Muscle-specific protein 300 kDa: Biological Overview | References
Gene name - Muscle-specific protein 300 kDa
Synonyms - Nesprin
Cytological map position - 25C6-25C10
Function - multifunctional protein
Keywords - cytoskeletal protein, muscle, component of the outer nuclear membrane, transport of mRNAs from the nucleus to postsynaptic sites during synaptic maturation, controls glutamate receptor density at the neuromuscular junction - component of LINC complex that links the nuclear cytoskeleton with the actin-based cytoplasmic cytoskeleton
Symbol - Msp300
FlyBase ID: FBgn0261836
Genetic map position - chr2L:5,100,973-5,208,189
Classification - KASH: Nuclear envelope localisation domain, spectrin repeats
Cellular location - nuclear envelope and cytoskeletal associated protein
|Recent literature||Titlow, J., Robertson, F., Jarvelin, A., Ish-Horowicz, D., Smith, C., Gratton, E. and Davis, I. (2020). Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation. J Cell Biol 219(3). PubMed ID: 32040548
Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. This study shows that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.
|Chakravarti Dilley, L., Szuperak, M., Gong, N. N., Williams, C. E., Saldana, R. L., Garbe, D. S., Syed, M. H., Jain, R. and Kayser, M. S. (2020). Identification of a molecular basis for the juvenile sleep state. Elife 9. PubMed ID: 32202500
Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, this study identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.
|Zheng, Y., Buchwalter, R. A., Zheng, C., Wight, E. M., Chen, J. V. and Megraw, T. L. (2020). A perinuclear microtubule-organizing centre controls nuclear positioning and basement membrane secretion. Nat Cell Biol 22(3): 297-309. PubMed ID: 32066907
Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. This study reports a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor gamma-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.
An important mechanism underlying synapse development and plasticity is the localization of mRNAs that travel from the nucleus to synaptic sites. This study demonstrates that the giant nuclear-associated Nesprin1 (dNesp1 - FlyBase name Muscle-specific protein 300 kDa) forms striated F-actin-based filaments, which were dubbed "railroad tracks," that span from muscle nuclei to postsynaptic sites at the neuromuscular junction in Drosophila. These railroad tracks specifically wrap around immature boutons formed during development and in response to electrical activity. In the absence of dNesp1, mRNAs normally localized at postsynaptic sites are lacking and synaptic maturation is inhibited. This dNesp1 function does not depend on direct association of dNesp1 isoforms with the nuclear envelope. It was also show that dNesp1 functions with an unconventional myosin, Myo1D, and that both dNesp1 and Myo1D are mutually required for their localization to immature boutons. These studies unravel a novel pathway directing the transport of mRNAs from the nucleus to postsynaptic sites during synaptic maturation (Packard, 2015).
A crucial property of synaptic connections is their ability to change, which is thought to be at the core of adaptive processes, such as learning and memory and the refinement of connectivity. A key feature of long-term changes in synaptic structure and function is the requirement for new protein synthesis. In hippocampal neurons, ribonucleoprotein (RNP) granules are transported to the base of dendritic spines, and following plasticity-eliciting stimuli, result in RNP translocation to activated spines and induction of protein synthesis (Packard, 2015).
An important, yet poorly understood question is: How are RNPs directed to their precise destinations once they exit the nucleus? Studies in several systems provide evidence for directed trafficking of RNPs by binding to kinesin and dynein motors, thus supporting a role for microtubules in this process. However, studies also implicate actin filaments or actin-based motors, such as MyosinV/Didium, in the translocation of RNPs to dendritic spines or the posterior pole of the Drosophila oocyte. In the oocyte, the precise posterior localization of oskar mRNA, required to establish the anterior-posterior axis, requires both the activities of microtubules and actin-based motors. In this process MyosinV/Didium interacts with Kinesin heavy chain, suggesting an interplay between the actin and microtubule cytoskeleton. It is proposed that microtubules could mediate long-range movements of RNPs from the nucleus to the periphery, but that precise localization of RNPs requires short-range interactions between RNPs and the actin-based cytoskeleton. However, these long versus short-range interactions are still ill defined (Packard, 2015).
To determine a potential role of the actin cytoskeleton in the postsynaptic localization of RNPs, this study focused on the actin-binding protein MSP300/Drosophila Nesprin-1 (dNesp1; also known as Syne1), a component of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex (Kim, 2015: Volk, 1992). The LINC complex links the nuclear cytoskeleton with the actin-based cytoplasmic cytoskeleton. dNesp1 is a giant transmembrane protein of the spectrin superfamily (Rajgor, 2013), which is associated with a variety of musculoskeletal disorders, such as X-linked Emery-Dreifuss muscular dystrophy (EDMD), movement disorders such as autosomal recessive cerebellar ataxia type 1 (ARCA1), bipolar disorder, and it is a risk gene for schizophrenia and autism (Rajgor, 2013; Shinozaki, 2014). The largest isoform(s) of dNesp1 is embedded in the outer nuclear membrane (ONM) via its transmembrane domain. The C-terminal tail, containing a Klarsicht/Anc1/Syne Homology (KASH) domain, faces the nuclear intermembrane space (also referred as to the perinuclear space) between the ONM and the inner nuclear membrane (INM) and interacts with the INM Sad1/Unc84 (SUN) domain-containing proteins, thus connecting ONM and INM proteins. Its giant N-terminal domain faces the cytoplasm and contains multiple spectrin-type repeats as well as two calponin actin-binding domains. However, other dNesp1 isoforms lack the KASH domain and thus likely are not directly linked to the nuclear envelope (Packard, 2015).
