The Actin Cytoskeleton of the Cell by Laura Feeley and Jeremy Keys

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Fluorescently Labeled Cell showing actin filaments in green, the nuclear membrane in blue, and viniculin in red [1]

Background

Representations of a. G-actin and b. F-actin. The two colors in G-actin represent the inner and outer domains of the actin monomer. These refer to the relative positions of the ends of the structure when it is integrated into the F-actin helix. Each color within the helical model represents an individual G-actin which has bonded to the filament [3]

Cytoskeleton Overview

The cytoskeleton is an array of proteins in the cytoplasm which forms the structural framework of all eukaryotic cells and most prokaryotic cells. The cytoskeleton is composed of three primary components: microtubules, actin microfilaments, and intermediate filaments. Each of these components provides a specific set of functions to regulate the overall behavior of the cell. Microtubules, composed of tubulin, are straw shaped structures which are approximately 20 nm in diameter and are the largest of the cytoskeletal components. Actin filaments are the smallest of the three components and are composed of a helical strand of actin monomers averaging around 6 nm in diameter. Intermediate filaments are twisted groups of proteins which provide tensile strength to the cell and are 10-20 nm in diameter. Collectively the cytoskeleton works to control cell shape, add structural strength, produce motility, and aid in cell division.

a) Normal actin polymerization is displayed as monomers are added to the positive end of the filament. b) ARP2/3 complex causes a branching of the filament by 70 degrees and allows for more polymerization sites. c) Cofillin splits actin chains and produces a second barbed end for more polymerization [4].

Actin

Actin is the most plentiful intercellular protein in eukarotic cells, accounting for 10% of cellular protein in muscle cells and 1-5% in most non-muscle cells. Initially recognized for its role in muscle contraction, actin is a primary component of the cellular cytoskeleton and plays a key role in cell motility, cell division, and cell-surface and cell-cell interactions.

Actin exists primarily in two forms: filamentous (F-actin) and globular actin (G-actin). Under physiological conditions, as much as 99% G-actin will naturally polymerize into F-actin [2]. Actin is often classified among three isotypes: alpha-actin, beta-actin, and gamma-actin. Alpha-actin is found primarily in muscle cells, being responsible for muscle contraction along in cooperation with myosin, while beta-actin and gamma actin are regularly associated with the cytoplasm of non-muscle cells. In addition to these three isotypes, there are numerous small variations on actin structures between organisms. While there is relatively little diversity among actin's isotypes, actin can observe a broad array of behaviors due to its interactions with actin-binding proteins (ABPs). As over 150 ABPs have been identified (which accounts for 25% of cellular proteins), actin's functions within the cytoskeleton are very diverse.

A linear actin polymer is the basis of all cytoplasmic actin. Two actin strands are non-covalently bound to each other in a helical shape. The actin polymers are made up of actin monomers. These polymers are sysnomous with F-actin whereas G-actin indicates only the monomeric actin form. A strand with greater than 6-7 monomers allows the actin polymer to bend. The helix is formed with 13 monomers, six left handed turns and an overall diameter of 5-9nm. (source) The actin protein itself has a globular shape with 6 subunits. The joining of the subunits in the middle yields an ATP or ADP binding site. Binding of different states yields changes in the structure of the protein. Focusing on the filamentous actin, it is a polar structure which has a negative (or sharp) end and a positive (or barbed) end. Three phases of actin polymerization have been identified: lag period, elongation phase and equilibrium phase. (image) Actin polymerization begins with a collection of actin monomers (G-actin) called a “nucleus.” The polymerization is not a spontaneous process because in order for it to begin stabilization of the nucleus or “nucleation” has to be conducted. In The addition of monomers yields a growth ate of about 1micrometer/second. The polymerization of actin is said to be in equilibrium where monomers are beings added to both the sharp and barbed end but addition is primarily favored on the barbed (positive) end and disassociation is primarily found at the negative (sharp) end,. The addition and disassociation of actin from subsequent ends of the polymer yields a phenomenon called treadmilling. Treadmilling can be seen when a monomer is added to the barbed (positive) end and ultimately moves down the polymer until it eventually disassociates at the pointed (negative) end. This polarity of the polymer is what drives forward motion of the cell. The role of actin binding proteins on polymerization also has a significant impact on how quickly a cell is able to move. As previously mentioned, polymerization requires nucleation which needs nucleators. A common nucleator is, actin related protein complex 2/3 (ARP2/3). This actin binding protein can also bind to existing filaments and cause branching of the actin filament at each of its binding sites [4]. Each branch consists of a Y-shape at a 70 degree angle from the original filament. By branching the actin, a second barbed end is formed and therefore the polymerization rate may be doubled provided there is enough G-actin available. Cofillin is another actin binding protein which produces a similar effect by splitting the polymer in half, thus producing a second barbed end and increasing the polymerization rate [4]

