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Unconventional myosin VI (MVI) is an ATP-dependent actin-binding molecular motor that participates in numerous cellular and tissue functions, including striated muscle physiology. Lack of MVI expression significantly aberrates myogenesis and skeletal muscle metabolism, and alters myoblast adhesion, fusion, and cytoskeletal organisation. Concomitantly, MVI knockout mice display functional and structural cardiac defects. Here, for the first time, we investigate the impact of MVI on neuromuscular junctions (NMJs), the peripheral synapses crucial for skeletal muscle contraction. We show that MVI is enriched at the postsynaptic machinery of developing and adult NMJs. We analyse the morphology of NMJs of MVI knockout mice (Snell’s waltzer, SV) during early developmental remodelling and show that MVI deficiency delays NMJ maturation in fast- and slow-twitch muscles. It also reduces the NMJ size of the soleus muscle, as demonstrated by the decreased morphological parameters of both presynaptic and postsynaptic compartments. Simultaneously, synaptic elimination remains unaffected after MVI knockout, suggesting that the observed phenotypes are innervation-independent. Lastly, depletion of MVI impairs the grip strength of both female and male SV/SV mice. In summary, our studies show that MVI is an important regulator of NMJ size and maturation, controls muscle performance, and its impact is independent of innervation and sex.
Introduction
Myosins are highly conserved actin-based molecular motors, expressed across Eukaryota1 and participating in cytokinesis, cell migration, adhesion and fusion, endo- and exocytosis, intracellular trafficking, transcription, chromatin organisation and DNA damage repair2, 3, 4, 5, 6, 7, 8, 9–10. Muscle myosin heavy chain isoforms serve as muscle fiber type-specific molecular markers, important to discern the mechanisms of myogenic development and to understand muscle function in health and disease. Based on their ability to form filaments and their origin, myosins are divided into conventional (class II) and unconventional, classified in humans into 11 classes (families)11, 12–13. Out of forty genes encoding myosin heavy chains in humans, almost two-thirds encode unconventional ones, but their function is still less understood than conventional ones.
Unconventional myosin VI (MVI) is a unique member of the myosin family that moves backwards, towards the minus end of actin filaments14,15. Snell’s waltzer (SV) mice are natural MVI knockouts carrying a spontaneous null mutation that causes deafness, circling, and hyperactivity16. Additionally, structural defects in kidneys, brain, and testes have been reported in SV/SV mice17, 18–19. Given the multiple roles of MVI, it is localised in various cell compartments and organelles, such as the sarcoplasmic reticulum, Golgi apparatus, intercalated discs, cell nucleolus and nucleus, and around mitochondria9,20, 21, 22, 23, 24, 25–26. Importantly, MVI is a crucial regulator of striated muscle development and physiology. It is involved in heart organisation and dysfunction of this protein entails left ventricular cardiomyopathy22,27, 28–29. Growing evidence underlines the importance of MVI in myogenesis and skeletal muscle function. Studies of our research group show that loss of MVI results in aberrated myoblast adhesion, fusion, metabolism, and actin organisation in myoblasts in vitro8. MVI controls expression of crucial myogenic regulators, Pax7 (Paired Box 7), MyoD (Myogenic Differentiation 1), and myogenin, as well as adhesion proteins, and fusogens, myomaker and myomerger8. We also determined that lack of MVI increases the muscle/body weight ratio and significantly affects the morphology of the murine hindlimb muscles. Myosin VI knockout causes a 2.5-fold increase of thin muscle fibres with a cross-sectional area below 100 µm2. This effect, observed as early as at birth, is the most evident in the slow-twitch muscle soleus and is maintained throughout the animals’ life span30.
Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal muscle fibers. Compromised NMJ integrity and function is a hallmark of neuromuscular disorders, such as Duchenne muscular dystrophy, myasthenia gravis, congenital myasthenic syndromes (CMS), Charcot-Marie-Tooth disease (CMTD) or amyotrophic lateral sclerosis (ALS)31, 32, 33, 34, 35, 36–37. Despite the central role of NMJs in the onset and progression of neuromuscular disorders, the molecular mechanisms underlying these processes are still poorly understood. Thorough characterisation of the genetic models manifesting neuromuscular symptoms can bring valuable insights into their pathology and help to develop more effective therapies.
