Role of endocytic proteins in muscle: what we know so far

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The skeletal muscle

Function

With roughly 600 muscles in the body, muscle mass accounts for 40% of a person’s weight. Muscle tissue is classified in one of three major categories: skeletal, cardiac, and smooth muscle. Cardiac muscle is responsible for beating of the heart and pumping blood. Smooth muscles provide majorly involuntary functions such as gut and urinary bladder movements, vessel contraction and childbirth. Striated skeletal muscles are the most common type and produce purposeful movements of the skeleton. They are necessary for voluntary movements, such as walking, but also respiration and more subtle actions like eye movement and facial expressions. They are also responsible for posture and body heat (Frontera and Ochala, 2015).

Development and Myofibers

The muscle is held together by an ensemble of envelopes of connective tissue. Muscle fibers are delimited the endomysium. Several muscle fibers are wrapped in a fascicle delimited by the perimysium. Hundreds to thousands of muscle fibers are wrapped in an epimysium and connected to the bone via a tendon SEER Training Modules, Anatomy and Physiology module. U. S. National Institutes of Health, National Cancer Institute. <https://training.seer.cancer.gov/anatomy/muscular/>.
Skeletal muscle is a peculiar tissue for a number of reasons. Myogenesis, the process of muscle formation, is not, in and of itself, typical. It involves a proliferation of committed myoblasts, which “On endocytic proteins in muscle”
differentiate into post-mitotic myocytes, and those myocytes then fuse into multi-nucleated myotubes. Maturation of the myotubes will give them their main function, with the development of a contractile apparatus. Further maturation and differentiation will induce very specialized and organized, multi-nucleated fibers (nuclei migrating to the periphery of muscle cells) (Bentzinger et al., 2012; Tajbakhsh, 2009). Structurally, the skeletal muscle is resistant and very organized, held together by an ensemble of envelopes of connective tissue. Each skeletal fiber is composed of one muscle cell delimited by the muscle cell plasma membrane – or sarcolemma – and surrounded by the endomysium. Several muscle fibers are wrapped in a fascicle – or fasciculus – delimited by the perimysium. Hundreds to thousands of muscle fibers are thus finally wrapped in an epimysium and connected to the bone via a tendon (Figure I.1).

A striated pattern of contractile units

In its adult state, skeletal muscle fibers have some abilities but also constraints. A great part of the cytoplasm is occupied by the contractile apparatus, which implies a different organization inside the cell compared to any other cell (Figure I.2).
Colored scanning electron micrograph of a skeletal, or striated, muscle fiber. It consists of a bundle of smaller fibers called myofibrils, which are crossed by transverse tubules (green) that mark the division of the myofibrils in to contractile units (sarcomeres).
Specialized contractile cells exhibit a banded pattern corresponding to the organization of the contractile cytoskeleton, composed of thick filaments of myosin interacting with thin filaments of actin (Figure I.3).
Actin microfilaments are polymers of globular actin (or G-actin) assembled to form filamentous actin (or F-actin). There exists six genes coding for different actin isoforms. Two are worth mentioning in this part covering the skeletal muscle. ACTG1 codes for γ-actin, located to specific parts of the pattern. ACTA1 gene encodes α-actin, a major component of the skeletal contractile apparatus.
Sarcomeres are composed of filaments of actin and filaments of myosin that slide past each other when a muscle contracts or relaxes. Z-bands anchor actin filaments. Both the I- and H-band shorten when the sarcomeres contract.
Myosins form a family of ATP-dependent motor proteins responsible for actin-based motility. Although they are crucial for muscle contractility, they are not restricted to muscle tissue. Most myosins are composed of a head domain that binds to actin and generates force through ATP hydrolysis, a neck domain acting as a lever, and a tail domain that can bind cargo or other myosins.
Thick filaments of myosin and thin filaments of actin are arranged in repeating units called sarcomeres that form the contractile apparatus of muscle cells and appear as dark and light bands under the microscope. A single muscle cell can contain thousands of sarcomeres that are defined as the segment between two neighboring Z-lines (Z-disks, Z-discs, Z-bands). I-band (for isotropic, a clear band) contains thin filaments that are not superimposed by thick filaments. The A-band (for anisotropic, a dark band) corresponds to the entire length of a thick filaments. The H-band (for “heller” meaning “brighter” in German) is defined by thick filaments that are not superimposed by thin filaments. And finally, in the middle of the sarcomere is the M-line (Figure I.3).
Z-lines define the lateral boundaries of sarcomeres. They are electron dense bands of varying size by electron microscopy (EM). Early works attributed the presence of α-actinin to Z-lines (Masaki et al., 1967; Stromer and Goll, 1972; Yamaguchi et al., 1983). This protein belongs to the spectrin gene superfamily and is an actin crosslinking protein. The exact composition of the complex and densely packed Z-lines was later deciphered, notably with the discovery of titin (or connectin) (Maruyama et al., 1977) and nebulin (Horowits et al., 1986; Wang and Wright, 1988). Proteins like titin span a large portion of the sarcomere and many other proteins interact with Z-lines, making it one of the most complex macromolecular structures in biology (Zou et al., 2006). This explains why the integrity of Z-lines is so vital to the function of the muscle and why defects in any of these numerous proteins can induce “discopathies” (Knöll et al., 2011). Z-lines are not only essential to maintain muscle’s structure, but it appeared progressively that they could also be involved in mechanotransduction and signaling to the nucleus (Clark et al., 2002; Frank et al., 2006) (cf I.1-c Mechanotransduction).