History of the Actin Cytoskeleton

While actin’s presence in muscle cells was understood as early as the late 19th century and the idea of a structural framework for cells was proposed as early as 1903, the role of the actin in the cytoskeleton of eukaryotic cells was not well understood until the advent of fluorescent labeling techniques in the early 1970s. A brief summary of key discoveries regarding the actin cytoskeleton include the following:

• 1887- Actin is first purified by W.D. Halliburton
• 1950- Role of Actin in muscle contraction first proposed (Straub)
• 1953- Earliest studies of cell motility (Abercrombie)
• 1968- Actin filaments observed in non-muscle cells (Holtzer, Ishikawa)
• 1970- Actin first implicated in cell motility (Wessels, Spooner, Yamada)
• 1974- Actin network is first visualized in non-muscle cells (Lazarides, Weber)
• 1980- Reproduction of actin cytoskeleton through “freeze-etching” (Heuser)
• 1992- Role of Rho GTPases in cytoskeletal regulation (Hall)
• 1995- First actin mutation is associated with skeletal muscle myopathy (Liang)
• 1999- Reconstruction of in vivo complete motile system (Louisel, Carlier). [5]

Cell Motility

a) The lammelipodium is formed as accelerated polymerization at the front of the cell pushes the membrane forward. b) Filopodia form focal adhesions at the substrate surface. c) The actin filaments reorganize into bundles to act as stress fibers as tension in the cell increases. d) The tension in the cell body breaks focal adhesions at the cell rear as the whole cell body is pulled forward

The most significant role of the actin cytoskeleton is its ability to control cell locomotion. Cell motility is driven by the rapid polymerization of actin filaments at the front of the cell coordinated with the gradual disassembly of actin filaments towards the cell rear. Polymerization at the front of the cell, which is driven by Rho GTPases, causes a large forward protrusion of a “lammelipodium” [2]. The lammelipodium is formed as forward extension of actin proteins stretches out the cell membrane. Smaller, finger-like protrusions called “filopodia” then form focal adhesions with the substrate beneath the lammelipodium [6]. As the extended actin begins to retract toward its rear, tension begins to form in the cell which activates LIM-proteins to aid in the “bundling” of actin filaments to form stress fibers [6]. As these stress fibers are formed, the cell begins to retract and focal adhesions at the rear of the cell begin to detach as the whole cell body is pulled forward.

Role in Cancer Metastasis

Metastasis, or the spread of cancer from one location of the body to another, is dependent on the ability of its cells to migrate throughout the body. In order to accomplish this, cancer cells are highly reliant on the cytoskeletal processes of motility in order to intravasate (or invade) other tissues. It has been demonstrated in some strains of breast cancer cells that the production of ARP2/3 and cofilin are upregulated while Eva/VASP proteins are shut down [4]. Eva/VASP proteins are associated with the formation of filopodia. Therefore, these cancer cells effectively increase the rate of polymerization to push forward lamellipodia faster, and eliminate the formation of filopodia in order to increase the surface area which the front of the cell attaches to; each of these results in a much faster moving cell. While not all cancers modify the regulation of the cell's motile mechanisms in the same way, many change these structures in some way in order to improve their chances of survival and spreading.

