[4][25] This means that the genetic information of each individual contains instructions that generate actin variants (called isoforms) that possess slightly different functions. This, in turn, means that eukaryotic organisms express different genes that give rise to: α-actin, which is found in contractile structures; β-actin, found at the expanding edge of cells that use the projection of their cellular structures as their means of mobility; and γ-actin, which is found in the filaments of stress fibres.[26] In addition to the similarities that exist between an organism's isoforms there is also an evolutionary conservation in the structure and function even between organisms contained in different eukaryotic domains. In bacteria the actin homologue MreB has been identified, which is a protein that is capable of polymerizing into microfilaments;[4][21] and in archaea the homologue Ta0583 is even more similar to the eukaryotic actins.[27]

Cellular actin has two forms: monomeric globules called G-actin and polymeric filaments called F-actin (that is, as filaments made up of many G-actin monomers). F-actin can also be described as a microfilament. Two parallel F-actin strands must rotate 166 degrees to lie correctly on top of each other. This creates the double helix structure of the microfilaments found in the cytoskeleton. Microfilaments measure approximately 7 nm in diameter with the helix repeating every 37 nm. Each molecule of actin is bound to a molecule of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) that is associated with a Mg2+ cation. The most commonly found forms of actin, compared to all the possible combinations, are ATP-G-Actin and ADP-F-actin.[28][29]

G-Actin

Scanning electron microscope images indicate that G-actin has a globular structure; however, X-ray crystallography shows that each of these globules consists of two lobes separated by a cleft. This structure represents the “ATPase fold”, which is a centre of enzymatic catalysis that binds ATP and Mg2+ and hydrolyzes the former to ADP plus phosphate. This fold is a conserved structural motif that is also found in other proteins that interact with triphosphate nucleotides such as hexokinase (an enzyme used in energy metabolism) or in Hsp70 proteins (a protein family that play an important part in protein folding).[30] G-actin is only functional when it contains either ADP or ATP in its cleft but the form that is bound to ATP predominates in cells when actin is present in its free state.[28]

Ribbon model of actin extracted from the striated muscle tissue of a rabbit after Graceffa and Domínguez, 2003. The four subdomains can be seen, as well as the N and C termini and the position of the ATP bond. The molecule is oriented using the usual convention of placing the - end (pointed end) in the upper part and the + end (barbed end) in the lower part.[1]

The X-ray crystallography model of actin that was produced by Kabsch from the striated muscle tissue of rabbits is the most commonly used in structural studies as it was the first to be purified. The G-actin crystallized by Kabsch is approximately 67 x 40 x 37 Å in size, has a molecular mass of 41,785 Da and an estimated isoelectric point of 4.8. Its net charge at pH = 7 is -7.[15][31]

Primary structure

Elzinga and co-workers first determined the complete peptide sequence for this type of actin in 1973, with later work by the same author adding further detail to the model. It contains 374 amino acid residues. Its N-terminus is highly acidic and starts with an acetyled aspartate in its amino group. While its C-terminus is alkaline and is formed by a phenylalanine preceded by a cysteine, which has a degree of functional importance. Both extremes are in close proximity within the I-subdomain. An anomalous Nτ-methylhistidine is located at position 73.[31]

Tertiary structure — domains

The tertiary structure is formed by two domains known as the large and the small, which are separated by a cleft centred around the location of the bond with ATP-ADP+Pi. Below this there is a deeper notch called a “groove”. In the native state, despite their names, both have a comparable depth.[15]

The normal convention in topological studies means that a protein is shown with the biggest domain on the left-hand side and the smallest domain on the right-hand side. In this position the smaller domain is in turn divided into two: subdomain I (lower position, residues 1–32, 70–144, and 338–374) and subdomain II (upper position, residues 33–69). The larger domain is also divided in two: subdomain III (lower, residues 145–180 and 270–337) and subdomain IV (higher, residues 181–269). The exposed areas of subdomains I and III are referred to as the “barbed” ends, while the exposed areas of domains II and IV are termed the “pointed" ends. This nomenclature refers to the fact that, due to the small mass of subdomain II actin is polar; the importance of this will be discussed below in the discussion on assembly dynamics. Some authors call the subdomains Ia, Ib, IIa, and IIb, respectively.[32]

Other important structures

The most notable supersecondary structure is a five chain beta sheet that is composed of a β-meander and a β-α-β clockwise unit. It is present in both domains suggesting that the protein arose from gene duplication.[16]