Excitation-contraction coupling

In skeletal muscle, the excitation-contraction coupling (ECC) machinery mediates the communication between electrical events transmitted by the motor neuron and intracellular calcium release by the sarcoplasmic reticulum (SR), inducing muscle contraction. ECC requires a highly specialized membrane structure, the triad, composed of a central plasma membrane (PM) invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Propagation of the action potential in the T-tubule leads to the detection of membrane potential changes by dihydropyridine receptors, connected to ryanodine receptors on the SR membrane. The SR releases Ca2+ which initiates muscle contraction (Calderón et al., 2014). The process of muscle contraction is explained with the sliding filament theory (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954). With muscle contraction, Z-lines come closer together while the A-bands remain the same size. This is achieved by the sliding of myosin heads over actin thin filaments which are tethered to Z-lines. Myosin performs a “molecular dance” on actin filaments. ADP bound to the globular end of myosin – or S1 segment – form a cross-bridge with actin filaments (Hynes et al., 1987). They undergo a powerstroke: the myosin filament changes its angle, pulling back on the actin and releasing ADP in the process. This causes the sarcomere to shorten. Myosin detaches from actin when a new ATP molecule binds to it. ATPase catalyzes the breakdown of ATP in ADP, returning myosin heads in the resting state (Lorand, 1953).

Costameres

Muscle is a tissue which undergoes an enormous amount of deformation each times it is solicited. Therefore a strong cohesion between the different parts of this complex arrangement is needed to ensure muscular maintenance and proper function.
Scientists observed contracting cells and identified regularly spaced “wrinkles” that appeared at very specific sites. Pardo and colleagues (Pardo et al., 1983a) coined the name “costameres” from Latin costa, rib, and Greek meros, part. Costameres are specialized muscular focal adhesion-like complexes associated with Z-lines, which gives them this rib-like appearance. Focal adhesions (FAs) represent the major site of cell attachment where a structural and functional link between the extracellular matrix (ECM) and the intracellular actin cytoskeleton occurs. They are composed of macromolecular complexes formed by transmembrane receptors, structural and signaling proteins. At costameres, this complex of numerous proteins which appears dense on EM pictures, ensures structural integrity of muscle fibers and force transmission (Danowski et al., 1992; Pardo et al., 1983b, 1983a; Shear and Bloch, 1985).
In recent years, function and molecular composition of costameres have been further specified. They are composed of several large membrane protein complexes that are linked to the contractile apparatus by intermediate filaments, playing both a mechanical and signaling role during contraction (Figure I.4). Two complexes are typically described at the costamere. The dystrophin-glycoprotein complex (DGC) involves dystrophin and a series of proteins such as sarcoglycans and dystroglycans. This complex provides structural support to the sarcolemma by linking contractile elements to the ECM. The vinculin-talin-integrin complex is essential for the linkage of actin to the PM via vinculin and talin, while integrins α and β mediate signaling. γ-actin is localized to Z-discs and costameres of skeletal muscle and is responsible for force transduction and transmission in muscle cells. As such, costameres physically couple force-generating sarcomeres with the sarcolemma, and also form a two-way signaling highway between Z-lines and the extracellular matrix (ECM) (Peter et al., 2011).
Force transmission in the muscle can occur longitudinally (from one sarcomere to the other) or perpendicularly (between costameres until the ECM is reached). Perpendicular – or lateral – force transmission accounts for up to 80% of the force generated by sarcomeres and sustained by costameres (Peter et al., 2011). Early on, costameres were identified as being major sites for force transmission (Craig and Pardo, 1983; Danowski et al., 1992), and it was already thought that small changes or malfunctions in any of their components could have dramatic consequences for muscle integrity (Shear and Bloch, 1985).
Two laminin receptors, a dystrophin/glycoprotein complex and an integrin receptor complex are among the sarcolemmal structures that link the contractile apparatus of muscle fibres with the surrounding basal lamina. Components of both receptors co-localize in subsarcolemmal complexes which connect through γ-actin and intermediate filaments to the Z-disc of skeletal muscle fibers.
Since then, many mutations have been identified in key components of costameres and associated with muscle diseases, such as Duchenne muscular dystrophy. Costameres provide structural and functional support to the entire muscle fiber, thus they are of critical importance and have been considered the “Achilles’ heel” of muscles (Ervasti, 2003; Peter et al., 2011).