In addition, cancer cells hijack the ability to form actin-driven protrusions in order to form specialized protrusions called "invadopodia" [4]. Invadopodia are crucial for metastasis because they push into the surrounding extracellular matrix (ECM) to open up holes, and also work to break down the ECM [4]. For cancer cells which are contained in otherwise isolated regions of the body, the formation of invadopodia are their means of spreading into other tissues.

(Left to Right) A human breast cancer cell is shown with its stress fibers and lammelipodium labeled. Note the lack of filopodia protruding from the lammelipodium. In the center, a colon cancer cell displays a unique pseudopod structure with concentrated actin structures towards its end. An endothelial carcinoma cell shows numerous filopodia extending away from its cell membrane. Such an adaptation could be useful for latching onto other tissue cites in the body if it were to find its way into the circulatory system. [4]

Actin Mutations

Diseases associated with specific mutations to actin isoforms in humans are shown [7]

Mutated actins have been associated with muscular dystrophy and other myopathies since 1995 [5]. Alterations to actin structures can have adverse impacts on cell contractility, binding potential with myosin, cell motility, cell division, and cell strength, among other factors [7,8]. Each of these defects can manifest itself as dystrophies, atrophied muscle, developmental defects, mental disabilities, and numerous other illnesses [7]. Notably, associations between cardiomyopathies and muscular dystrophy have come under investigation for potential treatments.

Potential Actin Cytoskeleton Targeted Therapies

Cancer

In 2013, a group of researchers in New South Wales, Australia led by Dr. Peter Gunning and Dr. Justine Stehn completed several successful animal trials of a drug which targets tropomyosin, an actin binding protein associated with motility, in tumor cells [9]. By affecting the cells motility, the ability of tumor cells to seek out nutrients and spread throughout the body is significantly reduced, ideally leading to the cells starving themselves. The drug, named TR100 was slated to enter human trials in a group of 12 children suffering from advanced neuroblastoma in 2015. An advantage of the drug is that it is potentially applicable to many types of cancer because it targets structures which is necessary for all cancer cells to thrive.

Challenges

Drugs which target the cytoskeleton in cancer cells have been investigated in the past, but the difficulty with such drugs is controlling them in such a way that they exclusively impact cancer cells. Tropomyosin is critical for the function of many cells in the body, particularly in skeletal and cardiac muscle. It is for this reason that most researchers have not continued investigation of targeting cancers in this manner [9]. The researchers have stated that they will be closely monitoring the muscular and cardiac health of their patients, however it was notable that these negative side effects were not observed in their animal models. [9]

Gene Editing for Dystrophy

Genetic defects to actin which cause dystrophies have had no comprehensive treatments in the past, however recent mouse trials utilizing adeno-associated virus (AAV) delivery were successful in recovering the missing gene necessary for the formation of dystrophin (actin binding protein which is missing in patients suffering from dystrophy) [10]. If these processes can be shown to continue to be successful, gene editing could represent a major change in how dystrophies are treated.

Ethical Concerns

Gene editing has faced considerable controversy due to its potential applications outside of treating disease. Specifically, many are concerned with the ethical ramifications of a tool which might be used towards "improving" humans; treating conditions or modifying ones genes in order to reach a biological ideal. These, of course, have associations with ideas such as eugenics which has put the idea of gene editing under scrutiny.

Other factors impacting gene editing's success include a group of 3 deaths which occurred during human trials of gene editing treatments between 1999 and 2007. While there is some debate over whether or not all of these deaths were actually a result of the treatment, skepticism towards its viability as a treatment has risen.

Additionally, due to the lack of human testing of gene editing, there is still little known about the long term effects of gene editing in humans.