We have previously identified MVI as a postsynaptic protein at NMJs in rats, however, the role of MVI in NMJ function remains unexplored21. Here we show that MVI is involved in NMJ development and maintenance. MVI is a protein abundant at NMJs during developmental remodelling and adulthood and is localised postsynaptically in both slow and fast-twitch muscles. The knockout of MVI delays NMJ maturation, however, independently of synaptic elimination. Moreover, loss of MVI decreases the size of both pre- and postsynaptic apparatuses, in accordance with the overall reduction of the body, muscle, and muscle fiber size in SV/SV mice30. The significantly diminished grip strength of both female and male MVI knockouts suggests that MVI depletion has universal functional consequences for muscle, regardless of sex.
Results
MVI is enriched at NMJs during developmental remodelling and maintenance
Mature neuromuscular junctions develop from simple oval acetylcholine receptor (AChR) assemblies during plaque-to-pretzel transition. After the first week of life, plaques become increasingly perforated and reshape into pretzel-like structures by P21, when maturation is mostly completed (Fig. 1a)38. Subsequently, NMJs grow in size until the mouse reaches adulthood (P90). Another type of NMJ remodelling is induced by aging which starts around 14 months of age (Fig. 1a)39,40. We assessed the localisation of MVI in fast-twitch tibialis anterior at the time of intense developmental remodelling (P10), and NMJ maintenance in mature adult (P120) and middle-aged (P365) mice. MVI was enriched at NMJs at all analysed stages, but distributed differently (Fig. 1b). At the stage of NMJ maturation (P10) MVI was dispersed at the NMJ postsynaptic compartment (Fig. 1b). At mature NMJs (P120, P365) MVI mainly occupied the domains between AChR-rich areas, and was also clustered at the vicinity of AChRs (Fig. 1b, orthogonal views at P120, Fig. 1c and Supplementary Fig. 1). The enrichment of MVI along the entire postsynaptic machinery was characteristic for NMJs of adult muscles with different fiber composition and function: fast-twitch diaphragm and slow-twitch triangularis sterni and soleus (Supplementary Fig. 2). Our results show that MVI is localised postsynaptically at NMJs of various skeletal muscles throughout the mouse lifespan, during developmental reorganisation and maintenance of NMJs.
Fig. 1 [Images not available. See PDF.]
Myosin VI is localised at the postsynaptic compartment during NMJ development and maintenance. (a) Maturation of murine NMJs. Immature NMJs take the form of unperforated plaques. After around a week from birth first perforations in AChR assemblies start to appear, and they expand and fuse to form mature pretzel-like NMJs by P21. This complex structure is maintained throughout adulthood (P90) until the aging-related degeneration occurs at NMJs (P547). Timepoints of the analysis are marked in pink. Created in BioRender. Gawor, M. (2025) https://BioRender.com/uk9u03o. (b) Representative MVI localisation at NMJs in tibialis anterior muscle at time points depicted in (a). MVI is enriched at NMJs at all analysed stages and is specific to the postsynaptic machinery (orthogonal views in the lower and right panels at P120). P10 represents the stage of NMJ maturation, and P120 and P365 NMJ maintenance during adulthood and middle-age, before the age-dependent structural changes appear. Postsynaptic AChRs (green) are labelled with bungarotoxin (α-BTX), and motoneurons (blue) with anti-neurofilament (NF) and anti-synaptophysin (Syn.) antibodies. Antibody control – control of secondary antibody unspecific binding. Scale bar = 20 µm. (c) Measurement of the fluorescence intensity from a single plane of the optical cross-section from the NMJ at P120 (on the right, white line). Red peaks show MVI labelling fluorescence intensity and green and blue AChRs and motoneuron, respectively. Maximal intensity for MVI (black arrowheads) overlaps with the lowest for AChRs, confirming that MVI is abundant between AChR-rich areas, as shown in (b).
Knockout of MVI delays NMJ maturation in slow- and fast-twitch muscles
Since MVI is present at NMJs during developmental remodelling, we tested its role in postsynaptic maturation. To this end, we determined the ratio of perforated NMJs upon MVI knockout at P10 in two types of muscles: fast-twitch tibialis anterior (TA) and slow-twitch soleus (Fig. 2a–f). The number of maturing NMJs decreased in both analysed muscles by 34% (TA) and 36% (soleus) (Fig. 2e and f, on the left). Simultaneously, we did not observe changes in the proportion of NMJs with different numbers of perforations (Fig. 2f, on the right), which suggests that MVI expression rather affects the induction of postsynaptic maturation than its progression. Overall, these results show that MVI has a broad effect on NMJ development and the lack of this protein delays NMJ maturation both in fast- and slow-twitch muscles.