Intermediate filaments

Intermediate filaments (IFs) can vary in composition but are a type of cytoskeletal filaments that share common structural and sequence features. They measure about 10 nm in diameter. Their oligomerization and composition vary depending on the tissue they are situated in, thus harboring different functions depending on the tissue’s specific needs. Different types of intermediate filaments are defined by a certain similarity between one another in terms of sequences and structure. For example, keratins bind to each other to form type I and II intermediate filaments, which help epithelial cells resist mechanical stretch. Neurofilaments are type IV intermediate filaments that are incorporated in the axon of developing neurons and are thought to provide structural support for their growth. Most types of IFs are cytoplasmic but lamins are nuclear, type V IFs, providing structural and transcriptional regulation to the nucleus.
In skeletal muscle, the IF cytoskeleton is mainly composed of the type III IF protein desmin. Desmin is composed of three major domains, an alpha helix rod, a variable non alpha helix head, and a carboxy-terminal tail, and desmin proteins are able to assemble to form homopolymers (Goldfarb et al., 2008). Two parallel monomers form a dimer, which then align with another dimer to form a tetramer. Two sets of antiparallel tetramers will then form a protofilament. A protofilament bundling with seven other protofilaments give the final 10 nm wide IF (Figure I.5).
Copyright © 2010 Division of Life Sciences, Komaba Organization for Educational Excellence, College of Arts and Sciences, University of Tokyo.
Muscle specific desmin IFs span the entire width of the myofiber, from the sarcolemma to the nucleus (Figure I.6) (Capetanaki et al., 2015).
Figure I.6. Schematic representation of the desmin intermediate filament network The intermediate filament cytoskeleton of skeletal muscle is mainly composed of the type III IF protein desmin. They are connected to the plasmalemma via the costamere and interacting with the entire length of the Z-disk, and enclosing intracellular organelles like mitochondria and the nucleus.
Although they are not necessary for myogenic commitment or differentiation, IFs are essential for cell architecture, force transmission and organelle positioning (Li et al., 1997; Paulin and Li, 2004; Shah et al., 2004), and ablation of desmin by gene targeting induces major defects in the architectural and functional integrity of skeletal muscles (Li et al., 1996). Striated muscle of mice lacking muscle-specific desmin IFs exhibits numerous structural and functional abnormalities (Li et al., 1997; Milner et al., 1996). Most notably, desmin−/− muscle is weak and fatigued more easily (Sam et al., 2000) and lateral force transmission is greatly diminished (Boriek et al., 2001).
Mutations in the desmin (DES) gene (Dalakas et al., 2000), or that of its chaperone protein αB-crystallin (CRYAB) (Vicart et al., 1998), cause several myopathies belonging to the desmin-related myopathy (DRM) group of IF diseases (Omary et al., 2004). At least 53 mutations in the DES gene have been identified, the majority being inherited missense mutations, mostly located in the 2B domain that affect the correct assembly of desmin filaments (Goldfarb et al., 2008; van Spaendonck‐Zwarts et al., 2011).
Desminopathies are mostly characterized by weakness in the leg, facial, trunk, and respiratory muscles, but mostly also by cardiac phenotypes, with cardiomyopathy accompanied by cardiac conduction defect (Dalakas et al., 2000; Kostera-Pruszczyk et al., 2008). The pathophysiological implications of DES mutations are still being studied. Mostly, its inability to interact with key Z-disks partners could trigger disease development. Another hypothesis states that some mutations could directly alter the intrinsic biophysical properties of desmin filaments (Bär et al., 2010). However, the most common phenotype at the cellular level is the disruption of the desmin network and accumulation of dense inclusion bodies, granulofilamentous in nature, that stain positive for desmin (Figure I.7). Large intracellular aggregates of desmin interfere with a broader spectrum of cellular functions such as organelle positioning and signaling (Clemen et al., 2013).
Mutations in the desmin chaperone protein αB-crystallin gene (CRYAB) are also responsible for DRMs, and the first missense mutation identified in CRYAB was identified in a muscle biopsy from a DRM patient showing intracellular inclusions positive for desmin but with normal desmin expression. αB-crystallin belongs to the small heat shock protein family and is found in large quantities in the lens but also in a lesser extent in cardiac and skeletal tissue (Dubin et al., 1990). By binding to desmin, it inhibits its assembly, and is paramount for the equilibrium between soluble and filamentous desmin (Nicholl and Quinlan, 1994). Defective in its chaperone function, mutations in αB-crystallin therefore lead to abnormal desmin aggregates accumulating in muscle fibers (Bova et al., 1999).
Muscle pathology in a patient with homozygosity for the p.Arg173_Glu179del DES mutation. Prominent eosinophilic masses located under the sarcolemma and sometimes associated with basophilic granular material (A), displaying strong desmin immunoreactivity (B). By electron microscopy, the masses are composed of a matrix of dense granulofilamentous material (C). Cryostat sections, original magnification in A and B × 200; C × 5000. Figure by Dr. Ana Cabello, from (Goldfarb et al., 2008) In fact, most of the IFs present in skeletal muscle span the Z-lines. Other IF proteins identified and present in smaller quantities can form copolymers with desmin. We will cite synemin and paranemin (Granger and Lazarides, 1980; Price and Lazarides, 1983). Other IF proteins, nestin and syncoilin, are expressed and especially important at a specialized sites of neuron-muscle contact sites called the neuromuscular junction (Carlsson et al., 1999; Newey et al., 2001). Adult-muscle IFs also contain members of the cytokeratin family, namely cytokeratins 8 and 19. Present at costameres, they link the contractile apparatus to dystrophin (Ursitti et al., 2004).
Generally, IFs link the contractile apparatus via adaptors such as plectin which allow the interaction with actin, however the exact nature of these interactions remain elusive.
Recently, M. Palmisano and colleagues (San Diego, California) showed that a desmin-null model displayed a decrease in nuclear deformation following cell stretching and lower mechanical function, evidenced by a lower force drop after high-stress contractions (Palmisano et al., 2015). Thus, by linking the nucleus to the PM, desmin IFs are thought to be major players in the transmission of signals from the ECM to the nucleus. This process of transmission of mechanical signals and translation into cellular responses concerns mechanotransduction.

