Actin filaments are long chains of G-actin formed into two parallel polymers twisted around each other into a helical orientation with a diameter between 6 and 8nm.
From: Cellular Migration and Formation of Neuronal Connections, 2013
James M. Crawford, … Prodromos Hytiroglou, in Macsween's Pathology of the Liver (Seventh Edition), 2018
Microfilaments are double-stranded molecules of polymerized fibrous (F) actin; the monomeric form of the protein is globular (G) actin; and these two forms exist in equilibrium in the cell. The microfilaments are present in bundles and form a three-dimensional (3D) intracellular meshwork. There is extensive intracellular binding and cross-linking with other intracellular proteins, such as myosin, lamin and spectrin. The filaments are mainly located at the cell periphery; they attach to the plasma membrane and extend into microvilli. They are particularly concentrated in the pericanalicular zone, forming a pericanalicular web,177 and attach to the junctional complexes which limit the canaliculus. Four main functions are postulated for the contractile microfilaments of the hepatocyte: (1) translocation of intracellular vesicles implicated in bile secretion, especially by insertion and removal of canalicular plasma membrane transport proteins; (2) coordinated contraction, producing peristaltic movement in the canaliculus;178 (3) with microtubules, transmembrane control over the topography of intrinsic proteins in the phospholipid bilayer of the cell membrane, thus influencing the protein mosaic and functional differentiation of a particular membrane domain;179 and (4) possible modulation of the structure and tightness of the ‘tight junction’, thus regulating the permeability of the paracellular pathway.180,181 The functional roles for microfilaments involve cell membrane motility, endo- and exocytosis, secretion and vesicle transfer.
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Duncan de Souza, … Victor C. Baum, in Smith's Anesthesia for Infants and Children (Eighth Edition), 2011
An intracellular construction of microtubules and microfilaments links the contractile elements, T-tubules, sarcolemma, mitochondria, and nucleus. This scaffolding organizes the subcellular components that participate in cell signaling and allows transmission of the force of contraction to be applied to the myocyte. Mutations in several of these can be responsible for several familial cardiomyopathic conditions. The cytoskeleton not only undergoes modification with myocardial development, but microfilaments also play a role in the adaptive response to mechanical loading of the heart (Small et al., 1992; van der Loop et al., 1995; Schroder et al., 2002). One of the most important roles of the cytoskeleton is to link the thick filaments. Titin, the largest protein in the human, extends from the Z-disc to the M-line of the sarcomere. It both aligns the thick filament and has a spring-like function that determines passive tension. Titin isoforms are under developmental regulation, with fetal myocardium having the more compliant N2BA isoform, which is then replaced with the stiffer isoforms. The conversion of isoforms is species dependent, but it correlates with the shift from the more compliant fetal myocardial cells (when studied removed from the surrounding matrix) to the less compliant cells of the adult.
Christophe Laumonnerie, David J. Solecki, in Cellular Migration and Formation of Axons and Dendrites (Second Edition), 2020
22.214.171.124 The role of microtubule-actin cross talk
Although the potential for microtubule–microfilament interactions in neuronal cell migration has been postulated since the earliest EM studies in the field (Rakic, 1971; Gregory et al., 1988), the assessment of the precise mode of such interactions has been slowed by the lag in applying cell biological tools to the primary nervous system models. One of the first well-characterized examples of a regulatory interaction between the microtubule and actin cytoskeletons came from a careful analysis of CGNs from animals that were haploinsufficient for the PAFAH1BI gene (e.g., lacking one PAFAH1BI allele, PAFAH1BI+/−) (Kholmanskikh et al., 2003). Unexpectedly, phalloidin staining revealed that PAFAH1BI+/− CGNs possessed shorter neurites and had fewer actin-labeled structures in their leading process than did normal CGNs, and this was associated with diminished Rac and Cdc42 activity but elevated levels of RhoA activity. Surprisingly, RhoA inhibition normalized Rac and Cdc42 activity and migration to near wild-type levels, indicating that PAFAH1BI+/− deficiency unexpectedly fed back onto the Rho GTPases that have been shown through genetic studies to be critical regulators of actin polymerization levels in migrating neurons. Further functional studies showed that Lis1 forms a complex with IQGAP1, a protein that maintains Rac and Cdc42 in the active state at the leading edges of fibroblasts and the tips of extending neurites, and Clip170, a protein that tethers microtubules to the leading edge (Kholmanskikh et al., 2006). Taken together, these results suggest that Lis1 maintains leading-process extension by boosting Rac1 and Cdc42 activity and maintains appropriate levels of actin polymerization through leading-edge IQGAP1 recruitment. At the same time, Lis1–Clip170 interactions maintain appropriate levels of microtubule recruitment at the leading edge of growing neurites.