Fig. 2 [Images not available. See PDF.]
NMJ maturation is delayed in the absence of MVI. Effective MVI knockout validated with animal morphology assessment (a), genotyping (b) and Western blotting (c). (a) Body size of SV/SV mice at P10 is reduced, analogous to the results shown for other ages 19,29,30,91. (b) Representative cropped gel showing SV mice genotyping. SV/ + , controls, heterozygotes (WT allele = 230 bp, SV allele = 318 bp). SV/SV, MVI knockouts, homozygotes (SV allele = 318 bp). M, DNA molecular weight marker. Original gel presented in Supplementary Fig. 5. (c) MVI protein expression is effectively knocked out in the soleus muscles of SV/SV mice. Representative cropped blot showing MVI protein levels in SV/SV an SV/ + mice. MVI and GAPDH protein levels are from different regions of the same blot. Original blot presented in Supplementary Fig. 6. Lack of MVI localisation at NMJs of SV/SV mice (negative control) is presented in Supplementary Fig. 3. (d) Representative images of NMJ classes analysed in (f): immature, unperforated plaques and perforated NMJs (1, 2 and > 2 perforations). Arrowheads depict perforated NMJs. Scale bar = 10 µm. (e) Representative images of NMJs in tibialis anterior (TA, fast-twitch, top panel) and soleus (slow-twitch, bottom panel) muscles of SV/ + and SV/SV mice at P10. NMJs are labelled with α-BTX. Arrowheads show maturing NMJs. Scale bar = 10 µm. (f) MVI knockout decreases the percentage of maturing NMJs in both TA and soleus muscles. Quantification of the percentage of plaques vs. perforated NMJs (left) and three classes of perforated NMJs (1, 2 and > 2 perforations, right) in TA (top) and soleus (bottom) of SV/ + and SV/SV mice. Two-way ANOVA with Sidak’s multiple comparisons test, N = 7 (TA) and N = 9 (soleus), ± SD (left), ± SEM (right),*p ≤ 0.05, ***p ≤ 0.001.
Lack of MVI reduces the size of the NMJ pre- and postsynaptic domains, but does not affect innervation
Postsynaptic maturation is tightly regulated by innervation, and pre- and postsynaptic compartments morphologically mirror each other to collaborate optimally. Initially, NMJs are polyinnervated, and during muscle development, they gradually lose surplus axonal inputs in a process called synaptic elimination, completed by the end of the second week after birth41, 42, 43–44. We assessed whether the observed delay in postsynaptic maturation upon MVI knockout at P10 is accompanied by changes in NMJ innervation (Fig. 3a–d). Since soleus is the muscle where MVI expression is the most abundant45 and its effect on NMJ maturation was more statistically significant (Fig. 2f), we focused on this muscle in our subsequent analyses. We determined the ratio of mono- and polyinnervated NMJs, considered the presynaptic indicator of NMJ maturation (Fig. 3a). A higher ratio of polyinnervated NMJs would indicate delayed synaptic elimination. We also assessed the number of denervated NMJs and NMJs with degenerating nerves, another two indicators of synapse elimination (Fig. 3b). The overall number of NMJs was increased by 30% after MVI knockout (Fig. 3c). However, there were no significant differences in innervation, suggesting that the delayed postsynaptic maturation was independent of synaptic elimination (Fig. 3d). Thus, we analysed the morphology of the motoneuron terminals of SV/SV mice (Fig. 3e–m). MVI depletion decreased nerve terminal perimeter and area by 14,5% and 27%, respectively (Fig. 3f and g), as well as the total and average length of axonal branches by 19% (Fig. 3k) and by 21% (Fig. 3l), respectively. The changes in presynaptic morphology coincided with reduced size of the postsynaptic compartment and diminished SV/SV mice body weight (Fig. 4a-m). MVI knockout decreased AChR area by 11% (Fig. 4c), and endplate perimeter by 5% (Fig. 4e), and area by 11% (Fig. 4f). Since MVI knockout muscles are characterised by thinner muscle fibres, we verified whether the observed reduction in NMJ size is correlated with the muscle fibre diameter30. Indeed, endplate area when normalised to the muscle fibre diameter was similar in SV/SV and SV/ + mice, indicating that NMJs size is correlated with thinner muscle fibres (Supplementary Fig. 4). Parameters, such as number of AChR clusters, an indicator of endplate fragmentation, or overlap, showing endplate presynaptic coverage were similar (Fig. 4j and l). This result supports our conclusion that the synaptic elimination is not impaired in MVI knockouts (Fig. 3d). In summary, our observations confirm that MVI knockout impacts pre- and postsynaptic morphology of motor terminals, independently of denervation and degeneration characteristic for the loss of motoneurons during development.