  • The adenosine nucleotide binding site is located between two beta hairpin-shaped structures pertaining to the I and III domains. The residues that are involved are Asp11-Lys18 and Asp154-His161 respectively.
  • The divalent cation binding site is located just below that for the adenosine nucleotide. In vivo it is most often formed by Mg2+ or Ca2+ while in vitro it is formed by a chelating structure made up of Lys18 and two oxygens from the nucleotide's α-and β-phosphates. This calcium is coordinated with six water molecules that are retained by the amino acids Asp11, Asp154, and Gln137. They form a complex with the nucleotide that restricts the movements of the so-called "hinge" region, located between residues 137 and 144. This maintains the native form of the protein until its withdrawal denatures the actin monomer. This region is also important because it determines whether the protein's cleft is in the "open" or "closed" conformation.[1][32]
  • It is highly likely that there are at least three other centres with a lesser affinity (intermediate) and still others with a low affinity for divalent cations. It has been suggested that these centres may play a role in the polymerization of actin by acting during the activation stage.[32]
  • There is a structure in subdomain 2 that is called the “D-loop” because it binds with DNase I, it is located between the His40 and Gly48 residues. It has the appearance of a disorderly element in the majority of crystals, but it looks like a β-sheet when it is complexed with DNase I. It has been proposed that the key event in polymerization is probably the propagation of a conformational change from the centre of the bond with the nucleotide to this domain, which changes from a loop to a spiral.[1] However, this hypothesis has been refuted by other studies.[33]

F-Actin

F-actin; surface representation of a repetition of 13 subunits based on Ken Holmes' actin filament model[17]

The classical description of F-actin states that it has a filamentous structure that can be considered to be a single stranded levorotatory helix with a rotation of 166° around the helical axis and an axial translation of 27.5 Å, or a single stranded dextrorotatory helix with a cross over spacing of 350–380 Å, with each actin surrounded by four more.[34] The symmetry of the actin polymer at 2.17 subunits per turn of a helix is incompatible with the formation of crystals, which is only possible with a symmetry of exactly 2, 3, 4 or 6 subunits per turn. Therefore, models have to be constructed that explain these anomalies using data from electron microscopy, cryo-electron microscopy, crystallization of dimers in different positions and diffraction of X-rays.[21][22] It should be pointed out that it is not correct to talk of a “structure” for a molecule as dynamic as the actin filament. In reality we talk of distinct structural states, in these the measurement of axial translation remains constant at 27.5 Å while the subunit rotation data shows considerable variability, with displacements of up to 10% from its optimum position commonly seen. Some proteins, such as cofilin appear to increase the angle of turn, but again this could be interpreted as the establishment of different structural states. These could be important in the polymerization process.[35]

There is less agreement regarding measurements of the turn radius and filament thickness: while the first models assigned a length of 25 Å, current X-ray diffraction data, backed up by cryo-electron microscopy suggests a length of 23.7 Å. These studies have shown the precise contact points between monomers. Some are formed with units of the same chain, between the "barbed" end on one monomer and the "pointed" end of the next one. While the monomers in adjacent chains make lateral contact through projections from subdomain IV, with the most important projections being those formed by the C-terminus and the hydrophobic link formed by three bodies involving residues 39–42, 201–203, and 286. This model suggests that a filament is formed by monomers in a "sheet" formation, in which the subdomains turn about themselves, this form is also found in the bacterial actin homologue MreB.[21]

The F-actin polymer is considered to have structural polarity due to the fact that all the microfilament's subunits point towards the same end. This gives rise to a naming convention: the end that possesses an actin subunit that has its ATP binding site exposed is called the "(-) end", while the opposite end where the cleft is directed at a different adjacent monomer is called the "(+) end".[26] The terms "pointed" and "barbed" referring to the two ends of the microfilaments derive from their appearance under transmission electron microscopy when samples are examined following a preparation technique called "decoration". This method consists of the addition of myosin S1 fragments to tissue that has been fixed with tannic acid. This myosin forms polar bonds with actin monomers, giving rise to a configuration that looks like arrows with feather fletchings along its shaft, where the shaft is the actin and the fletchings are the myosin. Following this logic, the end of the microfilament that does not have any protruding myosin is called the point of the arrow (- end) and the other end is called the barbed end (+ end).[36] A S1 fragment is composed of the head and neck domains of myosin II. Under physiological conditions, G-actin (the monomer form) is transformed to F-actin (the polymer form) by ATP, where the role of ATP is essential.[37]