Mechanotransduction

Cells constantly integrate information from the physical nature of their environment via different stimuli. These mechanical stimuli need to be converted into signals that can be passed on through the cell all the way to the nucleus which will send an adaptive response. This translation of mechanical into chemical message and cytoskeletal reorganization is the process of mechanotransduction. At the cellular level, mechanical forces influence cytoskeletal organization, gene expression, proliferation and survival. It is of critical importance in skeletal muscle since converting mechanical cues into signals that the cell can interpret is necessary both for development and maintenance of the muscle, an organ that is highly subjected to mechanical stress.
Costameres constitute a major site of force transmission via dystroglycans and integrins. Following mechanical stimulation, several major cellular signaling cascades get activated, through mechanisms that are still largely unknown (Burkholder, 2007).

Integrin-mediated mechanotransduction

Integrin-mediated mechanotransduction is a wide field of research, and not confined to skeletal muscle. By linking the ECM to the intracellular cytoskeleton at adhesion sites, integrin-mediated adhesions are intrinsically mechanosensitive and present everywhere in the body.
Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit. There exists at least 18 α subtypes and 8 β subtypes that can generate 24 different binding pairs, allowing a certain adaptation and specialization of cell adhesions depending on the needs (Hynes, 2002). Mechanical-induced conformational changes produce a shift between low- to high-affinity binding for ligands. Upon ligand binding, integrins are able to recruit various proteins that differ depending on the subcellular location of the adhesion structures and the tissue. Via integrins, mechanical stretch on integrins can activate cell proliferation, migration, and direct remodeling of the cytoskeleton.
Major cellular and biophysical studies have focused on α5β1 integrins. The change in conformation subsequent to mechanical stimulation promotes talin binding, bridging the actin cytoskeleton to focal adhesion sites (Martel et al., 2001). The formation of focal adhesion is under the control of small GTPase Rho (Ridley and Hall, 1992). The stabilized actin-integrin-talin complex then allows binding of signaling proteins to integrin tails, such as kinase family members Focal Adhesion Kinase (FAK) (Schaller et al., 1992), paxillin (Mofrad et al., 2004), Src-family kinases (SFK), and zyxin (Beckerle, 1997; Yi et al., 2002) (Figure I.8).
Adhesions initially contain integrins, talin (tal), paxillin (pax) and low levels of vinculin (vinc) and focal adhesion kinase (FAK). The recruitment of vinculin along with talin promotes the clustering of activated integrins, forming a flexible bridge between the receptors and the actin network. Through a tension-dependent process, the maturation of stress fibers induces a redistribution of zyxin (zyx) to thicken them and regulates adhesion reinforcement.
FAK is necessary for inactivation of Rho, a small signaling G protein involved in cytoskeletal regulation, and promotion of focal adhesion turnover (Ren et al., 2000). FAK binding to Src and subsequent phosphorylation cascades also initiate multiple downstream signaling pathways (Zhao and Guan, 2011). SFK induced phosphorylation of p130Cas subsequently serves as a docking hub for downstream signaling molecules (Sawada et al., 2006; Tamada et al., 2004).
Importantly, activation of these mechanosensitive proteins control, activate and modulate the formation of branched actin networks, Rho-Rock-dependent contractile actomyosin bundles (Sun et al., 2016) and vinculin-based protrusion and force generation (Hirata et al., 2014). As we will see in the next parts, remodeling of the actin cytoskeleton can have dramatic effects on cell regulation even at the transcriptional level.