Fig. 3 [Images not available. See PDF.]
MVI knockout impairs the size of motoneuron terminals, but not innervation. (a) Representative images of lower motoneurons (green) labelled with anti-neurofilament (NF) and anti-synaptophysin (Syn.) antibodies and postsynaptic AChRs (magenta) labelled with α-BTX (soleus). Full arrowheads show monoinnervated NMJs, asterisks polyinnervated NMJs, and empty arrowhead NMJ that undergoes synaptic elimination. Magnification of the presynaptic compartment from the area marked with a dotted line is shown in (e). Scale bar = 10 µm. (b) Representative images of denervated NMJs and NMJs with degenerating nerve. Scale bar = 3,5 µm. (c) Soleus muscles of the SV/SV mice are characterised by a higher number of NMJs at P10. Two-tailed unpaired t-test, N = 9, ± SD, * p ≤ 0.05. (d) MVI has no impact on NMJ innervation at P10 (soleus). Quantification of the NMJ classes presented in (a) and (b). Two-way ANOVA with Sidak’s multiple comparisons test, N = 9, ± SEM, not significant. (e) Representative image of the motor nerve terminals (green, axon identified by full arrowhead and axon terminal by empty arrowhead) presented in (a). (f–m) Motoneuron terminals of SV/SV mice are smaller with shorter branches (P10, soleus), as shown by the morphometric analysis. Two-tailed unpaired t-test, N = 13, ± SD, *p ≤ 0.05, **p ≤ 0.01.
Fig. 4 [Images not available. See PDF.]
Loss of MVI expression reduces the size of the postsynaptic machinery. (a) Magnification of the postsynaptic AChRs (magenta) corresponding to the NMJs shown in Fig. 3e. (b–m) MVI regulates the size of motor endplates (P10, soleus), as shown by the morphometric analysis of the postsynaptic apparatus. (m) Reduced body weight of SV/SV mice at the stage of NMJ maturation (P10). Two-tailed unpaired t-test, N = 13, ± SD, *p ≤ 0.05, **** p ≤ 0.0001.
MVI is necessary to maintain proper muscle strength regardless of sex
Next, we determined whether the observed changes in the NMJ morphology alter muscle performance. To this end, we performed grip strength test with adult SV/ + and SV/SV mice of both sexes (Fig. 5a). Since both female and male MVI knockouts have reduced body weight17, we normalised the obtained average force values to the body weight of mice used in our behavioral studies (Fig. 5b and c).
Fig. 5 [Images not available. See PDF.]
SV mice exhibit reduced muscle strength. (a) Experimental design. The grip strength of adult SV/ + and SV/SV mice (males and females) was tested at P90 (marked in pink). The time when NMJ maturation is mostly completed (P21) is marked in grey. Created in BioRender. Gawor, M. (2025) https://BioRender.com/eyshv76. (b) Lack of MVI expression alters muscle performance. The force of the grip (N) normalised to body weight (g) shown in (c) dropped for both female and male SV/SV mice. Two-way ANOVA with Tukey’s multiple comparisons test, N = 11 (females, SV/ + and SV/SV) and N = 8 (male, SV/ +), and N = 9 (male, SV/SV), whiskers show min. to max. (b) or ± SD (c), *p ≤ 0.05, *** p ≤ 0.001, ****p ≤ 0.0001.
Both female and male SV/SV mice had weaker muscles in comparison to control animals, as shown by the 56% and 44% drop in their muscle strength, respectively (Fig. 5b). Interestingly, the bigger impact of MVI knockout on female performance was not associated with the more pronounced decrease of their body weight, since MVI depletion caused more significant reduction of the male body weight (Fig. 5c).