The helical F-actin filament found in muscles also contains a tropomyosin molecule, which is a 40 nanometre long protein that is wrapped around the F-actin helix.[22] During the resting phase the tropomyosin covers the actin's active sites so that the actin-myosin interaction cannot take place and produce muscular contraction. There are other protein molecules bound to the tropomyosin thread, these are the troponins that have three polymers: troponin I, troponin T, and troponin C.[38]

Folding

Ribbon model obtained using the PyMOL programme on crystallographs (PDB: 2ZDI​) of the prefoldin proteins found in the archaean Pyrococcus horikoshii. The six supersecondary structures are present in a coiled helix “hanging” from the central beta barrels. These are often compared in the literature to the tentacles of a jellyfish. As far as is visible using electron microscopy, eukariotic prefoldin has a similar structure.[39]

Actin can spontaneously acquire a large part of its tertiary structure.[40] However, the way it acquires its fully functional form from its newly synthesized native form is special and almost unique in protein chemistry. The reason for this special route could be the need to avoid the presence of incorrectly folded actin monomers, which could be toxic as they can act as inefficient polymerization terminators. Nevertheless, it is key to establishing the stability of the cytoskeleton, and additionally, it is an essential process for coordinating the cell cycle.[41][42]

CCT is required in order to ensure that folding takes place correctly. CCT is a group II chaperonin, a large protein complex that assists in the folding of other proteins. CCT is formed of a double ring of eight different subunits (hetero-octameric) and it differs from group I chaperonins like GroEL, which is found in Eubacteria and in eukaryotic organelles, as it does not require a co-chaperone to act as a lid over the central catalytic cavity. Substrates bind to CCT through specific domains. It was initially thought that it only bound with actin and tubulin, although recent immunoprecipitation studies have shown that it interacts with a large number of polypeptides, which possibly function as substrates. It acts through ATP-dependent conformational changes that on occasion require several rounds of liberation and catalysis in order to complete a reaction.[43]

In order to successfully complete their folding, both actin and tubulin need to interact with another protein called prefoldin, which is a heterohexameric complex (formed by six distinct subunits), in an interaction that is so specific that the molecules have coevolved. Actin complexes with prefoldin while it is still being formed, when it is approximately 145 amino acids long, specifically those at the N-terminal.[44]

Different recognition sub-units are used for actin or tubulin although there is some overlap. In actin the subunits that bind with prefoldin are probably PFD3 and PFD4, which bind in two places one between residues 60–79 and the other between residues 170–198. The actin is recognized, loaded, and delivered to the cytosolic chaperonin (CCT) in an open conformation by the inner end of prefoldin's "tentacles” (see the image and note).[40] The contact when actin is delivered is so brief that a tertiary complex is not formed, immediately freeing the prefoldin.[39]

Ribbon model of the apical γ-domain of the chaperonin CCT

The CCT then causes actin's sequential folding by forming bonds with its subunits rather than simply enclosing it in its cavity.[45] This is why it possesses specific recognition areas in its apical β-domain. The first stage in the folding consists of the recognition of residues 245–249. Next, other determinants establish contact.[46] Both actin and tubulin bind to CCT in open conformations in the absence of ATP. In actin's case, two subunits are bound during each conformational change, whereas for tubulin binding takes place with four subunits. Actin has specific binding sequences, which interact with the δ and β-CCT subunits or with δ-CCT and ε-CCT. After AMP-PNP is bound to CCT the substrates move within the chaperonin's cavity. It also seems that in the case of actin, the CAP protein is required as a possible cofactor in actin's final folding states.[42]

The exact manner by which this process is regulated is still not fully understood, but it is known that the protein PhLP3 (a protein similar to phosducin) inhibits its activity through the formation of a tertiary complex.[43]

ATPase’s catalytic mechanism

Actin is an ATPase, which means that it is an enzyme that hydrolyzes ATP. This group of enzymes is characterised by their slow reaction rates. It is known that this ATPase is “active”, that is, its speed increases by some 40,000 times when the actin forms part of a filament.[35] A reference value for this rate of hydrolysis under ideal conditions is around 0.3 s−1. Then, the Pi remains bound to the actin next to the ADP for a long time, until it is cooperatively liberated from the interior of the filament.[47][48]

