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Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that mus...
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... show these spots to be at MT inter- sections. The mean growth rate of EB3-GFP in muscle fibers is 8.6 ± 0.1 µm/min (Table 1), similar to that in Drosophila mela- nogaster neuronal dendrites (Ori-McKenney et al., 2012). ...
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... Tables 1 and S1 for data quantitation and technical parameters. Bars: (A-E) 10 µm; (insets) 2 µm; (kymograph vertical time axes) 60 s. ...
Citations
... Unique to striated muscle and neurons, centrosomal proteins are recruited to the NE by association with AKAP6, which together with AKAP9, are anchored by nesprin-1α2 to form an NE-MTOC [52,53]. Centrosomal proteins and MT nucleation are additionally observed at the Golgi apparatus which is uniquely distributed around the nucleus of striated muscle cells, yet the integration and contribution NE-MTOC activity at the Golgi is poorly understood [54][55][56]. ...
Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Giant nesprin-1 and -2 localise to the outer nuclear membrane, interact with SUN (Sad1p/UNC-84) domain-containing proteins at the inner nuclear membrane to form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, which, in association with lamin A/C and emerin, mechanically couples the nucleus to the cytoskeleton. Despite ubiquitous expression of nesprin giant isoforms, pathogenic mutations in nesprin-1 and -2 are associated with tissue-specific disorders, particularly related to striated muscle such as dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy. Recent evidence suggests this muscle-specificity might be attributable in part, to the small muscle specific isoform, nesprin-1α2, which has a novel role in striated muscle function. Our current understanding of muscle-specific functions of nesprin-1 and its isoforms will be summarised in this review to provide insight into potential pathological mechanisms of nesprin-related muscle disease and may inform potential targets of therapeutic modulation.
... 79,80 Apart from the main Golgi apparatus surrounding the nucleus, additional small Golgi elements are scattered around the cell, which also act as MT-organizing centres. 81 Hence, the MT-organizing centres at the Golgi apparatus and Golgi elements allow distribution of proteins across the cell via MTs. The MT minus-end is relatively static, and MT dynamic behaviour is therefore largely restricted to the other end of the MT: the MT plus-end. ...
The cardiac sodium channel NaV1.5 is an essential modulator of cardiac excitability, with decreased NaV1.5 levels at the plasma membrane and consequent reduction in sodium current (INa) leading to potentially lethal cardiac arrhythmias. NaV1.5 is distributed in a specific pattern at the plasma membrane of cardiomyocytes, with localization at the crests, grooves, and T-tubules of the lateral membrane, and particularly high levels at the intercalated disc region. NaV1.5 forms a large macromolecular complex with and is regulated by interacting proteins, some of which are specifically localised at either the lateral membrane or intercalated disc. One of the NaV1.5 trafficking routes is via microtubules (MTs), which are regulated by MT plus-end tracking proteins (+TIPs). In our search for mechanisms involved in targeted delivery of NaV1.5, we here provide an overview of previously demonstrated interactions between NaV1.5 interacting proteins and +TIPs, which potentially (in)directly impact on NaV1.5 trafficking. Strikingly, +TIPs interact extensively with several intercalated disc- and lateral membrane-specific NaV1.5 interacting proteins. Recent work indicates that this interplay of +TIPs and NaV1.5 interacting proteins mediates the targeted delivery of NaV1.5 at specific cardiomyocyte subcellular domains, while also being potentially relevant for the trafficking of other ion channels. These observations are especially relevant for diseases associated with loss of NaV1.5 specifically at the lateral membrane (such as Duchenne muscular dystrophy), or at the intercalated disc (for example, arrhythmogenic cardiomyopathy), and open up potential avenues for development of new anti-arrhythmic therapies.
... In the myofiber, microtubules form a grid that contains longitudinal components that are parallel along the axis of the fiber, and perpendicular transverse components across the fiber. This organization of microtubules into lattice-like arrays is depicted in Figure 1a,b [1,2]. Microtubule assembly nucleates at microtubule-organizing centers (MTOCs) at the microtubule minus-end. ...
... The presence of transverse microtubules, intersecting with longitudinal microtubules, suggests an additional nucleation center beyond the well-defined MTOC that surrounds the nuclear envelope. Golgi bodies within myofibers are located along the intersections of transverse and longitudinal microtubules [1], as well as along stabilized microtubules [42]. Additionally, Golgi bodies wrap around the nucleus and associate with gamma-tubulin and pericentrin, both of which are components of the MTOC. ...