Despite the definition of the individual contributions of the microtubule- and actin-based motor proteins to two-stroke motility, our knowledge of how the two systems function together in migrating neurons was fragmentary for an extended period. Pharmacological studies showing that the simultaneous inhibition of myosin II and cytoplasmic dynein caused a complete halt of all two-stroke motility parameters, as compared to the slowed movement with single inhibition, led to a search for a physical linkage between the microtubule and actin cytoskeletons in the proximal portion of the leading process (Trivedi et al., 2017). The drebrin protein that binds the sides of F-actin filaments and microtubule plus ends through direct interaction with the neuronal + TIP protein end-binding protein 3 (EB3) proved a useful reporter for establishing the sites of microtubule–actomyosin interaction in migrating CGNs, as it is enriched in these cells during their motile phase (Trivedi et al., 2017). Super-resolution microcopy examination of drebrin localization revealed that (1) drebrin dynamically localizes to the proximal leading process before or during cell body translocation in a myosin II–dependent manner and (2) a layer of drebrin and F-actin intervenes between the plasma membrane and elements of the microtubule cytoskeleton during CGN migration, making drebrin the most specific reporter for anterograde leading-process actomyosin flow yet found (Fig. 19.6B). Inhibiting drebrin function or blocking drebrin-microtubule end binding resulted in a lack of microtubule advance into the proximal leading process, the randomization of the direction of centrosome and cell body motility, and ultimately, the prevention of CGN migration to the IGL. Thus, the physical interaction between microtubules and microfilaments driven by the leading-process flow of drebrin coordinates the activities of these two cytoskeletal elements to produce the saltatory, polarized movement of cytoplasmic organelles and the neuronal cell body seen in two-stroke migration.
Magdalini Sachana, … Alan J. Hargreaves, in Reproductive and Developmental Toxicology (Second Edition), 2017
The cytoskeleton is a complex interconnected protein filamentous meshwork, comprising three distinct interconnected arrays of microtubules (MTs), microfilaments (MFs), and intermediate filaments (IFs). It plays a key role in a variety of developmentally important phenomena in the nervous system, including the regulation of mitosis, cell differentiation, cell migration, and neurite outgrowth (Hargreaves, 1997; Bezanilla et al., 2015). These roles, in turn, are dependent on the regulation of the integrity of the cytoskeleton. MTs and MFs are formed by the polymerization of tubulin dimers or actin monomers in a nucleotide-dependent fashion (Hargreaves, 1997; Bezanilla et al., 2015). In mammalian cells, MTs exhibit a property known as dynamic instability, whereby some MT subpopulations may rapidly shrink, whereas others undergo rapid growth, maintaining a constant polymer mass (Mitchison and Kirschner, 1988). GTP binding is required for MT assembly; its hydrolysis to GDP occurs shortly after incorporation of tubulin dimers and the growing ends of MTs are stabilized by a “cap” of tubulin subunits with nonhydrolyzed GTP (Carlier et al., 1984). MF assembly and dynamics, on the other hand, are dependent on the binding and hydrolysis of ATP, respectively (Gungabissoon and Bamburg, 2003).
In neural development, MTs, MFs, and their associated functions are modulated by interactions with a number of accessory proteins that can stabilize, destabilize, act as motor proteins, or link MTs and MFs to other cytoskeletal elements and membranes. Developmentally important MT-associated proteins (MAPs) include MAP 1b, MAP 2, tau, and stathmin, which stabilize growth cones, dendritic and axonal MTs in developing neurons or increase MT dynamics by upregulating GTP hydrolysis at the GTP cap, respectively (Kosik and Finch, 1987; Mack et al., 2000; Ohkawa et al., 2007). The binding of MAPs to MTs is regulated by various protein kinases including MT affinity regulating kinases (MARKs) and calmodulin kinase (Biernat et al., 2002; Ohkawa et al., 2007). The motor proteins kinesin and dynein also play key roles in the regulation of MT-dependent phenomena, including formation of the mitotic spindle, chromosome alignment/segregation, intracellular transport (e.g., axonal transport), and neurite outgrowth (Schliwa and Woehlke, 2003). As the roles of MTs are dependent on the correct regulation of MT dynamics and MAP interactions, neurotoxins that interfere with this process might be potential developmental toxins.
Of the actin-binding proteins, cofilin is of particular neurodevelopmental importance as it regulates actin dynamics in the growth cone of developing neurites, the binding of which is blocked when phosphorylated by the neurodevelopmentally important LIM kinase and Slingshot phosphatase (Endo et al., 2003). The dynamic properties of MFs are closely regulated throughout neural development, enabling them to perform key developmental functions such as the formation of the contractile ring at the end of mitosis, the regulation of cell migration and growth cone advance.