Discussion
We show that MVI is ubiquitously expressed at NMJs of various muscles at different stages of the development (Fig. 1 and Supplementary Fig. 2). At the analysed stage of adulthood (P120) MVI is mainly adjacent to AChRs and present in the domain devoid of AChRs (Supplementary Fig. 1). However, analysis of MVI localisation at the other stages of NMJ development and in other types of muscles reveals a more dispersed distribution along the entire synapse (Fig. 1 and Supplementary Fig. 2). Given the broad role of MVI in endo- and exocytosis, it can be involved in synaptic signalling at NMJs19,46, 47, 48, 49–50 and its localisation may change depending on the external factors, such as type of stimuli or intrinsic factors, such as accessibility of other proteins or metabolic footprint of the muscle fibre, among others. In the central nervous system, MVI plays a role in synaptic transmission and plasticity51,52. MVI expression increases in slow- and fast-twitch muscles upon denervation, and the protein localises to the entire muscle fibre, in contrast to its peripheral localisation in innervated muscles21. This observation indicates that MVI expression is regulated by synaptic activity. Drosophila melanogaster MVI loss-of-function mutants display defects in NMJ morphology, synaptic vesicle distribution, and basal synaptic transmission, accompanied by the impaired locomotor activity of the mutant larvae53. Moreover, MVI is an important regulator of the proper organisation of synaptic vesicles at Drosophila NMJs and anchors them at the specific regions of synaptic domains54. MVI can play a similar role at mouse NMJs and participate in synaptic signalling during development. However, further studies identifying MVI molecular partners specific to peripheral synapses and the spatiotemporal regulation of their distribution at NMJs are necessary to unravel the function of this protein in the described context.
The diminished percentage of perforated NMJs in both fast- and slow-twitch muscles of SV/SV mice indicates a wide-range delay in synapse maturation (Fig. 2e and f). However, this impairment appears not to be caused by stalled synaptic elimination (Figs. 3d and 4i and l). One of the possible explanations is that MVI is required for AChR clustering and redistribution during the formation of synaptic perforations. It has been shown that MVI is a part of the complex regulating Rac1 (Rac Family Small GTPase 1) and Cdc42 (Cell Division Cycle 42) Rho GTPases, rearranging the actin cytoskeleton and vesicle trafficking, which can subsequently impact AChR turnover and degradation55, 56–57. Moreover, Rho GEF ephexin1 is the effector protein of the MuSK-Dok7 complex, crucial for AChR dispersal57,58. Thus, Rho GTPases are potential MVI downstream targets that control AChR recycling at the maturing NMJs. Simultaneously, MVI can support the formation of synaptic perforations by anchoring AChRs and restricting their localisation to certain NMJ areas, which facilitates the formation and propagation of perforations. MVI anchoring role at NMJs has been confirmed in studies of Drosophila MVI mutants. Normally, synaptic vesicles are concentrated at the outer ring of the synaptic boutons. Upon MVI knockout, this selective localisation is lost, and they occupy the entire area of the boutons53. Similarly, loss of MVI stabilisation can result in AChR dispersal and delay the formation of perforations at mouse NMJs.
We observed the increase in the overall number of NMJs (Fig. 3c). This confirms our previous observations showing that SV/SV mice are characterised by a higher number of muscle fibres30. Both pre- and postsynaptic endplate size parameters were reduced in MVI knockouts (Figs. 3f, g, k and l and 4c, e and f). It was shown previously by our group that soleus muscles of SV/SV mice are characterised by a larger number of fibres with decreased cross-sectional area30. In agreement with the impact of the nuclear content on the myofiber size, the number of myonuclei was also decreased after MVI knockout30,59. Endplate areas normalised to the corresponding muscle fibre diameters showed similar values for SV/ + and SV/SV mice (Supplementary Fig. 4), suggesting a relationship between NMJ and muscle fibre size. Some studies showed that these parameters are correlated60,61, however, other groups reported that muscle fibre size is not a main contributor to NMJ morphology62. This discrepancy can be explained by the different developmental stages, muscle types and species analysed60, 61, 62–63. Another factor that can influence NMJ size is the fibre type62. Given the role of MVI in myogenesis and the significant increase in the number of thinner fibres in MVI knockout mice8,30, it would be interesting to perform fibre type analyses and the measurements of the size of corresponding NMJs in these knockouts. It was recently reported that the absence of MVI expression in skeletal muscles causes glycolytic-to-oxidative fibre-type switch45. However, the relationship between the muscle fibre metabolic type and the size of NMJs is yet to be determined, due to mixed results from different types of muscles62,64,65. Nevertheless, impairments in muscle structure of MVI knockouts are accompanied by changes in their metabolism. The levels of phosphorylated PKA (Protein Kinase A) and CREB (cAMP Response Element-Binding) proteins, regulators of glucose and lipid metabolism and mitochondrial function, are decreased in MVI-devoid soleus muscles at birth30. In adult mice, loss of MVI impairs ATP production and mTOR-dependent signalling, major contributors to muscle growth45,66. Moreover, loss of MVI disrupts adhesion, fusion, and differentiation of myotubes in vitro8. Altogether, these alterations can delay muscle fibre development and growth, undermining the muscle’s ability to support the reorganisation of developing NMJs.