At the mammalian neuromuscular junction (NMJ) Nesp1, is involved in interactions with the acetylcholine receptor (AChR) clustering molecule muscle-specific kinase (MuSK). In the central nervous system CPG2, an isoform of Syne1, participates in the trafficking of glutamate receptors (GluRs). Studies in Drosophila and mice show that Nesp1 is required for normal nuclear localization in muscle cells (Volk, 2013 and Zhang, 2010) and the integrity of muscle cell insertion sites into the cuticle (Volk, 1992). Recently, reports suggest that dNesp1 isoforms lacking the KASH domain are also required for normal Drosophila larval locomotion, selective localization of GluRIIA and synaptic function at the NMJ, independent of its nuclear localization role (Morel, 2014). However, its potential involvement in the localization of synaptic mRNAs has not been investigated (Packard, 2015).
This study reports that interfering with dNesp1 isoforms at the Drosophila NMJ disrupts the postsynaptic localization of mRNAs in muscle, and thus the localization of the proteins encoded by these mRNAs at the postsynaptic region. In addition, mutations in dnesp1 alter synapse development and activity-dependent plasticity. In these mutants, mRNAs accumulate in the cytoplasm at the nuclear periphery, suggesting that the defect likely originates from abnormal transport of these mRNAs to synaptic sites and not from the nuclear export of these mRNAs. Strikingly in wild-type muscles, dNesp1 protein is organized into long striated filaments, dubbed 'railroad tracks,' which extend all the way from the nucleus to the periphery of the NMJ. dNesp1 railroad tracks are the first postsynaptic elements found to associate specifically with immature synaptic boutons formed during NMJ expansion or upon spaced stimulation. This study showed that dNesp1 binds to a synaptically localized RNA. In addition, dNesp1 colocalizes and cosediments with F-actin, confirming its relationship with the actin cytoskeleton. Furthermore, its exclusive localization around nascent synaptic boutons is similar to the distribution of the unconventional actin motor, Myo31DF, the Drosophila ortholog of human Myo1D. Null mutations in myo31DF mimic the phenotypes of the severe hypomorphic dnesp1sZ75 mutant, and both dNesp1 and Myo31DF are required for each other's localization. These studies unravel a novel filamentous network connecting the nucleus to nascent synaptic boutons, and this network functions with actin motors for proper localization of postsynaptic RNPs (Packard, 2015).
mRNA localization and local translation are critical for the formation and plasticity of synaptic connections. However, the exact mechanisms involved in precisely localizing mRNAs are still unclear. This study provides evidence for a novel mechanism of mRNA delivery at the Drosophila larval NMJ, from the muscle nucleus to developing postsynaptic sites. F-actin-associated dNesp1 railroad tracks, which run through the muscle cell cortex, bridge the distance from the nuclear envelope to the NMJ. At the NMJ, these railroad tracks enwrap immature synaptic boutons becoming the first identified proteins localized to boutons, which until this point lack postsynaptic proteins. Thus, dNesp1 railroad tracks provide a pathway of communication between the nucleus and sites of synapse formation. The results suggest that dNesp1 railroad tracks serve to transport mRNAs required to build the postsynaptic machinery because severe reduction in dNesp1 results in accumulation of postsynaptically enriched transcripts at the nuclear periphery and their depletion from the NMJ. Consistent with the association of dNesp1 railroad tracks with F-actin suggested by labeling body wall muscles and by the finding that dNesp1 cosediments with F-actin, it was found that a myosin1 motor, Myo31DF, colocalizes with dNesp1. Absence of Myo31DF mimicked the synaptic phenotypes of dnesp1sZ75 mutants. In addition, Myo31DF is required for normal association of dNesp1 with immature boutons and with transport of postsynaptic transcripts. Taken together, it is proposed that dNesp1 railroad tracks form a pathway for the polarized transport of mRNAs to immature synapses during development of postsynaptic structures. Furthermore, based on the known properties of the Myosin1 family, it is proposed that this motor is required to either anchor dNesp1 railroad tracks to the membrane in their pathway to ghost boutons, to locally polymerize actin, or serve as a motor to specifically transport RNPs to maturing postsynaptic sites (Packard, 2015).
dNesp1 filaments can go all the way from the nuclear envelope to sites of postsynaptic maturation. dNesp1 is part of the LINC complex linking the nucleoskeleton to the cytoskeleton (Kim, 2015). However, many dNesp1 isoforms lack the transmembrane and KASH domain. Whether these isoforms are still linked to the nuclear envelope through dimerization with transmembrane and KASH domain-containing isoforms is not known, but there is evidence that Nesprins can associate with each other and form filaments as observed in other proteins of the spectrin family. Particularly prominent is the giant cytoplasmically localized N-terminal rod domain of about 300-500 nm, which projects into the cytoplasm. The long rod domain contains multiple spectrin repeats similar to other proteins of this family, such as spectrin, α-actinin, dystrophin, and utrophin. Of these, α-actinin has been shown to form F-actin-based striated filaments with staggered F-actin and α-actinin striations at a similar periodicity (0.5 μm) to those described in this study for F-actin and dNesp1. If dNesp1 does behave as an antiparallel dimer, as observed with %alpha-actinin and suggested in vitro for dNesp1, the actin-binding CH domains located at each end of the dimer could bind to F-actin, in a repeated manner, forming striations. Similar striated filaments have been observed in the case of actomyosin filaments (containing MyosinII) in several cell types and believed to convey elastic properties to the cells. The current studies were unable to determine if F-actin also formed these arrangements with Myo1 because the fixation conditions to examine both proteins with antibodies and fungal toxins were incompatible. This study demonstrates that these dNesp1 striated filaments can extend all the way from the nuclear envelope to sites of postsynaptic maturation, and enwrap these sites (Packard, 2015).