The exact molecular details of the catalytic mechanism are still not fully understood. Although there is much debate on this issue, it seems certain that a "closed" conformation is required for the hydrolysis of ATP, and it is thought that the residues that are involved in the process move to the appropriate distance.[35] The glutamic acid Glu137 is one of the key residues, which is located in subdomain 1. Its function is to bind the water molecule that produces a nucleophilic attack on the ATP's γ-phosphate bond, while the nucleotide is strongly bound to subdomains 3 and 4. The slowness of the catalytic process is due to the large distance and skewed position of the water molecule in relation to the reactant. It is highly likely that the conformational change produced by the rotation of the domains between actin's G and F forms moves the Glu137 closer allowing its hydrolysis. This model suggests that the polymerization and ATPase's function would be decoupled straight away.[21][22] The "open" to "closed" transformation between G and F forms and its implications on the relative motion of several key residues and the formation of water wires have been characterized in molecular dynamics and QM/MM simulations.[49][50]

Actin Structure articles: 104

Genetics

Principal interactions of structural proteins are at cadherin-based adherens junction. Actin filaments are linked to α-actinin and to the membrane through vinculin. The head domain of vinculin associates to E-cadherin via α-catenin, β-catenin, and γ-catenin. The tail domain of vinculin binds to membrane lipids and to actin filaments.

Actin has been one of the most highly conserved proteins throughout evolution because it interacts with a large number of other proteins. It has 80.2% sequence conservation at the gene level between Homo sapiens and Saccharomyces cerevisiae (a species of yeast), and 95% conservation of the primary structure of the protein product.[4]

Although most yeasts have only a single actin gene, higher eukaryotes, in general, express several isoforms of actin encoded by a family of related genes. Mammals have at least six actin isoforms coded by separate genes,[51] which are divided into three classes (alpha, beta, and gamma) according to their isoelectric points. In general, alpha actins are found in muscle (α-skeletal, α-aortic smooth, α-cardiac), whereas beta and gamma isoforms are prominent in non-muscle cells (β-cytoplasmic, γ1-cytoplasmic, γ2-enteric smooth). Although the amino acid sequences and in vitro properties of the isoforms are highly similar, these isoforms cannot completely substitute for one another in vivo.[52]

The typical actin gene has an approximately 100-nucleotide 5' UTR, a 1200-nucleotide translated region, and a 200-nucleotide 3' UTR. The majority of actin genes are interrupted by introns, with up to six introns in any of 19 well-characterised locations. The high conservation of the family makes actin the favoured model for studies comparing the introns-early and introns-late models of intron evolution.

All non-spherical prokaryotes appear to possess genes such as MreB, which encode homologues of actin; these genes are required for the cell's shape to be maintained. The plasmid-derived gene ParM encodes an actin-like protein whose polymerized form is dynamically unstable, and appears to partition the plasmid DNA into its daughter cells during cell division by a mechanism analogous to that employed by microtubules in eukaryotic mitosis.[53] Actin is found in both smooth and rough endoplasmic reticulums.

Actin Genetics articles: 18

Assembly dynamics

Nucleation and polymerization

Thin filament formation showing the polymerization mechanism for converting G-actin to F-actin; note the hydrolysis of the ATP.

Nucleating factors are necessary to stimulate actin polymerization. One such nucleating factor is the Arp2/3 complex, which mimics a G-actin dimer in order to stimulate the nucleation (or formation of the first trimer) of monomeric G-actin. The Arp2/3 complex binds to actin filaments at 70 degrees to form new actin branches off existing actin filaments. Arp2/3-mediated nucleation is necessary for directed cell migration.[54] Also, actin filaments themselves bind ATP, and hydrolysis of this ATP stimulates destabilization of the polymer.

The growth of actin filaments can be regulated by thymosin and profilin. Thymosin binds to G-actin to buffer the polymerizing process, while profilin binds to G-actin to exchange ADP for ATP, promoting the monomeric addition to the barbed, plus end of F-actin filaments.