The importance of the muscle intrinsic signals for NMJ remodelling and the role of MVI in muscle fibre development and myoblast fusion are well documented8,30,67, 68, 69–70. Lack of MVI alters the formation of myotubes and decreases γ-actin, focal adhesion kinase (FAK), and M-cadherin expression, all of which have been implicated in synapse formation and function8,71, 72, 73–74. Expression of the key myogenic regulators, such as Pax7 and myogenin, is also reduced in MVI knockout primary myoblasts8. Myogenin has been identified as an important regulator of NMJ size75, which is in accordance with the diminished pre- and postsynaptic area of the motor endplates observed in SV/SV mice (Figs. 3f,g and 4c, e and f). Importantly, myogenin is a myogenic regulatory factor (MRF) crucial for AChR clustering in myotubes in vitro, and its role cannot be replaced by other MRFs or AChR organisers, MuSK and rapsyn76. Thus, myogenin should be a primary focus in future analyses aiming to explain MVI-dependent mechanisms regulating NMJ morphology. Future investigation should also assess the MVI impact on localisation and function of terminal Schwann cells (TSCs) which are crucial for NMJ maturation and maintenance77,78. TSCs regulate the expression of postsynaptic genes and if their function is impaired, NMJ maturation can be delayed even in the presence of unaltered innervation (Fig. 3d). Altogether, these results and MVI emerging role in transcriptional regulation9,79,80, suggest this protein could be a regulator of transcription factors that control the activity of NMJ-related genes.
We show that MVI knockout causes a significant impairment in the muscle performance in both female and male mice (Fig. 5b). This can be a result of the reduced endplate and nerve terminal size, which limit synaptic transmission and weaken the muscles which cannot be compensated by the higher number of NMJs (Figs. 3 and 4). The size of nerve terminals impacts neurotransmitter release at NMJs of various species81, 82–83. The observed phenotype can also be a consequence of delayed maturation. A similar effect was reported for ephexin1 knockouts, where NMJs failed to acquire complex pretzel-like topology, which was accompanied by muscle weakening and impaired neurotransmission58. Finally, MVI binding partner Dock-7 (Dedicator of Cytokinesis 7) was shown to participate in the neuregulin-ErbB2 pathway, which regulates neurotransmission84, 85–86. Moreover, recent analyses performed by our research group show impaired mitochondria respiration and reduced ATP production upon MVI knockout which can also cause muscle weakening45. Future analyses should determine how lack of MVI affects synaptic transmission and neurotransmitter release, neurotransmitter receptor expression and function, as well as downstream signalling pathways controlling muscle performance and fatigability.
Methods
Ethics declarations
Procedures involving animals were approved by the 1st Local Ethical Committee for Experiments on Animals in Warsaw (resolutions 1311/2022 and 1639/2024) and were performed in accordance with the Act on the Protection of Animals Used for Scientific or Educational Purposes (2015) from the European Communities Council directives approved by the Polish Parliament. The researchers had individual permissions for the work involving mice granted by the Director of the Nencki Institute of Experimental Biology [218(W)/2024/IBD, 396(U)/2019/IBD, 396W/2020/IBD, 194P/2019/IBD, 194W/2020/IBD, 194/2021/IBD, 17(W)/2020/IBD, 27(W;P)/2020/IBD]. All experiments were performed in accordance with relevant guidelines and regulations and are reported according to ARRIVE guidelines.
Snell’s waltzer (SV) mice husbandry and genotyping
Snell’s waltzer (SV) mice (C57BL/6J genetic background), a gift from Dr. Folma Buss (Cambridge Institute of Medical Research, UK), and C57BL/6J mice were maintained in the animal house of the Nencki Institute of Experimental Biology. All analyses were performed on male mice except the grip strength test, which was performed using both sexes. The age of mice is reported in relevant description of the methods, figures and figure legends. The weight of mice representative of the age P10 (morphometric NMJ analysis) and P90 (grip strength test) is presented in Figs. 4m and 5c, respectively. Mice were euthanised with a lethal dose of isoflurane followed by cervical dislocation. SV mice tail clips (2–4 mm2) were used for genotyping with PCR Master Mix (Thermo Scientific) or KAPA Mouse Genotyping Kit (KAPA Biosystems) per manufacturer’s instructions. Primers for genotyping were as follows: SV1 5′-CTGACCCTGATCACTTAGCAGAGTTG-3′; SV2 5′-CATTGGGCCAGGTCACAGAAGTAAGC-3′; SV3 5′-GGTCCTCTGAAAGAGTAACC-3′ (SV/ + 318 and 230 bp, SV/SV 318 bp). Sex-matched littermates (SV/ +) were used as controls for the phenotype assessment of MVI knockouts (SV/SV).
