**3. Actomyosin cytoskeleton: the contractile machinery of muscle and nonmuscle cells**

The cytoskeleton constitutes a dynamic network of filaments that exists in the inner space of a cell. This network not only provides scaffolding but is also responsible for transporting organelles, generating and transducting mechanical forces. The cytoskeleton maintains cellular organization by linking together several cellular components in such a way that it mediates communication across the entire cell and, therefore, has a tremendous impact on cellular functions [64]. Three main filaments constitute the cytoskeleton, each one with its distinct protein composition and function: the microtubules, intermediate filaments, and microfilaments.

The actomyosin contractile machinery is relatively well conserved, despite some differences in organization and dynamics among different cell types. Actin filaments are polarized as barbed ends (fast-growing end) and pointed ends (slow-growing ends) and serve as scaffold for myosin filaments, which vary in size ranging from a few heads, in nonmuscle cells, to hundreds of heads in thick filaments of striated muscle cells. The myosin filaments drive the translocation of actin filaments toward their barbed ends. This event can trigger either the

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The most well understood example of a contractile actomyosin apparatus is found in striated muscles and is called sarcomere. The sarcomeres are what give the striated muscles their appearance. It is known that a sarcomere is defined as a segment between two neighboring Z-lines. The Z-lines (from the German "Zwischenscheibe") are "dark" lines that appear in both extremities of a sarcomere (**Figure 1A**). They act as anchoring points for actin filaments. Surrounding two Z-lines, there are two regions called I-bands, regions of actin filaments that are not superimposed by myosin filaments (**Figure 1A**). Between the two I-bands is an A-band, which contains the entire length of myosin filaments and part of actin filaments that extend from I-bands (**Figure 1A**). The barbed ends of actin filaments are localized at the Z-line. The myosin filaments are segregated toward the pointed ends of actin filaments. Several other proteins are present and allow the stability of a sarcomere. The interaction between actin and myosin filaments in the A-band of a sarcomere is responsible for muscle contraction (**Figure 1A**) [64].

The contraction starts when a motor neuron releases acetylcholine, a neurotransmitter that binds to a postsynaptic nicotinic acetylcholine receptor on the muscle fiber, causing a change in receptor conformation and allowing an influx of sodium ions followed by postsynaptic action potential initiation. The action potential travels through T-tubules until it reaches the sarcoplasmic reticulum, where it activates voltage-gated L-type calcium channels. The initial inward flow of calcium from the L-type calcium channels activates ryanodine receptors, which releases a huge amount of calcium ions from the sarcoplasmic reticulum toward the cytoplasm of muscle cells. This mechanism is called calcium-induced calcium-release [64]. Inside muscle cells, the protein tropomyosin covers the myosin binding sites of actin filaments in the sarcomere. In order to allow contraction, tropomyosin must be moved from its original place. Initially, tropomyosin is attached to the actin filaments, covering myosin binding sites. When calcium ions enter in the muscle cell cytoplasm, they immediately bind to troponin-C and trigger a change in the structure of tropomyosin. This change in conformation forces tropomyosin to reveal the myosin-binding sites on actin filaments and allows myosin filaments to pull antiparallel actin filaments together. Muscle contraction ends when calcium ions are pumped back from the muscle cell cytoplasm into the sarcoplasmic reticulum, allow-

The actomyosin cytoskeleton in nonmuscle and smooth muscle cells is organized in similar ways, both different from sarcomeres of striated muscles. Nonmuscle and smooth muscle cells use myosin to generate contractility during migration, cytokinesis, as well as cell-cell and cell-matrix junctions, for example [66]. Nonaligned actomyosin networks, with actin filaments and clusters of bipolar myosin filaments interacting with each other at their ends, represent the simplest contractile machinery in nonmuscle and smooth muscle cells, especially

contraction or extension of two actin filaments [64].

ing the contractile machinery to relax [64].