YAP/TAZ pathway

Two recently identified key mechanotransduction players are Yes-associated protein (YAP) (Sudol, 1994) and Transcriptional co-activator with PDZ-binding motif (TAZ) also named WW Domain-containing Transcription Regulator 1 (WWTR1) (Dupont et al., 2011; Kanai et al., 2000). Both are transcriptional cofactors that can shuttle between the cytoplasm and the nucleus in response to mechanical cues from the ECM (Aragona et al., 2013; Halder et al., 2012). They were originally described as parts of the Hippo pathway.
The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. It mainly regulates cell proliferation, differentiation and migration depending on different cues, such as cellular energy status, and mechanical or hormonal signals (Meng et al., 2016). When the Hippo pathway is inactive, YAP and TAZ are unphosphorylated and localized in the nucleus where they can bind to transcriptional enhancer factor TEF (TEAD) and activate target gene transcription (Zanconato et al., 2015). The Hippo pathway can be activated to phosphorylate MST1/2, which in turn phosphorylates LATS1/2. Phosphorylation of LATS1/2 leads to a phosphorylation of YAP and TAZ on S127 (S89 in TAZ) or S381 (S311 in TAZ), leading to 14-3-3-mediated YAP and TAZ cytoplasmic retention and/or YAP and TAZ degradation (Zhao et al., 2010). A simplified version of the pathway can be found in Figure I.9.
Although regulation of YAP/TAZ can be mediated by phosphorylation, it has come to light that inhibition of LATS1/2 only had a marginal effect on YAP/TAZ activation (Dupont et al., 2011), and that their reactivation after contact inhibition is possible by stretching (Pathak et al., 2014), suggesting that YAP/TAZ can be activated via purely mechanical means and independently of the Hippo pathway (Figure I.10).
Hippo signaling is an evolutionarily conserved pathway that controls organ size by regulating cell proliferation, apoptosis, and stem cell self-renewal. When the Hippo pathway is inactive, YAP and TAZ are unphosphorylated and localized in the nucleus to activate gene transcription. The Hippo pathway induces phosphorylation of kinase MST1/2, which in turn phosphorylates LATS1/2. Phosphorylation of LATS1/2 leads to a phosphorylation and subsequent inhibition of YAP and TAZ, by leading to 14-3-3-mediated YAP and TAZ cytoplasmic retention and/or degradation.
There is still a lot to learn about the mechanical regulation of YAP/TAZ. Although the activity of YAP/TAZ depends on F-actin structures enriched in adherent cells (Aragona et al., 2013; Das et al., 2016), the precise nature of the compartment that YAP and TAZ associate with is currently unknown. It has been described that YAP/TAZ mechanical regulation can be achieved by two means. Indeed, mechanical tension in cell layers can be split in two distinct structures: cell-cell contacts that link adjacent cells and cell-ECM adhesions. YAP and TAZ are generally nuclear in proliferating cells and excluded from the nucleus by cell-cell contact inhibition or by a change in ECM stiffness. Generally, tension through these structures exerts forces on cortical actomyosin fibers. Several teams suggested a role of actomyosin contractility on YAP activation. Using optogenetics, researchers showed that targeting of the small GTPase RhoA to the PM induced increased cellular traction and nuclear translocation of YAP. Reversely, YAP was translocated back to the cytoplasm when RhoA was inhibited (Valon et al., 2017).
Several regulatory pathways are at play. YAP/TAZ are cytoplasmic when cells are plated on a soft substrate (0.7 kPa) but inhibition is lifted when cells are able to spread on a stiffer surface (Dupont et al., 2011). On a soft substrate, YAP/TAZ allows cells to maintain their pluripotency potential by inhibition of the Notch pathway, a highly conserved signaling pathway regulating differentiation (Totaro et al., 2017), and it was shown that YAP/TAZ are inactivated to induce stem cell differentiation (Lian et al., 2010).