These studies demonstrate a specific association between dNesp1 railroad tracks and ghost boutons that are naturally occurring in wild-type NMJs, as well as those induced by patterned electrical stimulation. Ghost boutons are thought to represent a transient state of synaptic bouton maturation in which postsynaptic proteins have not yet been recruited. So far, dNesp1 and Myo31DF are the first proteins found to be localized at the postsynaptic region of ghost boutons. This is consistent with the model that these proteins participate in the earliest events during postsynaptic maturation, particularly the localization of specific postsynaptic mRNAs. Mutations that disrupt the maturation of ghost boutons result in NMJ arbors with fewer synaptic boutons and an overall accumulation of ghost boutons. Most of these mutations are associated with alterations in Wnt signaling, which is essential for postsynaptic maturation. Interestingly, mutations in the Caenorhabditis elegans Nesprin 1, ANC-1, also led to defects in synapse formation through interaction with Wnt signaling molecules (Tulgren, 2014; Packard, 2015 and references therein).
In mammals, the first Nesprin 1 isoform (Syne1) was isolated in a yeast two-hybrid screen using the MuSK as bait (Apel, 2000). MuSK is a protein required for postsynaptic differentiation. Interestingly, Syne1 was found to be exclusively associated with synaptic muscle nuclei, the subset of nuclei that transcribe synaptic genes needed for postsynaptic assembly (Apel, 2000). Subsequent studies at mammalian central glutamatergic synapses revealed that CPG2, an activity-dependent brain-specific isoform of Syne-1 was present at the postsynaptic region of excitatory synapses (Cottrell, 2004). Altering CPG2 levels resulted in abnormal dendritic spine size and disrupted constitutive endocytosis of AMPA receptors (Cottrell, 2004), which is linked to synaptic plasticity. Notably, mutations in syne-1 have been linked to autosomal recessive cerebellar ataxia, Emery Dreifuss muscular dystrophy, autism, and bipolar disorder (Rajgor, 2013; Shinozaki, 2014), suggesting its importance in nervous system function (Packard, 2015).
At the Drosophila NMJ, dNesp1 is also involved in regulating the subunit composition of glutamate receptors (GluRs), synaptic transmission, and larval locomotion (Morel, 2014). However, in these studies, the authors used a single dNesp1 mutation lacking the KASH domain. The current studies revealed that the KASH domain is not required for the regulation of bouton number or the localization of Par6 protein. Thus, the GluR phenotypes are most likely to represent a different function of dNesp1 in later stages of synaptic bouton maturation (Packard, 2015).
Myo31DF is a conserved protein belonging to the Myosin ID family of unconventional myosins. Class 1 myosins are monomeric and can interact with membranes through their C-terminal Tail Homology 1 (TH1) domain containing a Pleckstrin Homology (PH) lipid-binding domain. In addition, they bind to actin through their N-terminal ATPase motor head. Connecting the C- and N-terminal domains is the neck region, which binds to Calmodulin and behaves as a lever arm for force generation and membrane deformation. The monomeric nature of Myo1D makes it unlikely to function as a processive motor for cargo transport. However, MyoI ensembles have been shown to generate directed membrane movements when anchored to actin filaments. In rats, Myo1D is believed to mediate vesicular transport and fly Myo31DF interacts with dynamin. Studies in the fly have also suggested that Myo1D regulates contacts between cells because mutations in myo31DF lead to defective left-right asymmetry, a process highly dependent on adherens junctions. In the mammalian nervous system, Myo1D is found in dendrites and axons during development. As in the case of Nesprin 1, human Myo1D has also been linked to autism (Packard, 2015).
Similar to dNesp1, this study found that Myo31DF was enriched at ghost boutons, was required for activity-dependent ghost bouton formation and maturation, and was needed for proper localization of par6 and magi mRNA at the postsynaptic region of the NMJ. The remarkable similarity between the phenotypes, as well as the colocalization of the proteins at ghost boutons suggest that Nesp1 and Myo31DF function in the same early process of bouton maturation. Supporting this conclusion is the observation that dNesp1 and Myo31DF were required for each other's localization at ghost boutons and that both genes genetically interact. In myo31df mutants, cytoplasmic dNesp1 filaments were still observed, but they no longer associated with ghost boutons. Considering the properties of members of the myosin I family, it is possible that Myo31DF serves to direct and anchor dNesp1 railroad tracks to the postsynaptic membrane apposed to newly formed ghost boutons. Alternatively, or in addition, Myo31DF might be required for F-actin polymerization and thus the formation of dNesp1 railroad tracks around newly formed ghost boutons. Interestingly, Myo31DF binds to Calmodulin light chains and dNesp1 contains Calmodulin-binding sites, which might serve as a site for direct interaction (Packard, 2015).
Recently studies have determined that par6 and magi mRNAs exit the nucleus as part of large RNPs that exit the nucleus through a mechanism of budding at the nuclear envelope (Speese, 2012). Two lines of evidence suggest that the phenotypes observed in this study are unlikely to result from blocking nuclear envelope budding. First, the dnesp1Δ KASH mutation, lacking the C-terminal region required to associate dNesp1 with the nuclear envelope, had normal Par6 protein levels at the NMJ and did not display the morphological NMJ defects associated with the severe hypomorphic dnesp1sZ75 mutant. Second, in dnesp1sZ75 mutants par6 and magi RNAs were observed in the cytoplasm, suggesting that they are exported from the nucleus. However, they accumulated around the nucleus and were not transported to postsynaptic sites. It is proposed that in the absence of dNesp1 railroad tracks in the severe hypomorphic dnesp1sZ75 mutant, megaRNPs fail to be transported in a polarized manner to the postsynaptic region of the NMJ (Packard, 2015).