Whole-mount muscle fibre immunostaining
Soleus or tibialis anterior (TA) hindlimb muscles were isolated at time points reflecting different stages of NMJ development and maintenance (P10, P120, P365 for C57BL/6J and P10 for SV mice). Muscles were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) at room temperature (RT) for 15–80 min., depending on the muscle size and washed three times for 15 min. in PBS. For each analysis, muscle fibres were isolated from randomly chosen parts spanning the whole muscle. Pieces comprising approximately 10–30 muscle fibres were dissected, incubated for 15–30 min. in 0.1 M glycine in PBS, and rinsed in PBS and 0.5% Triton X-100. Prior to incubation in glycine, fibres for MVI detection in SV/ + and SV/SV muscles were additionally permeabilised in PBS with 2% Triton X-100 for 2 h. Then, muscles were incubated in blocking buffer [2–3% bovine serum albumin, 2–5% normal goat serum, 0.05–0.5% Triton X-100, 0.02% NaN3 in PBS] at RT for minimum 30 min. to overnight. Next, fibres were incubated in a sample shaker with primary antibodies diluted in blocking buffer at 4°C overnight, and washed three times with PBS or PBS with 0.2% Triton X-100 for 5 min. After washing, specimens were incubated with secondary antibodies diluted in blocking buffer for 1–2 h, washed with PBS, and stained with α-bungarotoxin (BTX) diluted in PBS at RT for 15 min. Control stainings omitting primary antibody were performed to test secondary antibody non-specific binding. Whole-mount preparations were mounted in Fluoromount Aqueous Mounting Medium (Sigma) with DAPI (4′,6-diamidino-2-phenylindole) or Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories). Antibodies and fluorescent reagents used are listed in Supplementary Table S1. Fluorescence intensity was measured using ZEN Blue 2012 or 3.1 software.
Confocal imaging
Images were collected using an Axio Observer Z.1 inverted microscopes: Spinning Disc equipped with CSU-X1 spinning disc unit (Yokogawa, Japan) and Evolve 512 EMCCD camera (Photometrics, USA) or LSM780 (Zeiss, Germany) using 40 × /1.2 Water and 63 × /1.4 Oil Plan Apochromat DIC objectives. Optical Sects. (1024 pixels × 1024 pixels × 8 or 12-Bit/pixel) were acquired at 0.5 μm Z-spacing with ZEN Blue 2012, 2.3 or 3.1 software (Zeiss, Germany). Images were further processed using FijiJ distribution of ImageJ software87 or ZEN Blue software.
NMJ morphology analysis
Maturation of NMJs was assessed independently by two researchers using the ZEN 2012 and Blue 3.1 software. BTX-labelled NMJs were analysed from maximum intensity projections (0.5–1 μm interval). On average, 76 NMJs per mouse were analyzed from 9 mice per genotype for soleus (1290 NMJs total) and 7 mice per genotype for TA (1094 NMJs total).
For pre- and postsynaptic morphometric analysis, NMJs were co-labelled with anti-neurofilament (2H3) and anti-synaptophysin antibodies or α-BTX, respectively. ImageJ software (https://imagej.nih.gov/ij/) combined with the aNMJ-morph macro88 was used to measure 20 individual pre- and postsynaptic morphological parameters (‘core variables’, ‘derived variables’, and ‘associated nerve and muscle variables’). Nerve terminal ‘complexity’ was calculated as log10 (number of terminal branches x number of branch points x total length of branches). Endplate ‘compactness’ was calculated as (AChR area/endplate area) × 100. The ‘overlap’ of presynaptic and postsynaptic structures was calculated as (area of synaptic contact/total area of AChRs) × 100. On average, 24 en face NMJs with clearly visible pre-synaptic axons and terminals were assessed per mouse. Thirteen mice were analysed per genotype (631 NMJs total). All analyses were performed using maximum intensity projections. Two thresholding methods (‘Huang’ and default method) provided the most accurate binary representation of the original raw NMJ images (Supplementary Fig. 4). The ‘Huang’ method was used for 96,4% of NMJs, and the default method for 3,6% of NMJs. For the measurement of muscle fibre diameter, fibres were labelled with anti-dystrophin antibody as described above and analysed using ZEN Blue 2.3 software.