Drugs Targetting Actin Polymerization

Three classes of pharmalogical drugs have been used to interfere with polymerization

Class I interfere with actin polymerization. Cytochasin inhibits elongation at the barbed (positive) ends of the filament while Latrunculin (1987) inhibits monomers aggregation and therefore stops polymerization. Class II interfere with the disassembly of the polymer. Jasplakinolide minimizes the disassociation rate at J is a cyclic peptide derived from a marine sponge and aims to stabilize the actin filament, inhibit depolymerization and promote polymerization. J also minimizes the dissociateion rate constant for the barbed () end which ultimately incorporating all free actin. Side effects of this drug caused cells to develop malformed patterns, changes in cell shapes, abnormal protrusions in cells and binucleated cells when it was applied during mitosis. In vitro tests displayed J as stabilizing actin filaments but in vivo testing induced polymerization of monomeric actin into amorphous masses ().

Class III interfere with the rearrangement of the network to favor contraction rather than protrusion. Myosin II is also important in dissambly of the entire actin network. The third class of drugs includes blebbistain which inhibits Myosin II and Rho kinase (ROCK) inhibitor which inhibits an activator for Myosin II.

Triple-drug Cocktail (three classes of drugs) to inhibit actin dynamics

A triple-drug cocktail containing jasplakinolide, latrunculin B, and Y27632, (JLY) was created to inhibit actin depolymerization, actin polymerization, and myosin II-based contractility respectively, without influencing polymer assembly, disassembly, or rearrangement. HL-60 cells which were claimed to be neutrophil like were used because they exhibit rapid actin polymerization. Specific doses of the JLY cocktail was found to “freeze” cell morphology which means that no polymerization, depolymerization or rearrangement occurred. JLY was also found to keep the polymer at steady state through no additions or deletions of monomers.

Disruption of the actin filament through targeting isoforms of tropomyosin

Recently research has shifted to developing drugs that target regulatory proteins rather than targeting actin directly. These actin targeting drugs cannot specificically target actin in tumor cells and therefore are very toxis to healthy cells. In 2013, researchers from __, began targeting isoforms of tropomyosin as a method for actin filament disruption. Tropomyosin is an important coiled -coil protein within actin filaments that is located within the alpha helix “groove” and interacts along the filament. In the cytoskeleton there are two possible actin filament isoforms and over 40 tropomyosin isoforms. The researchers developed an anti-tropomyosin compounds with the most successful being TR-100. This work was unique in that it deterred tumor growth but did not effect cardiac function in vivo. They identified a particicle isoform, Tm5NM1, that was continuously found in tumor cells. Since Tm5NM1 reduced F-actin depolymerization, they studied the rate of depolymerization in F-actin and in F-actin coated with Tm5NM1. TR100 eliminated the reduction of polymerization caused by Tm5NM1 therefore displaying its efficient inhibitory abilities. Studies were conducted in both neroblastoma and melanoma cancer cells. TR100 had a significant impact on actin but did not disrupt the microtubules within the cytoskeleton. There is not clear evidence that Tr100 can specifically target low or high molecular weight tropomyosins. It was also determined that TR100 would preferentially target actin cytoskeleton filaments rather than muscle actin filaments.

Diseases caused by Actin binding protein Defects

Actin-related primary immunodeficiencies (PIDs) The actin cytoskeleton plays important roles in all aspects of a functioning immune system; hempatopoiesis, immune cell development, recruitment, migration, intercellular signaling, and activation of immune responses.

WASp Wiskott-Aldrich syndrome (WAS)

WAS is an X-linked recessive disorder first identified in 1937. Symptoms of WAS are low platlet count, eczema, bloody diarrhea, recurrent fever, and ear infections. The gene that is responsible for this disorder is on chromosome 25 which controls the WASp family that regulate Arp 2/3, a common actin nucleator. There are over 300 possible disease causing mutations in WAS. Classic WAS is caused by complete loss of WASp function. X-linked thrombocytopenia (XLT), deficiency of platlets in blood causing bleeding in tissues and slow clotting, is caused by mutations that reduce WASp expression. X linked neutropenia, or low levels of neutrophils, is caused by a mutation within the GTPase binding domain (GBD) disrupting the autoinhibited conformation of WASp and yielding an active protein.