In some systems, such as the Drosophila embryo, RNA localization appears to be a major mechanism for the regulation of translation. The localization of mRNAs at postsynaptic sites allows a rapid and synapse-specific translation of plasticity related transcripts in response to appropriate patterns of electrical activity, which appear essential for long-term synaptic plasticity. Studies of RNA localization to synapses and other cellular regions have implicated both microtubules and kinesin motors, as well as F-actin and myosin motors, in transporting RNPs to their site of translation. It has been suggested that microtubules constitute a long-range transport mechanism for RNP transport to sites close to the membrane whereas microfilaments may serve as a short-range transporters at the cellular cortex, with the unconventional myosins V and VI and the conventional myosinII serving as motors. However, recent studies have demonstrated that actin can serve as tracts for long-range transport of vesicles (Schuh, 2011). The current studies uncover a novel acto-Nesprin filamentous pathway, dNesp1 railroad tracks, which serve as a long-range pathway for mRNA localization and synapse maturation during development and plasticity (Packard, 2015).
Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery-Dreifuss muscular dystrophy. This study describes a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. Larvae carrying a deletion of Msp-300 KASH domain (Msp-300ΔKASH) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. Msp-300ΔKASH mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. Msp-300ΔKASH locomotion phenotypes could be rescued by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, this study found that Msp-300 controls GluRIIA density at the neuromuscular junction. Theses results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies (Morel, 2014).
This work describes a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction function. It was first shown that larvae carrying a deletion of Msp-300 KASH domain present a locomotion defect suggestive of a myasthenia, and the importance of muscle Msp-300 for this phenotype was demonstrated using tissue-specific RNAi knock-down. It was then shown that Msp-300 ΔKASH mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. Finally, Msp-300 ΔKASH locomotion phenotypes could be rescued by GluRIIA overexpression, suggesting that the locomotion defects associated with the KASH domain deletion are partly due to a reduction in junctional GluRIIA (Morel, 2014).
Biological evidence is presented supporting previous bioinformatics prediction that Msp-300 is a Nesprin-1, thus validating the use of Drosophila to study LINC complex and Nesprin-1-related diseases. Msp-300 forms filaments with a 'beads on a string' pattern, which seems to assemble as sheets at the level of Z-discs and form a web closely apposed to nuclei. This perinuclear localization requires the presence of the KASH domain, showing that the predicted KASH is functional. A strong nuclear clustering associated with the KASH domain deletion was documented, in agreement with nuclei anchoring defects recently reported by Elhanany-Tamir (2012) in larvae carrying a genomic deletion removing the 3' half of Msp-300 gene. Nesprin-1 was shown to be an important player of nuclear positioning in mouse muscles, where the KASH domain deletion causes nuclei mislocalization with the occurrence of extrasynaptic nuclei clustering and in C. elegans hypoderm syncitia, where loss of the Nesprin-1 homolog ANC-1 causes nuclei clustering. These observations therefore constitute biological evidence that Msp-300 is a bona fide Nesprin-1 (Morel, 2014).
Nuclei clustering is a striking feature of KASH domain deletion in proteins such as Msp-300/Nesprin1, ANC-1 and Klarsicht. Correlation between nuclei clustering and locomotion impairment in Syne-1 KO mice, klarsicht, and Msp-300 mutants together with the occurrence of nuclei abnormal localization in pathologies such as centronuclear myopathies raise the question of the possible contribution of nuclei clustering to the locomotion phenotype. Since there were no Msp-300 mutants presenting a locomotion/junctional phenotype without nuclei clustering, therefore establishing the contribution of Msp-300 alone, mutant conditions perturbing nuclear localization and presenting locomotion impairment were examined, and it was asked if these phenotypes occur independently of Msp-300. klarsicht mutations result in nuclei clustering and locomotion impairment but also Msp-300 mislocalization. They thus could not be used to discriminate between a role of nuclei clustering or an independent contribution of Msp-300 to the locomotion phenotype. On the other hand, ens swo mutants present altered locomotion, Msp-300 mislocalization, together with irregularly spaced nuclei, but no nuclei clusters. Based on these results, a contribution of fine nuclei position to the locomotion phenotype cannot be excluded. However, the locomotion defects together with the Msp-300 subcellular mislocalization observed in both ens swo and Msp-300 ΔKASH larvae independently of the presence or absence of nuclei clusters suggest that nuclei clustering itself is not responsible for the locomotion impairment and rather point toward a direct contribution of Msp-300 localization (Morel, 2014).
Interestingly, Z-disc localization is not affected by the KASH domain deletion. The antibody used in this work was generated using a partial cDNA of Msp-300. Blast analysis reveals that this cDNA 3' end aligns with all Msp-300 isoforms, the 5' end being shared by fewer isoforms. Msp-300 localizations observed in this study are thus likely to result from the superposition of several discrete localization patterns corresponding to different isoforms. It is proposed that KASH-containing isoforms are responsible for the perinuclear Msp-300 staining while the Z-disc staining corresponds to a different subset of Msp-300 isoforms, which could perform different tasks in the cell (Morel, 2014).
Msp-300 ΔKASH larvae present a clear locomotion defect, which is fully recapitulated in larvae with muscle-specific knock-down of Msp-300 KASH-containing isoforms. This locomotion defect can be explained by a decreased percentage of GluRIIA-containing receptors at the NMJ. Indeed, deletion of one copy of the gluRIIA gene results in identical locomotion defects while overexpression of GluRIIA in Msp-300 ΔKASH rescues the locomotion phenotype. Interestingly, Z-disc localization is not affected by the KASH domain deletion. The antibody used in this work was generated using a partial cDNA of Msp-300. Blast analysis reveals that this cDNA 3' end aligns with all Msp-300 isoforms, the 5' end being shared by fewer isoforms. Msp-300 localizations observed in this study are thus likely to result from the superposition of several discrete localization patterns corresponding to different isoforms. It is proposed that KASH-containing isoforms are responsible for the perinuclear Msp-300 staining while the Z-disc staining corresponds to a different subset of Msp-300 isoforms, which could perform different tasks in the cell (Morel, 2014).