Microfilaments, also known as actin filaments, are ~7 nm in width. They are primarily composed of actin, the most abundant protein in cells. Actin filaments can create a huge number of arrays, such as bundles, two-dimensional networks, and three-dimensional gels. These different structural organizations are controlled by several actin-binding proteins and are found, for example, at the leading edge of a moving cell, particularly in filopodia and lamellipodia (**Figure 1B**), which causes the actin filaments to be the primary cytoskeletal component to drive cell motility [64]. Actin filaments also allow the cell to probe or sense its microenvironment. More stable networks of actin filaments, known as stress fibers, allow cells to brace against the underlying surface [65]. Thus, microfilaments can either be alone, as simple filaments, or together with the myosin filaments, which are part of the actomyosin contractile apparatus, in muscle and nonmuscle cells. Myosin filaments, associated with actin filaments, use ATP hydrolysis to exert forces against stress fibers during cytoskeletal contractility [65].

**Figure 1.** Actomyosin cytoskeleton schematic of striated muscle cell (A) and smooth muscle/nonmuscle cells. Striated muscle cells have the sarcomeric organization, which is shown in A and consists of actin and myosin filaments overlapping. Smooth muscle and nonmuscle cells (B) have different populations of actin stress-fibers that can be found in distinct parts of the cell. There are stress fibers that form filopodia (pink), lamellipodia (green), contractile transverse arcs (red), dorsal stress fibers (light-blue) and ventral stress fibers (orange) that terminate into one or two focal adhesions (navy-blue).

The actomyosin contractile machinery is relatively well conserved, despite some differences in organization and dynamics among different cell types. Actin filaments are polarized as barbed ends (fast-growing end) and pointed ends (slow-growing ends) and serve as scaffold for myosin filaments, which vary in size ranging from a few heads, in nonmuscle cells, to hundreds of heads in thick filaments of striated muscle cells. The myosin filaments drive the translocation of actin filaments toward their barbed ends. This event can trigger either the contraction or extension of two actin filaments [64].

The most well understood example of a contractile actomyosin apparatus is found in striated muscles and is called sarcomere. The sarcomeres are what give the striated muscles their appearance. It is known that a sarcomere is defined as a segment between two neighboring Z-lines. The Z-lines (from the German "Zwischenscheibe") are "dark" lines that appear in both extremities of a sarcomere (**Figure 1A**). They act as anchoring points for actin filaments. Surrounding two Z-lines, there are two regions called I-bands, regions of actin filaments that are not superimposed by myosin filaments (**Figure 1A**). Between the two I-bands is an A-band, which contains the entire length of myosin filaments and part of actin filaments that extend from I-bands (**Figure 1A**). The barbed ends of actin filaments are localized at the Z-line. The myosin filaments are segregated toward the pointed ends of actin filaments. Several other proteins are present and allow the stability of a sarcomere. The interaction between actin and myosin filaments in the A-band of a sarcomere is responsible for muscle contraction (**Figure 1A**) [64].

The contraction starts when a motor neuron releases acetylcholine, a neurotransmitter that binds to a postsynaptic nicotinic acetylcholine receptor on the muscle fiber, causing a change in receptor conformation and allowing an influx of sodium ions followed by postsynaptic action potential initiation. The action potential travels through T-tubules until it reaches the sarcoplasmic reticulum, where it activates voltage-gated L-type calcium channels. The initial inward flow of calcium from the L-type calcium channels activates ryanodine receptors, which releases a huge amount of calcium ions from the sarcoplasmic reticulum toward the cytoplasm of muscle cells. This mechanism is called calcium-induced calcium-release [64]. Inside muscle cells, the protein tropomyosin covers the myosin binding sites of actin filaments in the sarcomere. In order to allow contraction, tropomyosin must be moved from its original place. Initially, tropomyosin is attached to the actin filaments, covering myosin binding sites. When calcium ions enter in the muscle cell cytoplasm, they immediately bind to troponin-C and trigger a change in the structure of tropomyosin. This change in conformation forces tropomyosin to reveal the myosin-binding sites on actin filaments and allows myosin filaments to pull antiparallel actin filaments together. Muscle contraction ends when calcium ions are pumped back from the muscle cell cytoplasm into the sarcoplasmic reticulum, allowing the contractile machinery to relax [64].