DOCK8 deficiency

Dedicator of Cytokinesis 8 (DOCK8) are found in the edges during llamellipodia formation. DOCK 8 deficiencies are characterized by severe food and environmental allergies, otitis media, pneumonia, bronchitis, eczema, eosinophilia and severe cutaneuous viral infections sucha as human papilloma virus (HPV), herpes and varicella-zoster virus. The DOCK8 defiecncy is an autosomal recessive form of hyper IgE syndrome (AR-HIES)

Rac2

Causes epidermodysplasia verruciformis

Severe combined immunodeficiency with CORO1A mutation

Coronins which are actin regulatory proteins. Type I Coronins regulate actin branching by limiting nucleation through inhibition of Arp2/3 attachment or direct debranching. A mutation in type I corinins causes severe immunodeficiency, T-cell deficiency and susceptibility to EBV-induced lymphoproliferation. In mice the suppression of type I corinin, Coronin 1A, inhibited the immune reposonse to lupus.

Actin-accumulation myopathy

Actin found in muscle cells is also linked to specific disorders.

Actin-accumulation myopathy is found in skeletal muscles where the patient experiences severe muscle weakness and tone. This is usually first seen in infants who have trouble feeding, swallowing, controlling head movements, and breathing. Most patients with disease d not survive past infancy and if they do, delayed motor skills are usually observed.

Actin Polymerization Kinetics

The study of actin polymerization has been a popular subject for decades. Recent research has focused on developing kinetics models of actin polymerization with the application of a specific drug. Researchers at Columbia University are focusing on analyzing a single filament with non-steady state growth rates accounting for monomers added to the growing filament as ATP-actin, ATP being hydrolyzed to ADP and phosphate (Pi) and the release of Pi into the solution. They concluded through their model that growth rates and the critical concentrations on the barbed end are closely related to cap structure and dynamics. The cap structure refers to a three state region on the end of the filament. Changes in filament lengths were measured by length diffusion coefficient D, in terms of ATP-actin concentration. Length fluctuations above the critical concentration (when the mean growth rate at one end vanishes) were observed which indicates that growth is controlled by oligomer on-off events rather than monomer as previously thought.

References

[1] "Actin Staining Techniques." Cytoskeleton Inc. Cytoskeleton Inc, 2015. Web. 29 Feb. 2016.

[2] Hall A., (1998), Rho GTPases and the Actin Cytoskeleton. Science. 279: 509-514.

[3] Holmes K.C. (2009), Structural Biology: Actin in a Twist. Nature. 457: 389-390

[4] Olson MF, Sahai E (2009), The actin cytoskeleton in cancer motility, Clin Exp Metastasis, 26:273-287

[5] "Cytoskeleton Milestones Timeline." Nature Milestones. Macmillan Publishers Limited, 2016. Web. 29 Feb. 2016.

[6] Smith M.A., Hoffman L.M., and Beckerle M.C. (2014), LIM proteins in actin cytoskeleton mechanoresponse. Trends Cell Biol., 24: 575-583.

[7] Rubenstein P.A., Wen K.K., (2014), Insights Into the Effects of Disease-Causing Mutations in Human Actins. Cytoskeleton, 71:211-229. Doi: 10.1002/cm.21169

[8] Rahminov F and Kunkel L.M. (2013), Cellular and molecular mechanisms underlying muscular dystrophy. J. Cell Biol. 201: 499-510.

[9] Stehn JR, Haas NK, and Bonello T et al. (2013), A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells. Cancer Res. 73: 5169

[10] Tabebordbar M, Zhu K, and Cheng JK et al (2015), In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. doi: 10.1126/science.aad5177