When performing electrophysiological analysis, a decrease was detected in eEJC's amplitude and quantal content in Msp-300 ΔKASH larvae but no alteration of the mEJCs. This result was at first surprising knowing that these mutant larvae have a decreased GluRIIA density and that GluRIIA density somehow controls mEJPs amplitude. Indeed, increasing GluRIIA density by twofold in GluRIIB null background leads to an increase in mEJP amplitude. According to immunostainings, GluRIIA density was only decreased by 35% in Msp-300 ΔKASH larvae when compared to WT conditions. Thus, it is proposed that in Msp-300 ΔKASH larvae, the density of postsynaptic GluRIIA-containing receptors is sufficient to give a normal response to the spontaneous exocytosis of one neurotransmitter containing vesicle, hence the lack of modifications in the mEJCs. It is also proposed that the amount of GluRIIA-containing receptors would be limiting upon stimulation and release of several quanta of neurotransmitter, resulting in decreased eEJCs. This hypothesis is further supported by the observation that although larvae carrying a single copy of the gluRIIA gene present a clear locomotion defect (similar to Msp-300 ΔKASH larvae), larvae with a single copy of both gluRIIA and gluRIIB genes only present a small decrease in mEJPs. Both the GluR density measures and the electrophysiological analysis are thus in agreement with Msp-300 KASH domain deletion resulting in a decreased postsynaptic sensitivity to neurotransmitter release (Morel, 2014).
Investigations on the mechanisms underlying GluR synaptic localization have revealed that A- and B-type receptor localization are governed by different processes. Indeed, Discs-Large, a prototypical MAGUK protein localized in the subsynaptic reticulum (SSR), positively regulates B-type receptor NMJ targeting, without affecting A-type receptors while A-type, but not B-type, receptor targeting is under the control of Dorsal (NF-κB) and Cactus (IκB). This study has shown that neither Dorsal nor Cactus SSR localization are affected by Msp-300 KASH domain deletion. It can therefore be concluded that Msp-300 contributes to A-type GluR NMJ localization independently of either Dorsal or Cactus (Morel, 2014).
Several non-mutually exclusive hypotheses can be proposed to explain how Msp-300 KASH domain deletion alters GluR composition at the NMJ. Disconnecting Msp-300 from the nuclei could directly impact transcription of GluR subunits. Indeed, an increasing number of results suggest that the LINC complex controls gene expression: the LINC complex has been involved in chromatin organization in mammalian cells and S. cerevisiae and disconnecting the LINC complex from the actin cytoskeleton leads to altered cellular response to mechanical stress and abnormal gene expression. Msp-300 could also control GluR subunit proteins levels by controlling mRNAs access to the translation machinery or post-translational modification of GluRIIA. In agreement with this, human glutamate receptors undergo important post-translational modifications impacting their activity, trafficking, or localization. Similar modifications could occur on GluRIIA and control the assembly of functional postsynaptic glutamate receptors, their activity or localization at the synapse (Morel, 2014).
Is Msp-300 an organizer of the perinuclear region? Msp-300 size (13,000 aa for the CH and KASH domain-containing isoforms), the presence of numerous spectrin repeats (up to 52), the localization pattern observed at Z-band and in the cytoplasm surrounding nuclei, suggest that Msp-300 could be a scaffold organizing the perinuclear region. Several lines of evidence support that hypothesis, which could explain the phenotypes described in this work for Msp-300 ΔKASH mutants (Morel, 2014).
In higher eukaryotes, ER is seen as a highly dynamic continuum consisting of three different subcompartments, the rough ER, the smooth ER, and the nuclear envelope. ER dynamics is thought to play an important role in both the morphology and the functions of ER, and relies mostly on microtubules in mammals and Drosophila. Elhanany-Tamir (2012) documented ER and microtubule organization in WT Drosophila larval muscle and showed that astral microtubules are attached to the nuclear envelope from which they radiate, while ER localizes around myonuclei and at Z-bands. This organization is lost in Msp-300 mutants. In Msp-300 ΔKASH mutants, microtubules detach from the nucleus and form a loose perinuclear ring overlapping with Msp-300 ring. Elhanany-Tamir further reports an important disorganization of ER staining in Msp-300-3' deletion mutants (Morel, 2014).
Considering microtubules' important role in ER dynamics and shaping and their disorganization in Msp-300 ΔKASH, it is tempting to speculate that ER dynamics or fine subcellular organization might be altered upon Msp-300 KASH domain deletion (Morel, 2014).
Syne-1, mammalian Msp-300, was isolated in a screen for Golgi-specific spectrin repeats containing proteins. Observation of the subcellular localization of Syne-1 and Golgi in myoblasts and myotubes, together with comparison of physical distance between nuclei and Golgi apparatus and Syne-1 size, led to the idea that Syne-1 could physically couple Golgi, ER, and nuclei in muscle cells (Morel, 2014 and references therein).
It is therefore speculated that altering Msp-300 anchorage to nuclei could directly impact both ER and Golgi organization and localization with respect to myonuclei or dynamics, thus resulting in altered translation or post-translational modification of proteins including glutamate receptors. Modification of organelle subcellular organization upon Msp300 mutation could thus in turn impact NMJ function (Morel, 2014).