F-actin is both strong and dynamic. Unlike other polymers, such as DNA, whose constituent elements are bound together with covalent bonds, the monomers of actin filaments are assembled by weaker bonds.[55] The lateral bonds with neighbouring monomers resolve this anomaly, which in theory should weaken the structure as they can be broken by thermal agitation. In addition, the weak bonds give the advantage that the filament ends can easily release or incorporate monomers. This means that the filaments can be rapidly remodelled and can change cellular structure in response to an environmental stimulus. Which, along with the biochemical mechanism by which it is brought about is known as the "assembly dynamic".[6]

In vitro studies

Studies focusing on the accumulation and loss of subunits by microfilaments are carried out in vitro (that is, in the laboratory and not on cellular systems) as the polymerization of the resulting actin gives rise to the same F-actin as produced in vivo. The in vivo process is controlled by a multitude of proteins in order to make it responsive to cellular demands, this makes it difficult to observe its basic conditions.[56]

In vitro production takes place in a sequential manner: first, there is the "activation phase", when the bonding and exchange of divalent cations occurs in specific places on the G-actin, which is bound to ATP. This produces a conformational change, sometimes called G*-actin or F-actin monomer as it is very similar to the units that are located on the filament.[32] This prepares it for the "nucleation phase", in which the G-actin gives rise to small unstable fragments of F-actin that are able to polymerize. Unstable dimers and trimers are initially formed. The "elongation phase" begins when there are a sufficiently large number of these short polymers. In this phase the filament forms and rapidly grows through the reversible addition of new monomers at both extremes.[57] Finally, a stationary equilibrium is achieved where the G-actin monomers are exchanged at both ends of the microfilament without any change to its total length.[24] In this last phase the "critical concentration Cc" is defined as the ratio between the assembly constant and the dissociation constant for G-actin, where the dynamic for the addition and elimination of dimers and trimers does not produce a change in the microfilament's length. Under in vitro conditions Cc is 0.1 μM,[58] which means that at higher values polymerization occurs and at lower values depolymerization occurs.[59]

Role of ATP hydrolysis

As indicated above, although actin hydrolyzes ATP, everything points to the fact that ATP is not required for actin to be assembled, given that, on one hand, the hydrolysis mainly takes place inside the filament, and on the other hand the ADP could also instigate polymerization. This poses the question of understanding which thermodynamically unfavourable process requires such a prodigious expenditure of energy. The actin cycle, which couples ATP hydrolysis to actin polymerization, consists of the preferential addition of G-actin-ATP monomers to a filament's barbed end, and the simultaneous disassembly of F-actin-ADP monomers at the pointed end where the ADP is subsequently changed into ATP, thereby closing the cycle. This aspect of actin filament formation is known as “treadmilling”.

ATP is hydrolysed relatively rapidly just after the addition of a G-actin monomer to the filament. There are two hypotheses regarding how this occurs; the stochastic, which suggests that hydrolysis randomly occurs in a manner that is in some way influenced by the neighbouring molecules; and the vectorial, which suggests that hydrolysis only occurs adjacent to other molecules whose ATP has already been hydrolysed. In either case, the resulting Pi is not released; it remains for some time noncovalently bound to actin's ADP. In this way there are three species of actin in a filament: ATP-Actin, ADP+Pi-Actin and ADP-Actin.[47][60] The amount of each one of these species present in a filament depends on its length and state: as elongation commences the filament has an approximately equal amount of actin monomers bound with ATP and ADP+Pi and a small amount of ADP-Actin at the (-) end. As the stationary state is reached the situation reverses, with ADP present along the majority of the filament and only the area nearest the (+) end containing ADP+Pi and with ATP only present at the tip.[61]

If we compare the filaments that only contain ADP-Actin with those that include ATP, in the former the critical constants are similar at both ends, while Cc for the other two nucleotides is different: At the (+) end Cc+=0.1 μM, while at the (-) end Cc=0.8 μM, which gives rise to the following situations:[26]

  • For G-actin-ATP concentrations less than Cc+ no elongation of the filament occurs.
  • For G-actin-ATP concentrations less than Cc but greater than Cc+ elongation occurs at the (+) end.
  • For G-actin-ATP concentrations greater than Cc the microfilament grows at both ends.

It is therefore possible to deduce that the energy produced by hydrolysis is used to create a true “stationary state”, that is a flux, instead of a simple equilibrium, one that is dynamic, polar, and attached to the filament. This justifies the expenditure of energy as it promotes essential biological functions.[47] In addition, the configuration of the different monomer types is detected by actin binding proteins, which also control this dynamism, as will be described in the following section.