Concerning cell adhesion to the substrate, YAP regulation was linked to focal adhesions. Researchers identified the FAK-Src pathway as a negative regulator of YAP activation. Namely cell attachment to fibronectin induced YAP nuclear accumulation that was reduced by FAK or Src inhibition (Kim and Gumbiner, 2015). Regulation of FAK during durotaxis, a form of cell migration that depends on substrate rigidity, was linked to YAP translocation in a very recent study (Lachowski et al., 2017). Authors showed that in fact, YAP nuclear translocation in response to stiffness required FAK activity.
Figure I.10. Mechanical stimuli influencing YAP and TAZ subcellular localization and activity (A) When YAP and TAZ are mechanically activated, they translocate to the nucleus, where they interact with TEAD factors to regulate gene expression. (B) Schematics illustrating how different matrix, geometry and physical conditions influence YAP and TAZ localization and activity: the left panels show conditions in which YAP and TAZ are inhibited and localized to the cytoplasm, whereas the right panels show conditions that promote YAP and TAZ nuclear localization (indicated by bluer coloring of cell nuclei).
Regulation of YAP/TAZ through cell-cell contacts involves proteins that are part of adherens junctions. Adherens junction proteins such as cadherins and α-catenin form complexes on the PM that help sequestrate YAP/TAZ, thereby inhibiting their activity. H. Hirata and colleagues found a supplementary regulation of YAP in keratinocytes. They showed that tensile forces mediated by RhoA inhibited YAP-driven proliferation in layers of keratinocytes, and found this regulation especially important in multilayered cell systems (Hirata et al., 2017).
Another protein responsible for YAP inhibition at cell-cell contacts is Merlin, also called NF2. It is a FERM (Four point one, Ezrin, Radixin, Moesin)-domain containing protein involved in the establishment of cell polarity. Proteins with a FERM-domain are involved in the regulation of the dialogue between membrane complexes and cortical actin (Fehon et al., 2010). At adherens junctions, Merlin interacts with E-cadherin, β-catenin, α-catenin as well as cortical actin, and is thought to be a tumor-suppressor and an important regulator of YAP. Inactivation of Merlin induced abnormal cell proliferation that led to hepatocellular carcinoma and bile duct hamartoma, phenotypes that were alleviated by heterozygous deletion of Yap (Zhang et al., 2010).
Although the YAP/TAZ field is still in its infancy, there begins to be an important amount of data suggesting a mechanical regulation of YAP/TAZ independent of the Hippo pathway.
Muscle cells being highly sensitive to their environment and subjected to several stresses, one can ponder how are YAP/TAZ particularly regulated in muscle cells? It has been established that in proliferating myoblasts, YAP/TAZ are located in the nucleus where they act as coactivators for several transcription factors including TEAD factors and promote proliferation (Watt et al., 2010). However, during differentiation, both translocate from the nucleus to the cytoplasm thus inhibiting their transcriptional function (Watt et al., 2010), but mechanical cues such as cell stretching can cause YAP/TAZ translocation back into the nucleus (Fischer et al., 2016).
As such, YAP/TAZ mechanotransducers were first thought to be involved in muscle mass regulation. Skeletal muscle mass reflects a dynamic turnover between net protein synthesis and degradation. Fibers can also enlarge with an increased fusion of muscle stem cells called satellite cells. The Hippo-YAP pathway was introduced recently as a regulator of muscle mass through its links with the mTOR pathway (Csibi and Blenis, 2012). Even more recently, YAP was introduced as a direct regulator of muscle mass via its interaction with TEAD. It was shown to be required to maintain muscle size and also induce muscle repair after muscle injury (Watt et al., 2015). Interestingly, while certain studies suggested that expression of YAP could correct defects in hypotrophic muscles (Watt et al., 2015), another study suggested that constitutive expression of YAP in muscle induced hypotrophy and structural defects resembling that of centronuclear myopathies (Judson et al., 2013). Considering the growing evidence of a regulation of YAP/TAZ by mechanical factors and potential effects of their dysregulation on muscle, one can wonder the implications of this pathway regarding the development of myopathies.