Finally, Nesprin-1 was originally isolated in a yeast two-hybrid screen for MuSK interactors and called Syne-1 (for synaptic nuclear envelope-1) based on its enrichment at the nuclear envelope of synaptic nuclei. MuSK is a receptor tyrosine kinase involved in acetylcholine receptors clustering at the NMJ in mammals. Although acetylcholine receptor density or molecular architecture of the NMJ are not altered by the expression of a dominant negative form of Syne-1 in transgenic mice, effects of Syne-1 KASH domain deletion on NMJ organization were not described. Further investigation is thus necessary to exclude a potential contribution of Syne-1 to NMJ organization. The parallel between the potential involvement of Syne-1 together with MuSK in the clustering of the acetylcholine receptors and the role of Msp-300 in type-A glutamate receptor density at NMJ should nevertheless be kept in mind when investigating the molecular mechanisms of Msp-300/Nesprin-1 contribution to synapse function (Morel, 2014).
Mutations in Nesprin-1 have been associated with autosomal recessive cerebellar ataxia (ARCA1), EDMD, and autosomal recessive arthrogryposis diseases. ARCA1 is a neural disorder associated with cerebellar atrophy and impaired walking. Seven mutations were identified in ARCA1 patients, either in introns or exons, leading to a premature stop and resulting in Nesprin-1 C-terminus deletion. These were interestingly associated with mislocalized subsynaptic nuclei at the NMJ. Autosomal recessive arthrogryposis is a rare disease associated with congenital contractures. Analysis of two generations of a congenital family led to the identification of a mutation in nesprin-1 gene also resulting in a premature stop of the protein and deletion of its KASH domain. Finally, EDMD has been associated with mutations in LMNA, EMD , and nesprin-1 and 2 genes, all proposed to affect LINC complex organization. These three pathologies are associated with impaired muscle function, attributed either to neuromuscular or neuronal defects. In all cases, the nesprin-1 mutations identified result in Nesprin-1 disconnection from the LINC complex, often due to the KASH domain loss, explaining the increasing interest for the contribution of the LINC complex in muscle and neural functions (Morel, 2014).
This study has shown that Msp-300 ΔKASH larvae display obvious signs of locomotion defects that are not due to a lack of muscle contractility but rather to a defective synaptic function. Indeed, the results establish that Msp-300 is involved in the control of glutamate receptor density at the NMJ in a KASH-dependent manner. Considering the role of Msp-300 in controlling postsynaptic homeostasis, it is tempting to speculate that EDMD, ARCA1 and autosomal recessive arthrogryposis could all result from alterations of the postsynaptic fields associated with Nesprin-1 mutations (Morel, 2014).
Since Drosophila NMJ, being glutamatergic, is widely used as a model for central glutamatergic synapses, it is proposed that Drosophila is a new relevant model to study the function of Nesprin-1 in the accumulation of postsynaptic glutamate receptors and more generally to decipher the mechanisms by which Nesprin-1 impacts synapse physiology and understand its implications in neuromuscular and neuronal pathologies (Morel, 2014).
Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).
Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).
Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor . Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation. Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).
This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).
The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences, these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. The data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).
Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).
RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).
The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).
This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy , and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).
Striated muscle fibers are characterized by their tightly organized cytoplasm. This study shows that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300ΔKASH, or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, these results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance (Elhanany-Tamir 2012).
This paper shows cooperative, as well as unique, activities of the two Drosophila KASH proteins MSP-300 and Klar in promoting even spacing and anchoring of myonuclei in striated muscle fibers. A novel MSP-300 nuclear ring assembles and anchors the MTs to the nuclear envelope in a Klar- and MSP-300 KASH-dependent manner, mediating MT astral organization around each nucleus. It is suggested that the astral MT associated with each myonucleus, forming a basic unit, which in the steady-state holds each nucleus in place. However, during muscle fiber growth, each unit might change its relative position as a result of MT growth so that the distance between the myonuclei is maintained. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment is mediated exclusively by MSP-300, which maintains physical continuity between the nuclear ring and the Z-discs, presumably through dimerization of the spectrin repeats capable of forming filaments (Elhanany-Tamir 2012).
Recent results suggested that reducing KASH proteins from the nuclear membrane (by overexpressing the KASH domain) did not affect nuclear positioning in C2C12 cells. It is possible that residual KASH-dependent activity was present in these cells, capable of rescuing nuclei position (Elhanany-Tamir 2012).
Physiological measurements demonstrate the critical contribution of nuclear spacing to larval locomotion and muscle activity. Significantly, these studies further demonstrate that the contribution of MSP-300 to muscle function is more critical relative to that of Klar (as msp-300 mutant larvae were significantly slower than klar mutant larvae), presumably because MSP-300 affects the positioning of the myonuclei as well as that of the mitochondria and ER (Elhanany-Tamir 2012).
The MT network appears to possess the dynamic properties required for myonuclear organization. Consistently, recent data show the active involvement of opposing MT motors in driving robust nuclear dynamics in differentiating CTC12 cells. During embryogenesis, MT arrangement in myotubes is polarized with their plus ends close to the myotendinous junction (MTJ). However, after muscle sarcomerization, the MTs undergo a significant rearrangement into astral organization with their plus ends facing the nuclei. This suggests that nuclear positioning and spacing differs mechanistically between early embryonic and larval stages, i.e., before and after the establishment of sarcomeric architecture. In support of this model, this study shows that: (1) Klar is the only KASH protein required for nuclear positioning in embryonic myotubes, whereas MSP-300 is dispensable. (2) The polarity and distribution of the MT network changes during development: in embryos (stage 16), the plus ends are close to the MTJ, whereas in striated muscle fibers, the MT forms astral structures surrounding each nucleus with their plus ends facing the nuclear envelope. (3) The nuclei are arranged in distinct patterns at each stage; e.g., in the embryonic dorsal acute and oblique muscles, the nuclei are positioned in a typical half circle close to the MTJ, whereas in third instar larvae, the nuclei of the same muscles rearrange and are distributed evenly along the entire muscle fiber. Thus, it is suggested that sarcomerization involves a significant rearrangement of the MT network and the nuclei, at the end of which the nuclei are spaced evenly along the muscle fiber and attached to the Z-discs (Elhanany-Tamir 2012).