... Additionally, Golgi bodies wrap around the nucleus and associate with gamma-tubulin and pericentrin, both of which are components of the MTOC. Thus, it is not surprising that Golgi bodies have been shown to serve as additional microtubule nucleation sites in myofibers [1]. ...
The contractile cells of skeletal muscles, called myofibers, are elongated multinucleated syncytia formed and maintained by the fusion of proliferative myoblasts. Human myofibers can be hundreds of microns in diameter and millimeters in length. Myofibers are non-mitotic, obviating the need for microtubules in cell division. However, microtubules have been adapted to the unique needs of these cells and are critical for myofiber development and function. Microtubules in mature myofibers are highly dynamic, and studies in several experimental systems have demonstrated the requirements for microtubules in the unique features of muscle biology including myoblast fusion, peripheral localization of nuclei, assembly of the sarcomere, transport and signaling. Microtubule-binding proteins have also been adapted to the needs of the skeletal muscle including the expression of skeletal muscle-specific protein isoforms generated by alternative splicing. Here, we will outline the different roles microtubules play in skeletal muscle cells, describe how microtubule abnormalities can lead to muscle disease and discuss the broader implications for microtubule function.
... Unique to striated muscle and neurons, centrosomal proteins are recruited to the NE by association with AKAP6, which together with AKAP9, are anchored by nesprin-1α2 to form an NE-MTOC [52,53]. Centrosomal proteins and MT nucleation are additionally observed at the Golgi apparatus which is uniquely distributed around the nucleus of striated muscle cells, yet the integration and contribution NE-MTOC activity at the Golgi is poorly understood [54][55][56]. ...
... However, TubA treatment did not affect the overall α-tubulin abundance (P > 0.05 compared with mdx-veh mice; Fig. 1d). In healthy muscle, the microtubule network forms a grid lattice with longitudinal, transverse, and perinuclear microtubules [78][79][80] . Here, in WT-CTL mice, we observed that transverse and longitudinal microtubules are regularly spaced by ∼2 µm and ∼5 µm, respectively (see arrowheads Fig. 3a, and Supplementary Fig. 3c-e). ...
The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy. Here, authors show that Smad3 acetylation via HDAC6 inhibition reverses Duchenne muscular dystrophy-like symptoms in the mdx mouse model, suggesting a potential therapeutic target for the disorder.
... Desmin depletion, on the other hand, virtually eliminated the typical Z-disk bias for pauses, rescues, or fewer catastrophes. Initiations had a strong Z-disk bias regardless of intervention, which likely reflects nucleating events from microtubule organizing centers at Golgi outposts proximal to the Z-disk that are not affected by these manipulations [26]. ...
In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments, and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosination (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules. This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coincident with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment of cardiac disease.
... Golgi elementsanother name for Golgi outposts that was initially used in the 1980's to describe tubular Golgi along proximal dendrites in rodent brain immunostaining experiments [50]; also refers to Golgi outposts in muscle cells [51]. ...
Both neurons and glia in mammalian brains are highly rami-fied. Neurons form complex neural networks using axons and dendrites. Axons are long with few branches and form pre-synaptic boutons that connect to target neurons and effector tissues. Dendrites are shorter, highly branched, and form post-synaptic boutons. Astrocyte processes contact synapses and blood vessels in order to regulate neuronal activity and blood flow, respectively. Oligodendrocyte processes extend toward axons to make myelin sheaths. Microglia processes dynamically survey their environments. Here, we describe the local secretory system (ER and Golgi) in neuronal and glial processes. We focus on Golgi outpost functions in acentrosomal microtubule nucleation, cargo trafficking, and protein glyco-sylation. Thus, satellite ER and Golgi are critical for local structure and function in neurons and glia.
... Similarly, the MTOC proteins Pcnt and PCM1, which are normally localized to the cardiomyocyte NE (35), were also displaced in Syne1 Kfs/Kfs cardiomyocytes ( Fig. 2A and B). In rat neonatal cardiomyocytes, and mouse and human myotubes and muscle fibres, the Pcnt paralogue AKAP450 (38) also relocates to the NE and is essential for the recruitment of MTs to the myotube NE (31,36,39). Surprisingly, however, in both mature and neonatal wild-type and Syne1 Kfs/Kfs cardiomyocytes, Akap450 was not found at the NE. ...