MRTF-SRF

Serum response factor (SRF) is a ubiquitous transcription factor, functioning with myocardin-related transcription factors A and B (MRTFs), and is involved in the regulation of most cytoskeletal genes (Esnault et al., 2014; Olson and Nordheim, 2010; Yu-Wai-Man et al., 2017). MRTF-A and MRTF-B are bound to G-actin and thus respond to variations of G-actin concentration induced by Rho GTPase signaling (Vartiainen et al., 2007). F-actin polymerization occurring during mechanical stimulation reduces the amount of G-actin and releases MRTFs to activate SRF– mediated transcription (Miralles et al., 2003). Integrin-mediated force in fibroblasts was shown to induce RhoA activation and subsequent MRTF nuclear translocation (Zhao et al., 2007). SRF activation by MRTFs activates numerous essential gene responses including actin dynamics, lamellipodial/filopodial formation, integrin-cytoskeletal coupling, myofibrillogenesis, and muscle contraction (Esnault et al., 2014; Miano et al., 2007; Posern and Treisman, 2006). There exists a crosstalk between YAP/TAZ and MRTF/SRF pathways. Interestingly, an in-depth study of MRTF/SRF transcriptional target genes showed an overlap with YAP target genes, including CTGF
(connective tissue growth factor), CYR61 (cysteine-rich angiogenic inducer 61), and ANKRD1 (ankyrin repeat domain 1), and several actin genes that are frequently used as readouts for YAP activation (Esnault et al., 2014). One study found that MRTF/SRF and TAZ were actually downstream effectors of Heregulin β1, protein interacting with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) and promoting proliferation, angiogenesis and invasion of cancer cells (Liu et al., 2016). There was even proof of a direct interaction between MRTF family proteins and YAP (Kim et al., 2017).
While the MRTF/SRF pathway has been extensively studied in the context of cancer, it was also examined in muscle, where it was shown to activate certain regulatory – growth and differentiation – genes in skeletal, smooth, and cardiac muscle (Belaguli et al., 1997). Study of SRF in muscle cells under straining conditions showed that a moderate strain leads to rapid accumulation of MRTF in the nucleus (Montel et al., 2014). It was shown that actin dynamics are essential to activate this pathway in muscle, which in turn regulates vinculin, actin and SRF itself (Sotiropoulos et al., 1999).

Membranes and vesicular trafficking

The Cell Theory

Magnification technology allowed the crafting of the Cell Theory over centuries, starting in the 1600s. The first official appearance of the word cell occurred in Robert Hooke’s Micrographia (Hooke, 1667) (Figure I.11).
I took a good clear piece of Cork, and with a Pen-knife sharpen’d as keen as a Razor, I cut a piece of it off, and thereby left the surface of it exceeding smooth, then examining it very diligently with a Microscope, me thought I could perceive it to appear a little porous; […] Next, in that these pores, or cells, were not very deep, but consisted of a great many little Boxes, separated out of one continued long pore. […] me thinks, it seems very probable, that Nature has in these passages, as well as in those of Animal bodies, very many appropriated Instruments and contrivances, whereby to bring her designs and end to pass, which ’tis not improbable, but that some diligent Observer, if help’d with better Microscopes, may in time detect.