The mechanism regulating this rearrangement has not been elucidated. It is suggested that as part of this rearrangement, MSP-300 is translocated from the MTJ, where it accumulates during embryonic stages (Volk 1992) into the Z-discs at the end of embryogenesis. Presumably, during this latter stage MSP-300 isoforms containing KASH are produced, and collaborate with Klar to promote the attachment of the astral MT network to the nuclear envelope. Klar function has been previously linked to MTs as a regulator of the MT motors dynein (associated with MT minus ends) and/or kinesin (associated with MT plus ends). Therefore, Klar function in promoting the association of the MSP-300 nuclear ring and MT plus ends with the nuclear envelope might be mediated through its ability to recruit MT plus-end motors. Nesprin 1 and 2 were also shown to interact with dynein/dynactin and kinesin-1 in developing mouse neurons, and the spectrin repeats region of Nesprin 1 was shown to bind kinesin-2. Therefore, Klar can potentially mediate an interaction with MT, which is also consistent with the demonstration that kinesin heavy chain is essential for myonuclei positioning in Drosophila larval muscles. MSP-300 might be indirectly linked to this activity through its association with Klar (Elhanany-Tamir 2012).
Because muscle size increases dramatically during larval growth, and the number of nuclei remains constant, it is not clear how nuclear spacing is maintained during this growth process. It is suggested that similarly to nuclear spacing in preblastoderm embryos, the minus ends of the MT associate with each other, whereas the plus ends grow so that the distance between the nuclei remains equal. In this manner, nuclei are able to compare their relative position and maintain even spacing in all directions. A prerequisite for this mechanism is the anchoring of the astral MT to the nuclear membrane in a polarized fashion, and this function is provided by the MSP-300 ring, which might associate with MT in a klar-independent manner (Elhanany-Tamir 2012).
The current experiments suggest a major function for MSP-300 both in promoting nuclear spacing (cooperatively with Klar) and in mediating anchoring of the nuclei as well as other organelles to the muscle core myofibrillar domain. The spectrin repeats domain is critical for this latter function, as most of it is eliminated in the msp-300 mutant. Similarly to other proteins containing the spectrin repeats domain, the multiple (52) spectrin repeats are likely to promote formation of MSP-300 dimers that form elongated filaments capable of connecting between different organelles and the cytoskeleton. In striated muscle, it is suggested that such filaments connect between the various organelles and the Z-discs. The association of these filaments with the Z-discs requires the activity of d-Titin/Sallimus, another large protein that associates with the Z-discs. The function of the N-terminal domain of MSP-300 containing the calponin-homology (CH) actin-binding motifs is still elusive because of the early lethality of the homozygous mutants (Elhanany-Tamir 2012).
This analysis provides a mechanistic explanation for the severity of Nesprin-related diseases. It is suggested that as in Drosophila, Nesprin 1 and 2 and a mammalian klar orthologue (yet to be identified) cooperate to promote myonuclei positioning in vertebrate muscle fibers. Thus, elimination of the KASH domains of both nesprin1 and nesprin2 in mutant mice might still leave a putative mammalian Klar orthologue intact, thereby only partially affecting nuclear positioning in the mutant muscles. However, elimination of additional domains of Nesprin described in might abrogate MT organization more profoundly so that other Nesprins are incapable of complementing this situation. In addition, the position of other organelles might be disrupted as observed in the Drosophila msp-300 mutant, leading to a more severe phenotype in mice, and possibly also in humans (Elhanany-Tamir 2012).
A recent study suggested that the amyotrophic lateral sclerosis (ALS)-associated protein VAPB is secreted from motor neurons and promotes the correct positioning of mitochondria in muscle fibers. It would be interesting to test for a possible functional link between VAPB and MSP-300 activity in promoting mitochondrial positioning in striated muscles (Elhanany-Tamir 2012).
In summary, these studies provide a mechanistic explanation for the process of myonuclear positioning at distinct developmental stages in striated muscles. These studies may help in the prognosis of the severity of various Nesprin-related diseases in which distinct domains are missing, and provide a basis for future therapeutic approaches (Elhanany-Tamir 2012).
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. This study uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, a novel mechanism is proposed for maintaining myonuclear morphology, and its critical link to correct levels of nuclear factors in differentiated muscle fibers is revealed. These findings may shed light on the underlying mechanism of various muscular dystrophies (Wang, 2015).
Striated muscles contain a tightly ordered cytoplasm in which the shape and size of the nuclei are comparable and nuclear distribution is uniform. These features were recently shown to be essential for muscle function. In an attempt to elucidate mechanisms regulating the position and shape of myonuclei, this study analyzed the function of the two KASH proteins that are uniquely present in the Drosophila genome, MSP-300 and Klarsicht, both expressed in striated muscles. Both KASH proteins cooperate to construct a unique ring composed of MSP-300 protein that surrounds and attached to the nuclear envelope. The MSP-300 nuclear ring structure recruits and associates with a network of polarized astral microtubules that enables the dynamic movement and uniform spacing between the nuclei in each muscle fiber (Volk, 2013).