Innervation analysis
The analysis of NMJ innervation and the morphology of motoneuron terminals was performed in a blinded manner, using ZEN 2012 Blue software. Maximum intensity projections (0.5 μm interval) of BTX-, 2H3- and synaptophysin-labelled NMJs were used to count mono- and polyinnervated NMJs, NMJs with ruptured or absent nerve (denervated) and NMJs with degenerated nerves. Nine mice per genotype were scored with an average of 184 NMJs per mouse (3319 NMJs analysed total).
Western blotting
Muscles were homogenised with a Pro200 Double insulated tissue homogenizer (Bioeko) in 50 volumes of ice-cold lysis buffer per muscle weight [0,1 M K2HPO4, 0,1 M KH2PO4 pH 7.2, 1mM PMSF] and samples were boiled in Laemmli buffer for 10 min. Twenty micrograms of protein/well were separated with SDS-PAGE through 10% polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked in 3% non-fat milk in TBST [Tris-buffered saline (TBS) with 0.2% Triton X-100] at RT for 1 h, followed by overnight incubation with primary antibodies diluted in blocking buffer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. Then, membranes were incubated with HRP-conjugated (horseradish peroxidase) secondary antibodies at RT for 1h. The list of antibodies used can be found in Supplementary Table S1. The bands were visualised using Immobilon Western Chemiluminescent HRP substrate (Merck) per manufacturer’s instructions.
Grip strength
The forelimb grip strength of P90 male and female SV/SV mice was tested using a force meter (Bioseb, France) in a blinded manner. Prior to performing tests mice were habituated to the researchers and the environment. On the day of the test, mice were held closely to the grid of the force meter, allowing them to grasp it, and then they were pulled away horizontally. The force meter measured the peak force when the animal lost its grip. Five consecutive trials were performed with a few-minute intervals between them. The final performance was assessed as a mean from all five measurements and is presented as values normalised to body weight.
Statistical analysis
All statistical tests were performed using GraphPad Prism 7 (CA, USA). Normality of the data was tested with the D’Agostino-Pearson omnibus normality test or the Shapiro–Wilk normality test, depending on the sample size. Datasets were analysed with two-tailed unpaired t-test and Mann–Whitney test, two-way ANOVA followed by the Sidak’s or Tukey’s multiple comparisons test, and two-tailed Spearman correlation analysis. Sample size was determined based on previous similar analyses88, 89–90, represents biological replicates and is reported in figure legends. Error bars depict standard deviation (SD), minimum to maximum or standard error of the mean (SEM) as stated in the figure legends.
Acknowledgements
Confocal imaging was performed at the Laboratory of Imaging Tissue Structure and Function, which serves as an imaging core facility at the Nencki Institute of Experimental Biology and is part of the infrastructure of the Polish Euro-BioImaging Node. The authors thank Artur Wolny from the Laboratory of Imaging Tissue Structure and Function for technical support of automated image analysis. The anti-neurofilament monoclonal antibody (#AB_2314897) developed by T. M. Jessell and J. Dodd was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Illustrations created with Biorender.com.
Author contributions
P.M.B., M.J.R. and M.G.—conceptualisation; J.N., P.A.-S. and M.G.—investigation, validation, formal analysis; T.W. and R.Z.—investigation; J.N., P.A.-S. and M.G.—visualisation; M.J.R and M.G.—supervision; J.N. and P.A.-S.—Project administration and funding acquisition; M.G.—writing—original draft; J. N., P. A.-S., T. W., R. Z., P.M.B., M. J. R. and M. G.—writing—review and editing.
Funding
This work was supported by the Preludium 15 grant UMO-2018/29/N/NZ3/02682 (awarded to P. A.-S.) and Miniatura 8 enabling award UMO-2024/08/X/NZ4/00609 (awarded to J. N.), from the National Science Centre, Poland. Polish Euro-BioImaging Node is supported by the project co-financed by the Minister of Education and Science based on contract No 2022/WK/05 (Polish Euro-BioImaging Node “Advanced Light Microscopy Node Poland”).
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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