IFs are biochemically much more stable than MTs and MFs. Thus they play a more structural or supportive role. However, like MTs and MFs they are modulated to some degree by their phosphorylation state (Omary et al., 2006). IFs specific to the nervous system include:
Glial fibrillary acidic protein (GFAP) and peripherin, which are found mainly in astrocytes and peripheral neurons, respectively.
Neurofilaments (NFs), which comprise a triplet of polypeptides known as the neurofilament heavy (NFH; 200 kDa), medium (NFM; 120–150 kDa), and light (NFL; 70 kDa) chains, which are found in most neurons and enriched in axons.
In summary, the complexity and developmental importance of the cytoskeleton makes it a likely target for DNTs. Indeed, in vitro toxicity studies have shown that a variety of cytoskeletal proteins may be targeted by DNTs. This can occur in a number of ways, which are summarized in Table 15.2. The rest of this section will focus on cytoskeletal targets identified in cellular studies of neural cell differentiation.
Table 15.2. Ways in Which Developmental Neurotoxins Can Target the Cytoskeleton
Agents that may bind directly to the polymer-forming subunit and interfere with dynamics, integrity, or assembly of the network
Agents that affect the expression of nerve-specific cytoskeletal core proteins, such as β-type III tubulin, GFAP, and NFs
Agents that have the ability to modulate the cytoskeleton via disruption of Ca2+ homeostasis
|Cytoskeleton-associated proteins||Agents that affect the protein levels and/or gene expression of regulatory proteins such as MAPs and ABPs|
|Phosphorylation status of cytoskeletal proteins||Agents that affect the activities of kinases that modulate the binding of regulatory proteins and/or core proteins|
|Free SH groups||Agents that block
SH groups directly or induce their oxidation indirectly
ABP, actin binding protein; GFAP, glial fibrillary acidic protein; MAP, microtubule-associated protein; NF, neurofilament.
Reduced phosphorylation state of actin depolymerizing factor/cofilin, but no change in the levels of total cofilin or actin, was demonstrated in proteomic studies of differentiating primary cultures of mouse CGCs exposed to subcytotoxic levels of MeHg chloride (Vendrell et al., 2010). This would potentially result in enhanced binding of cofilin to actin and increased MF dynamics.
Various studies on mitotic tumor cell lines and purified MTs suggested that MeHg was capable of disrupting the MT network and preventing MT assembly, respectively (Vogel et al., 1985; Miura et al., 1999). Using in vitro development assays, MT disruption was found in cultured cells induced to differentiate into a neuronal phenotype (Graff et al., 1997) and the immunological detection of neuron-specific β-tubulin was applied to demonstrate the disruption of MTs and reduced numbers of neurons in MeHg-treated NSCs (Tamm et al., 2006).
Studies with organic lead compounds have also shown disruption of the MT network using polymerization assays with purified MTs and in vitro cellular models, suggesting a direct interaction that disrupts the assembly and/or distribution of MTs (Zimmermann et al., 1985a). Zimmermann et al. (1985b) also found that triethyl lead had a direct effect on purified NFs and disrupted NFs in cultured cells, although no such effect has yet been reported for MeHg. Furthermore, these authors detected no obvious effect on the MF network (Zimmermann et al., 1985a), suggesting that the two heavy metals may have some differences in their cytoskeletal toxicity.
It is known that many heavy metals are capable of disrupting the cytoskeleton in nonneural cultured cells (Chou, 1989), although not all of them have been tested in developing neural cell models. However, the demonstration that exposure to sublethal levels of arsenic inhibits the outgrowth of neurites by differentiating PC12 and N2a cells and that this involves upregulation of the mRNA levels for NFM and NFL but downregulation of those for tubulin and tau in the latter, suggests that cytoskeletal organization is a likely target for this toxin in developing neurons (Frankel et al., 2009; Aung et al., 2013).
In a further report it was also suggested that measurement of the levels of mRNA corresponding to specific cytoskeletal proteins could be a very sensitive method for detecting exposure to neurodevelopmentally toxic metals using in vitro models (Hogberg et al., 2010). However, it should be borne in mind that a detectable (or lack of) effect using this approach may not necessarily reflect the same change at the protein level and is unable to demonstrate changes due to posttranslational modifications such as proteolytic degradation and phosphorylation.