Myotube migration and the formation of muscle attachments are crucial events for the proper development of muscle patterning in the Drosophila embryo. This paper describes the identification of a new embryonic muscle-specific protein, MSP-300, in Drosophila. This protein is initially expressed by muscle precursors at muscle-ectoderm and muscle-muscle attachment sites. As development continues, MSP-300 becomes associated with muscle myofibrillar network. Studies of the subcellular localization of this muscle-specific protein in primary embryonic cultured myotubes show that MSP-300 decorates actin filaments, and that it is specifically enriched in sites where actin microfilaments are linked to the plasma membrane. Migrating myotubes exhibit high levels of this protein at their leading edge while, in myotubes that have already developed sarcomeric architecture, the protein is localized mainly at the Z-discs. Sequence of a partial 3.9 kb cDNA clone and molecular analysis of the predicted protein sequence of this protein indicates that it encodes a high relative molecular mass protein (approximately 300 x 103, which exhibits at least five spectrin-like repeats. Several properties are shared by MSP-300 and members of the spectrin superfamily: it is associated with actin microfilaments, its sequence exhibits spectrin-like repeats and it is localized at sites where actin is linked to the plasma membrane. This protein could have a developmental role in the formation of muscle-ectoderm attachments and may be involved in myotube migration on the ectoderm (Volk, 1992).
Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear Anchorage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in C. elegans, this study found that ANC-1 binds to the Regulator of Presynaptic Morphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called Pam/Highwire/RPM-1 (PHR) proteins that are important regulators of neuronal development. ANC-1, like RPM-1, regulates axon termination and synapse formation. This genetic analysis indicates that ANC-1 functions via the beta-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. Variable roles were identified for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. This study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions (Tulgren, 2014).
Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. This study identified the nuclear envelope protein nesprin-2 (see Drosophila Nesprin) as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, these results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion (Jayo, 2016).
Search PubMed for articles about Drosophila Nesprin
Apel, E. D., Lewis, R. M., Grady, R. M. and Sanes, J. R. (2000). Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J Biol Chem 275: 31986-31995. PubMed ID: 10878022
Beck, K. A. (2005). Spectrins and the Golgi. Biochim Biophys Acta 1744: 374-382. PubMed ID: 15921768
Cottrell, J. R., Borok, E., Horvath, T. L. and Nedivi, E. (2004). CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors. Neuron 44: 677-690. PubMed ID: 15541315
Elhanany-Tamir, H., Yu, Y. V., Shnayder, M., Jain, A., Welte, M. and Volk, T. (2012). Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules. J Cell Biol 198: 833-846. PubMed ID: 22927463
Jayo, A., Malboubi, M., Antoku, S., Chang, W., Ortiz-Zapater, E., Groen, C., Pfisterer, K., Tootle, T., Charras, G., Gundersen, G. G. and Parsons, M. (2016). Fascin regulates nuclear movement and deformation in migrating cells. Dev Cell 38: 371-383. PubMed ID: 27554857
Kim, D. I., Birendra, K. C. and Roux, K. J. (2015). Making the LINC: SUN and KASH protein interactions. Biol Chem 396: 295-310. PubMed ID: 25720065
McDermott, S. M., Yang, L., Halstead, J. M., Hamilton, R. S., Meignin, C. and Davis, I. (2014). Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction. RNA 20(10): 1593-606. PubMed ID: 25171822
Morel, V., Lepicard, S., A, N. R., Parmentier, M. L. and Schaeffer, L. (2014). Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions. Cell Mol Life Sci. 71(17): 3363-79.. PubMed ID: 24492984
Packard, M., Jokhi, V., Ding, B., Ruiz-Canada, C., Ashley, J. and Budnik, V. (2015). Nucleus to synapse Nesprin1 railroad tracks direct synapse maturation through RNA localization. Neuron 86(4): 1015-28. PubMed ID: 25959729
Rajgor, D. and Shanahan, C. M. (2013). Nesprins: from the nuclear envelope and beyond. Expert Rev Mol Med 15: e5. PubMed ID: 23830188
Schuh, M. (2011). An actin-dependent mechanism for long-range vesicle transport. Nat Cell Biol 13: 1431-1436. PubMed ID: 21983562
Shinozaki, G. and Potash, J. B. (2014). New developments in the genetics of bipolar disorder. Curr Psychiatry Rep 16: 493. PubMed ID: 25194313
Speese, S. D., Ashley, J., Jokhi, V., Nunnari, J., Barria, R., Li, Y., Ataman, B., Koon, A., Chang, Y. T., Li, Q., Moore, M. J. and Budnik, V. (2012). Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149: 832-846. PubMed ID: 22579286
Tulgren, E. D., Turgeon, S. M., Opperman, K. J. and Grill, B. (2014). The Nesprin family member ANC-1 regulates synapse formation and axon termination by functioning in a pathway with RPM-1 and beta-Catenin. PLoS Genet 10: e1004481. PubMed ID: 25010424
Volk, T. (1992). A new member of the spectrin superfamily may participate in the formation of embryonic muscle attachments in Drosophila. Development 116: 721-730. PubMed ID: 1289062
Volk, T. (2013). Positioning nuclei within the cytoplasm of striated muscle fiber: cooperation between microtubules and KASH proteins. Nucleus 4: 18-22. PubMed ID: 23211643
Wang, S., Reuveny, A. and Volk, T. (2015). Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1. J Cell Biol 209: 529-538. PubMed ID: 26008743
Zhang, J., Felder, A., Liu, Y., Guo, L. T., Lange, S., Dalton, N. D., Gu, Y., Peterson, K. L., Mizisin, A. P., Shelton, G. D., Lieber, R. L. and Chen, J. (2010). Nesprin 1 is critical for nuclear positioning and anchorage. Hum Mol Genet 19: 329-341. PubMed ID: 19864491
date revised: 20 October 2016
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