The actomyosin cytoskeleton in nonmuscle and smooth muscle cells is organized in similar ways, both different from sarcomeres of striated muscles. Nonmuscle and smooth muscle cells use myosin to generate contractility during migration, cytokinesis, as well as cell-cell and cell-matrix junctions, for example [66]. Nonaligned actomyosin networks, with actin filaments and clusters of bipolar myosin filaments interacting with each other at their ends, represent the simplest contractile machinery in nonmuscle and smooth muscle cells, especially

**Figure 1.** Actomyosin cytoskeleton schematic of striated muscle cell (A) and smooth muscle/nonmuscle cells. Striated muscle cells have the sarcomeric organization, which is shown in A and consists of actin and myosin filaments overlapping. Smooth muscle and nonmuscle cells (B) have different populations of actin stress-fibers that can be found in distinct parts of the cell. There are stress fibers that form filopodia (pink), lamellipodia (green), contractile transverse arcs (red), dorsal stress fibers (light-blue) and ventral stress fibers (orange) that terminate into one or two focal adhesions (navy-blue).

mediates communication across the entire cell and, therefore, has a tremendous impact on cellular functions [64]. Three main filaments constitute the cytoskeleton, each one with its distinct protein composition and function: the microtubules, intermediate filaments, and

Microfilaments, also known as actin filaments, are ~7 nm in width. They are primarily composed of actin, the most abundant protein in cells. Actin filaments can create a huge number of arrays, such as bundles, two-dimensional networks, and three-dimensional gels. These different structural organizations are controlled by several actin-binding proteins and are found, for example, at the leading edge of a moving cell, particularly in filopodia and lamellipodia (**Figure 1B**), which causes the actin filaments to be the primary cytoskeletal component to drive cell motility [64]. Actin filaments also allow the cell to probe or sense its microenvironment. More stable networks of actin filaments, known as stress fibers, allow cells to brace against the underlying surface [65]. Thus, microfilaments can either be alone, as simple filaments, or together with the myosin filaments, which are part of the actomyosin contractile apparatus, in muscle and nonmuscle cells. Myosin filaments, associated with actin filaments, use ATP hydrolysis to exert forces against stress fibers during cytoskeletal contractility [65].

microfilaments.

38 Cholesterol - Good, Bad and the Heart

in cytoskeletal regions that do not have stress fibers [67, 68]. Smooth muscle and nonmuscle cells also contain more organized actomyosin bundles, such as transverse arcs, radial stress fibers, peripheral bundles, and ventral stress fibers (**Figure 1B**) [65]. Transverse stress fibers or arcs (**Figure 1B**) are formed after reorganization of lamellipodial actin filaments [69] during lamellipodium retraction [70]. This process is driven by myosin filaments, which become co-aligned with actin filaments and form stacks separated by alfa-actinin [68]. Radial stress fibers (**Figure 1B**), on the other hand, are anchored to focal adhesions in one end. Myosin molecules are recruited to the tips of focal adhesions, where nascent radial stress fibers start to form [71]. Contractility of the radial-transverse-stress fiber network leads to the formation of ventral stress fibers (**Figure 1B**) attached to focal adhesions on both ends [72]. Also, actomyosin filaments from nonmuscle and smooth muscle cells are highly dynamic when compared to striated muscle cells. Both actin and myosin can frequently undergo turnover or cycles of assembly/disassembly [73, 74].

the overall cellular tension, which is a combination of the tension in the plasma membrane together with the tension in the underlying actin cytoskeleton [86]. Moreover, membrane tether pulling assays also bring information about the membrane itself and its attachment with the cortical cytoskeleton [86, 87]. During bleb formation, for example, a momentary

Role of Membrane Cholesterol in Modulating Actin Architecture and Cellular Contractility

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Biophysical methods show that the membrane-cortex attachment is the major determinant in cell surface tension [89, 90] and that different cells have different surface tension values, indicating that there may have different mechanisms to maintain surface tension homeostasis among cells [90]. Modifications of specific cross-linking proteins, whose function is to link the plasma membrane to the actin cortex, can induce changes in cell surface tension [91–94]. Also, actin filament disruption or myosin inhibition can reduce cell surface tension [90, 95–97]. Changes in membrane composition, particularly in cholesterol content, have also been shown to influence cell surface tension. MβCD causes an increase in tension in embryonic kidney cells [98], fibroblasts [9] and cardiomyocytes [99]. This increase is not only due to changes in membrane composition, but it also affects the actomyosin cytoskeleton. In fibroblasts, MβCD treatment shows an increase in stress fiber formation [9] whereas cardiomyocytes show sar-

separation between the plasma membrane and the actin cortex occurs [88].

comeric disorganization together with contraction abnormalities [99].