Microfilament formation by treadmilling has been found to be atypical in stereocilia. In this case the control of the structure's size is totally apical and it is controlled in some way by gene expression, that is, by the total quantity of protein monomer synthesized in any given moment.[62]

Associated proteins

An actin (green) - profilin (blue) complex.[63] The profilin shown belongs to group II, normally present in the kidneys and the brain.

The actin cytoskeleton in vivo is not exclusively composed of actin, other proteins are required for its formation, continuance, and function. These proteins are called actin-binding proteins (ABP) and they are involved in actin's polymerization, depolymerization, stability, organisation in bundles or networks, fragmentation, and destruction.[24] The diversity of these proteins is such that actin is thought to be the protein that takes part in the greatest number of protein-protein interactions.[64] For example, G-actin sequestering elements exist that impede its incorporation into microfilaments. There are also proteins that stimulate its polymerization or that give complexity to the synthesizing networks.[26]

  • Thymosin β-4 is a 5 kDa protein that can bind with G-actin-ATP in a 1:1 stoichiometry; which means that one unit of thymosin β-4 binds to one unit of G-actin. Its role is to impede the incorporation of the monomers into the growing polymer.[65]
  • Profilin, is a cytosolic protein with a molecular weight of 15 kDa, which also binds with G-actin-ATP or -ADP with a stoichiometry of 1:1, but it has a different function as it facilitates the replacement of ADP nucleotides by ATP. It is also implicated in other cellular functions, such as the binding of proline repetitions in other proteins or of lipids that act as secondary messengers.[66][67]
The protein gelsolin, which is a key regulator in the assembly and disassembly of actin. It has six subdomains, S1-S6, each of which is composed of a five-stranded β-sheet flanked by two α-helices, one positioned perpendicular to the strands and the other in a parallel position. Both the N-terminal end, (S1-S3), and the C-terminal end, (S4-S6), form an extended β-sheet.[68][69]

Other proteins that bind to actin regulate the length of the microfilaments by cutting them, which gives rise to new active ends for polymerization. For example, if a microfilament with two ends is cut twice, there will be three new microfilaments with six ends. This new situation favors the dynamics of assembly and disassembly. The most notable of these proteins are gelsolin and cofilin. These proteins first achieve a cut by binding to an actin monomer located in the polymer they then change the actin monomer's conformation while remaining bound to the newly generated (+) end. This has the effect of impeding the addition or exchange of new G-actin subunits. Depolymerization is encouraged as the (-) ends are not linked to any other molecule.[70]

Other proteins that bind with actin cover the ends of F-actin in order to stabilize them, but they are unable to break them. Examples of this type of protein are CapZ, which binds the (+) ends depending on a cell's levels of Ca2+/calmodulin. These levels depend on the cell's internal and external signals and are involved in the regulation of its biological functions).[71] Another example is tropomodulin (that binds to the (-) end). Tropomodulin basically acts to stabilize the F-actin present in the myofibrils present in muscle sarcomeres, which are structures characterized by their great stability.[72]

Atomic structure of Arp2/3.[73] Each colour corresponds to a subunit: Arp3, orange; Arp2, sea blue (subunits 1 and 2 are not shown); p40, green; p34, light blue; p20, dark blue; p21, magenta; p16, yellow.

The Arp2/3 complex is widely found in all eukaryotic organisms.[74] It is composed of seven subunits, some of which possess a topology that is clearly related to their biological function: two of the subunits, ARP2 and ARP3, have a structure similar to that of actin monomers. This homology allows both units to act as nucleation agents in the polymerization of G-actin and F-actin. This complex is also required in more complicated processes such as in establishing dendritic structures and also in anastomosis (the reconnection of two branching structures that had previously been joined, such as in blood vessels).[75]

Chemical inhibitors

Chemical structure of phalloidin

There are a number of toxins that interfere with actin's dynamics, either by preventing it from polymerizing (latrunculin and cytochalasin D) or by stabilizing it (phalloidin):

  • Latrunculin is a toxin produced by sponges. It binds to G-actin preventing it from binding with microfilaments.[76]
  • Cytocalasin D, is an alkaloid produced by fungi, that binds to the (+) end of F-actin preventing the addition of new monomers.[77] Cytocalasin D has been found to disrupt actin's dynamics, activating protein p53 in animals.[78]
  • Phalloidin, is a toxin that has been isolated from the death cap mushroom Amanita phalloides. It binds to the interface between adjacent actin monomers in the F-actin polymer, preventing its depolymerization.[77]

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