Mutations in LMNA, the gene encoding A-type lamins, cause laminopathies—diseases of striated muscle and other tissues. The aetiology of laminopathies has been attributed to perturbation of chromatin organization or structural weakening of the nuclear envelope (NE) such that the nucleus becomes more prone to mechanical damage. The latter model requires a conduit for force transmission to the nucleus. NE-associated LINC complexes are one such pathway. Using CRISPR to disrupt the Nesprin-1 KASH domain, we identified this LINC complex protein as the predominant nuclear envelope anchor for microtubule cytoskeleton components, including nucleation activities and motor complexes, in mouse cardiomyocytes. Loss of Nesprin-1 LINC complexes resulted in loss of microtubule cytoskeleton proteins at the nucleus and changes in nuclear morphology and positioning in striated muscle cells, but with no overt physiological defects. Disrupting the KASH domain of Nesprin-1 suppresses Lmna-linked cardiac pathology, likely by reducing microtubule cytoskeleton activities at the nucleus. Nesprin-1 LINC complexes thus represent a potential therapeutic target for striated muscle laminopathies.
... The cytoskeleton of muscle cells is highly organized and specialized with actin, myosin, and many associated proteins forming contractile sarcomeres (Henderson et al., 2017). Much less is known about microtubules, which arrange into grid-like arrays parallel to the long axis of sarcomeres in myofibers, the giant multinucleated cells of skeletal muscle, where they are thought to provide viscoelastic resistance, maintain nuclear integrity in the presence of vigorous contractile forces, and contribute to proper spacing of nuclei at the cell periphery (Azevedo & Baylies, 2020;Becker et al., 2020;Caporizzo et al., 2018;Heffler et al., 2020;Oddoux et al., 2013;Robison et al., 2016;Warren, 1974). Defects in microtubule associated proteins are related with nuclear mis-positioning that can impede muscle function, but the molecular mechanisms leading to the organization and function of the microtubule cytoskeleton in muscle cells remain poorly understood (Azevedo & Baylies, 2020). ...
... The microtubule cytoskeleton is dramatically reorganized during muscle cell differentiation (Abmayr & Pavlath, 2012;Becker et al., 2020). Microtubule depolymerization and re-growth assays have demonstrated that microtubules in differentiated muscle cells do not nucleate from centrosomes, but polymerize from non-centrosomal microtubule organizing centers (ncMTOCs) found in the cytoplasm and on the nuclear envelope (Gimpel et al., 2017;Kronebusch & Singer, 1987;Musa et al., 2003;Oddoux et al., 2013;Tassin et al., 1985). A key step in microtubule reorganization in these cells involves the expression of specific isoforms of the nesprin family of outer nuclear-membrane spanning, nucleoskeletal proteins (Apel et al., 2000;Duong et al., 2014;Randles et al., 2010;Zhang et al., 2001). ...
... However, our assay did not detect a relationship between ncMTOC area and total microtubule intensity, perhaps because microtubule polymerization was variable and/or our assay is not sensitive enough. The transient nature of nuclear envelope-associated microtubule nucleation is interesting and consistent with the pattern of microtubule growth in differentiating muscle, as perinuclear microtubules are thought to dissociate and rearrange into bundles parallel to the long axis of the cell ( Figure 2A) (Lu et al., 2001;Oddoux et al., 2013;Tassin et al., 1985;Warren, 1974). Analogous parallel microtubule organization was not observed in egg extracts at later time points, which is not surprising considering differences in composition and organization of the cytoplasm compared to muscle cells. ...
Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.
... In this context, studies have shown that Golgi apparatus can function as an important microtubuleorganizing center (MTOC) in many cell types, and unlike centrosomal MTOC, it can give rise to polarized MTs important for a number of cellular processes, including Golgi reassembly after mitosis (Maia et al., 2013) and polarized transport of post-Golgi carriers that are important for cell migration (Vinogradova et al., 2009(Vinogradova et al., , 2012Hurtado et al., 2011;Wu et al., 2016). In neuronal cell types, Golgi-anchored MTs have also been implicated in neurite outgrowth and branching (Oddoux et al., 2013;Yalgin et al., 2015). Thus, it is tempting to speculate that impaired activity of TBCK mutant protein over other Golgi resident vesicle transport regulators impact MT nucleation and Golgi-derived MT dependent processes that are essential for normal brain development and structural organization, such as cell division, migration, and neuronal morphogenesis (Ori-McKenney et al., 2012;Etienne-Manneville, 2013;Roccio et al., 2013;Borrell and Calegari, 2014;Maizels and Gerlitz, 2015;Garcin and Straube, 2019;Shokrollahi and Mekhail, 2021). ...
Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.