Solvents such as ethanol and toluene have also been shown to disrupt cytoskeletal proteins in cultured neural cells. For example, MF disassembly was shown to be involved in the ability of ethanol to inhibit NMDA receptor activity in primary neural cultures (Popp and Dertien, 2008). Toluene is also known to disrupt NMDA receptor activity but a similar effect on MFs has not yet been demonstrated (Bale et al., 2007). On the other hand, the enhancement of neurite outgrowth in differentiating PC12 cells by chronic ethanol exposure was associated with increased MT polymerization, although the precise mechanism remains unknown (Reiter-Funk and Dohrman, 2005). In a study with cultured mouse embryo cells, the ability of toluene to inhibit astrocyte differentiation was demonstrated by reduced GFAP expression following chronic exposure to environmentally relevant levels of solvent (Yamaguchi et al., 2002). By contrast, ethanol exposure was found to increase GFAP expression in cultured differentiating NSCs, consistent with enhanced differentiation/proliferation of astrocytes under the conditions tested (Tateno et al., 2005).
Exposure of differentiating neuronal and/or glial cells to sublethal neurite inhibitory concentrations of the organophosphorothioate pesticides DZN and CPF has been shown to affect the levels of cytoskeletal proteins. At a molecular level, exposure to DZN and CPF had no effect on MT organization or the levels of tubulin, but did cause reduced reactivity of antibodies with MAP1B and NFH, in addition to NFH aggregation in the cell body (Sachana et al., 2001, 2005; Flaskos et al., 2007). Furthermore, although DZN had no effect on the levels of actin detected on Western blots, it induced upregulation of the actin binding protein cofilin, which regulates MF dynamics in the advancing growth cone (Harris et al., 2009). The levels of phosphorylated cofilin (p-cofilin) detected immunologically were also upregulated but to a significantly lower extent than total cofilin, suggesting a reduction in the overall level of cofilin phosphorylation and increased MF dynamics under these experimental conditions. It is not yet known whether CPF induces the same effects. The neurite inhibitory effects of CPF on differentiating C6 glioma cells were associated with reduced levels of MAP1B but not MAP 2c (transiently expressed during early development) or tubulin (Sachana et al., 2008).
However, studies of the effects of the acutely toxic (in terms of acetylcholinesterase activity inhibition) oxon metabolites of DZN and CPF (DZO and CPO, respectively) on C6 cell differentiation suggest that both agents inhibit astrocyte differentiation, as determined by reduced levels of the astrocyte marker GFAP (Sidiropoulou et al., 2009b). In both cases, impaired neurite outgrowth was associated with reduced levels of antibody reactivity with α-tubulin and MAP1B, suggesting reduced synthesis and/or increased degradation of these MT proteins (Sachana et al., 2008; Sidiropoulou et al., 2009b). In contrast, the levels of MAP 2c in CPO- (Sachana et al., 2008) and DZO- (Sidiropoulou et al., 2009b) treated cells were not significantly affected. The validity of the neural cytoskeleton as a target for CPF and CPO was further strengthened by the demonstration of a direct binding interaction of both compounds with tubulin and by their ability to inhibit MT assembly and to interfere with kinesin-dependent MT motility assays in vitro (Gearhart et al., 2007; Prendergast et al., 2007). In differentiating N2a cells, neurite inhibitory concentrations of DZO had no effect on total tubulin or NFH levels but did induce a reduction in the levels of MAP1B and the neuron-specific βIII-tubulin isoform, together with increased phosphorylation of NFH (Sidiropoulou et al., 2009a; Sachana et al., 2014). On the other hand, exposure of differentiating N2a cells to CPO was associated with reduced levels of NFH but with the remaining NFH had a higher phosphorylation state than the control; GAP-43 protein levels were also reduced (Flaskos et al., 2011). These data suggest that, although cytoskeletal changes may represent good biomarkers of effect in cellular models of development, chemically related compounds may affect these proteins differently in a manner concomitant with their potency as a developmental toxin.
Few studies have been published showing direct effects of PCBs on the neural cytoskeleton using in vitro models. However, the ability of sublethal concentrations of several PCBs to (1) inhibit differentiation, induce cellular hypertrophy, and impair the formation of contractile filaments in a differentiating skeletal muscle myocyte cell line (Coletti et al., 2001), (2) perturb calcium homeostasis in cultured rat CGCs (Kodavanti et al., 1993), and (3) promote neurite outgrowth in differentiating PC12 cells (Angus and Contreras, 1994) imply underlying molecular effects on cytoskeletal targets. A study of the effects of PCBs on the proteome of primary cultures of CGCs revealed a number of congener-specific alterations in the levels of cytoskeletal proteins (Brunelli et al., 2012). For example, 13 days' exposure to 1 μM PCB180 led to approximately threefold reduction in the levels of actin-related protein 2/3 (ARP2/3) and a threefold increase in MAP2, compared with untreated control cells. The same study found an approximately 12-fold increase and a 1.5-fold decrease in the levels of α-actinin 1 and α-internexin, respectively, following exposure to 0.1 μM PCB138. These changes could reflect the disruption of different cytoskeleton-mediated events involved in the growth and development of neurites.