**pleiotropic effects of statins**

**4. Cellular contractility in nonmuscle cells: the role of Rho and** 

In order to divide, migrate, and undergo tissue morphogenesis, cells change shape and exert forces either on the substrate that they are attached to or on the neighboring cells. Nonmuscle cells generate contractile stresses via molecular motors, such as myosin, that are able to convert chemical energy into mechanical work [2]. Myosin activity is controlled through phosphorylation of its light chain via myosin light chain kinase (MLCK) [100, 101] which, in turn, is activated by Rho kinase ROCK and the small GTPase Rho A upstream. In 1985, the Rho gene was first isolated, from abdominal ganglia of the Aplysia, and identified as a member of the Ras family [102]. In 1990, after injecting a constitutively active form of Rho (Vall4rho), Paterson and collaborators verified that active Rho is able to cause changes in cellular morphology inducing formation of stress fibers [103]. In 1992, Ridley and Hall showed that active RhoA induces formation of stress fibers and focal adhesions upon growth factor stimulation [104]. Being a GTPase, Rho can switch back and forth between its active state, when bound to GTP, and its inactive state, when bound to GDP. The switching process is finely regulated by guanine nucleotide exchange factors (GEFs), which promote activation, and GTPase-activating proteins (GAPs), which promote inactivation. There are approximately 60 GEFs and 70 GAPs that were already identified in the human genome [105]. In order to be activated, Rho goes through some posttranslational modifications that are essential to induce Rho migration toward the plasma membrane, where it gets activated. Prenylation is a posttranslational modification that is known to be pivotal for Rho translocation to the membrane [106]. Protein prenylation is essentially an insertion of a prenyl group, which is a hydrophobic group, to the c-terminal of a protein. That way, the protein has a lipid anchor that allows

Based on all above-described features, the most striking differences between stress fibers and sarcomeres are: (1) the molecular composition is cell-specific [75], (2) stress fiber contraction is regulated by phosphorylation of myosin light chain (pMLC) (see pMLC regulation in Section 4), while sarcomere contraction is regulated by troponin switching [76], (3) stress fibers are approximately one order of magnitude thinner, less organized (with different directions and lengths) and with less coordinated contraction when compared to sarcomeres [75], (4) the magnitude of the force they produce is different, while the stress fiber contractile forces come from individual cells and are applied, through focal adhesions, to the extracellular environment in which these cells are located [77], the sarcomeres from striated muscles can transmit contractile forces over macroscopic lengths. Finally, (5) striated muscles can rapidly contract and relax based on action potentials and Ca2+ release [64, 78] while stress fibers from smooth muscle and nonmuscle cells respond much slower and do not depend solely on electrical pulses [78].

While striated muscle cells present different actomyosin organization and features when compared to nonmuscle and smooth muscle cells, these three cell types share a common actomyosin structure: the actin cortex, also known as cell cortex or actomyosin cortex. This is a thin and highly disordered contractile actomyosin network underling the plasma membrane of cells [79]. It was first discovered in large cells, like amoeba and animal eggs and subsequently, extrapolated to all animal cells [79]. Non-adherent cells [80], cells during mitosis [81], or cells performing amoeboid-like migration [82] present a well distributed and uniform actin cortex. Cells spread over flat surfaces, although more difficult to be observed, also present a cortical layer of actomyosin, as shown by electron microscopy [83]. The actomyosin appears as an isotropic network parallel (and some perpendicular) to the plasma membrane with a width of 20–250 nm [83, 84]. Numerous actin-binding proteins have already been described to be part of the actin cortex [84], most of them are classical actin-binding proteins; however, little is known about how actin cortex is assembled.