Table of contents :

I. Introduction
1- The skeletal muscle
a. Function
b. Development and Myofibers
i. A striated pattern of contractile units
ii. Excitation-contraction coupling
iii. Costameres
iv. Intermediate filaments
c. Mechanotransduction
i. Integrin-mediated mechanotransduction
ii. YAP/TAZ pathway
iii. MRTF-SRF
2- Membranes and vesicular trafficking
a. The Cell Theory
b. Membrane trafficking
c. Clathrin-mediated membrane traffic
i. Clathrin
ii. Adaptor proteins
iii. Dynamin
iv. Sequential recruitment of CCV formation actors
v. Role of actin filaments
d. Diversity of clathrin-coated structures
i. A very stable assembly of hexagons
ii. Clathrin plaques as… hotspots for endocytosis
iii. Clathrin plaques as… adherent structures
iv. Clathrin plaques as… signaling hubs
v. Clathrin plaques as… actin organisers
3- Role of endocytic proteins in muscle: what we know so far
a. Clathrin plaques are part of costameres
b. Clathrin, AP2 and DNM2 are required for α-actinin scaffold formation
c. Clathrin plaques are required for sarcomere maintenance in vivo
4- Centronuclear myopathies
a. X-linked myotubular myopathy (XLMTM)
b. Autosomal recessive centronuclear myopathy (AR CNM)
c. Autosomal dominant centronuclear myopathy (AD CNM)
i. Membrane trafficking hypothesis
ii. Focal adhesions hypothesis
iii. Cytoskeletal regulation hypothesis
iv. T-tubule hypothesis
v. A mouse model for AD CNM linked to DNM2
2- Unanswered questions – Aims of the study
a. Aim one: What are clathrin plaques made of and what are their dynamics?
b. Aim two: How are they involved in cytoskeletal scaffolding?
c. Aim three: What is their exact role in CNM pathophysiology?
II. Methods
1- Cell culture
a. Primary culture preparation
b. Primary cell culture subculturing
c. Cell differentiation
d. Extended differentiation protocol
e. Immortalized cell lines
f. siRNA transfection
g. Cell stretching
i. Flexcell apparatus
ii. Quantification of YAP/TAZ location
h. Transferrin uptake assay
2- Histomorphological and ultrastructural analyses
a. Fluorescence microscopy
i. Immunofluorescence
ii. AAV injection, live microscopy
b. Electron microscopy
i. Unroofing and preparation of metal replicas
ii. Making anaglyphs
iii. Thin-section EM
c. Histology
2- Biochemistry
a. Western blotting
b. Immunoprecipitation
3- RNA extraction and RT-qPCR
4- Statistical analysis
5- Study approval
III. Results
1- Ultrastructure and dynamics of clathrin plaques
a. Morphology of clathrin plaques
i. Regularly spaced structures along the PM
ii. AP2 and Dab2 are clathrin plaque adaptors
iii. Clathrin plaques act as scaffold for cytoskeletal anchoring
iv. Clathrin plaques scaffold intermediate filaments
v. Actin surrounding clathrin plaques organizes the cortical IF anchoring
b. Stable structures with a dynamic turnover
i. Clathrin plaques are stable structures in adherent myotubes
ii. In vivo plaque dynamics
2- Clathrin platforms are involved in mechanotransduction
a. Clathrin plaques respond to cyclic stretching
Agathe Franck – phD thesis 2018
“On endocytic proteins in muscle”
b. Clathrin is required for YAP/TAZ signaling
c. Clathrin platforms directly sequestrate YAP/TAZ mechanotransducers
d. DNM2 and TAZ biochemical interaction
e. DNM2 is required for YAP/TAZ cytoplasmic sequestration and translocation
f. Actin and IF remodeling is needed for YAP/TAZ activation
3- Costameric disorganization in CNM
a. DNM2-linked mutations impair plaque organization
b. DNM2 CNM mutation disorganizes the desmin network in vivo
c. CNM affects desmin distribution in patients
d. CNM mutation impairs YAP/TAZ location and expression
e. DNM2-linked CNM mutations delay plaque dynamics in vivo
IV. Discussion and future directions
1- Composition and regulation of clathrin plaques
2- Plaques, DNM2 and actin
3- Impact of clathrin plaques organization on mechanotransduction
a. YAP/TAZ
b. Intermediate filaments
4- Consequences for CNM pathophysiology
V. References
VI. Appendix
1- Movie legends and URLs
2- List of siRNA sequences
3- List of primers
4- Buffers
a. Mammalian Ringers (“extracellular” buffer)
b. Ca2+ free Ringers
c. KHMgE (“intracellular” buffer)
5- List of publications and presentations
Publications
Posters and oral presentations

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