The actin cortex plays a major role in cell mechanics as the main determinant of cell surface tension [79]. Biophysical methods like micropipette aspiration and membrane tether pulling assays (using either optical tweezers or atomic force microscopy) have been used to measure cell surface mechanics [85]. Micropipette aspiration is a suitable technique to measure the overall cellular tension, which is a combination of the tension in the plasma membrane together with the tension in the underlying actin cytoskeleton [86]. Moreover, membrane tether pulling assays also bring information about the membrane itself and its attachment with the cortical cytoskeleton [86, 87]. During bleb formation, for example, a momentary separation between the plasma membrane and the actin cortex occurs [88].

in cytoskeletal regions that do not have stress fibers [67, 68]. Smooth muscle and nonmuscle cells also contain more organized actomyosin bundles, such as transverse arcs, radial stress fibers, peripheral bundles, and ventral stress fibers (**Figure 1B**) [65]. Transverse stress fibers or arcs (**Figure 1B**) are formed after reorganization of lamellipodial actin filaments [69] during lamellipodium retraction [70]. This process is driven by myosin filaments, which become co-aligned with actin filaments and form stacks separated by alfa-actinin [68]. Radial stress fibers (**Figure 1B**), on the other hand, are anchored to focal adhesions in one end. Myosin molecules are recruited to the tips of focal adhesions, where nascent radial stress fibers start to form [71]. Contractility of the radial-transverse-stress fiber network leads to the formation of ventral stress fibers (**Figure 1B**) attached to focal adhesions on both ends [72]. Also, actomyosin filaments from nonmuscle and smooth muscle cells are highly dynamic when compared to striated muscle cells. Both actin and myosin can frequently undergo turnover or cycles of

Based on all above-described features, the most striking differences between stress fibers and sarcomeres are: (1) the molecular composition is cell-specific [75], (2) stress fiber contraction is regulated by phosphorylation of myosin light chain (pMLC) (see pMLC regulation in Section 4), while sarcomere contraction is regulated by troponin switching [76], (3) stress fibers are approximately one order of magnitude thinner, less organized (with different directions and lengths) and with less coordinated contraction when compared to sarcomeres [75], (4) the magnitude of the force they produce is different, while the stress fiber contractile forces come from individual cells and are applied, through focal adhesions, to the extracellular environment in which these cells are located [77], the sarcomeres from striated muscles can transmit contractile forces over macroscopic lengths. Finally, (5) striated muscles can rapidly contract and relax based on action potentials and Ca2+ release [64, 78] while stress fibers from smooth muscle and nonmuscle cells

While striated muscle cells present different actomyosin organization and features when compared to nonmuscle and smooth muscle cells, these three cell types share a common actomyosin structure: the actin cortex, also known as cell cortex or actomyosin cortex. This is a thin and highly disordered contractile actomyosin network underling the plasma membrane of cells [79]. It was first discovered in large cells, like amoeba and animal eggs and subsequently, extrapolated to all animal cells [79]. Non-adherent cells [80], cells during mitosis [81], or cells performing amoeboid-like migration [82] present a well distributed and uniform actin cortex. Cells spread over flat surfaces, although more difficult to be observed, also present a cortical layer of actomyosin, as shown by electron microscopy [83]. The actomyosin appears as an isotropic network parallel (and some perpendicular) to the plasma membrane with a width of 20–250 nm [83, 84]. Numerous actin-binding proteins have already been described to be part of the actin cortex [84], most of them are classical actin-binding proteins; however,

The actin cortex plays a major role in cell mechanics as the main determinant of cell surface tension [79]. Biophysical methods like micropipette aspiration and membrane tether pulling assays (using either optical tweezers or atomic force microscopy) have been used to measure cell surface mechanics [85]. Micropipette aspiration is a suitable technique to measure

respond much slower and do not depend solely on electrical pulses [78].

little is known about how actin cortex is assembled.

assembly/disassembly [73, 74].

40 Cholesterol - Good, Bad and the Heart

Biophysical methods show that the membrane-cortex attachment is the major determinant in cell surface tension [89, 90] and that different cells have different surface tension values, indicating that there may have different mechanisms to maintain surface tension homeostasis among cells [90]. Modifications of specific cross-linking proteins, whose function is to link the plasma membrane to the actin cortex, can induce changes in cell surface tension [91–94]. Also, actin filament disruption or myosin inhibition can reduce cell surface tension [90, 95–97]. Changes in membrane composition, particularly in cholesterol content, have also been shown to influence cell surface tension. MβCD causes an increase in tension in embryonic kidney cells [98], fibroblasts [9] and cardiomyocytes [99]. This increase is not only due to changes in membrane composition, but it also affects the actomyosin cytoskeleton. In fibroblasts, MβCD treatment shows an increase in stress fiber formation [9] whereas cardiomyocytes show sarcomeric disorganization together with contraction abnormalities [99].
