A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells

Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.

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SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins

Human Molecular Genetics ◽

10.1093/hmg/ddaa276 ◽

2020 ◽

Author(s):

Shiyu Luo ◽

Qifei Li ◽

Jasmine Lin ◽

Quinn Murphy ◽

Isabelle Marty ◽

...

Keyword(s):

Focal Adhesion ◽

Skeletal Muscles ◽

Striated Muscle ◽

Calcium Handling ◽

Intermediate Filament Protein ◽

Β1 Integrin ◽

Adhesion Proteins ◽

Focal Adhesion Proteins ◽

Early Endosomes

Abstract SPEG, a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amounts of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1, and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

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Myosin VI Localization and Expression in Striated Muscle Pathology

The Anatomical Record ◽

10.1002/ar.22967 ◽

2014 ◽

Vol 297 (9) ◽

pp. 1706-1713 ◽

Cited By ~ 5

Author(s):

Justyna Karolczak ◽

Serge Weis ◽

Elisabeth Ehler ◽

Biruta Kierdaszuk ◽

Mariusz Berdyński ◽

...

Keyword(s):

Striated Muscle ◽

Muscle Pathology ◽

Myosin Vi

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A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle

eLife ◽

10.7554/elife.47460 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Glenda Comai ◽

Eglantine Heude ◽

Sebastian Mella ◽

Sylvain Paisant ◽

Francesca Pala ◽

...

Keyword(s):

Skeletal Muscle ◽

Striated Muscle ◽

Striated Muscles ◽

Food Ingestion ◽

Loss Of Function ◽

Pharmacological Studies ◽

Posterior Migration ◽

Functional Relevance ◽

Genetic Signatures ◽

Genetic Hierarchy

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

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The Tail Domain of Myosin-VI Ensures the Directed Processive Movement

Biophysical Journal ◽

10.1016/j.bpj.2010.12.856 ◽

2011 ◽

Vol 100 (3) ◽

pp. 119a

Author(s):

Keigo Ikezaki ◽

Tomotaka Komori ◽

Mitsuhiro Sugawa ◽

So Nishikawa ◽

Atsuko Iwane ◽

...

Keyword(s):

Myosin Vi ◽

Tail Domain

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Loss of IGF-IEa or IGF-IEb Impairs Myogenic Differentiation

Endocrinology ◽

10.1210/en.2010-1279 ◽

2011 ◽

Vol 152 (5) ◽

pp. 1923-1934 ◽

Cited By ~ 25

Author(s):

Ronald W. Matheny ◽

Bradley C. Nindl

Keyword(s):

Splice Variants ◽

Myogenic Differentiation ◽

Horse Serum ◽

Myoblast Differentiation ◽

Proliferation And Differentiation ◽

Igf I ◽

Simultaneous Loss ◽

Functional Relevance ◽

Free Media ◽

The Impact

Actions of protein products resulting from alternative splicing of the Igf1 gene have received increasing attention in recent years. However, the significance and functional relevance of these observations remain poorly defined. To address functions of IGF-I splice variants, we examined the impact of loss of IGF-IEa and IGF-IEb on the proliferation and differentiation of cultured mouse myoblasts. RNA interference-mediated reductions in total IGF-I, IGF-IEa alone, or IGF-IEb alone had no effect on cell viability in growth medium. However, cells deficient in total IGF-I or IGF-IEa alone proliferated significantly slower than control cells or cells deficient in IGF-IEb in serum-free media. Simultaneous loss of both or specific loss of either splice variant significantly inhibited myosin heavy chain (MyHC) immunoreactivity by 70–80% (P < 0.01) under differentiation conditions (48 h in 2% horse serum) as determined by Western immunoblotting. This loss in protein was associated with reduced MyHC isoform mRNAs, because reductions in total IGF-I or IGF-IEa mRNA significantly reduced MyHC mRNAs by approximately 50–75% (P < 0.05). Loss of IGF-IEb also reduced MyHC isoform mRNA significantly, with the exception of Myh7, but to a lesser degree (∼20–40%, P < 0.05). Provision of mature IGF-I, but not synthetic E peptides, restored Myh3 expression to control levels in cells deficient in IGF-IEa or IGF-IEb. Collectively, these data suggest that IGF-I splice variants may regulate myoblast differentiation through the actions of mature IGF-I and not the E peptides.

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Splicing transitions of the anchoring protein ENH during striated muscle development

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.03.142 ◽

2012 ◽

Vol 421 (2) ◽

pp. 232-238 ◽

Cited By ~ 7

Author(s):

Jumpei Ito ◽

Taiki Hashimoto ◽

Sho Nakamura ◽

Yusuke Aita ◽

Tomoko Yamazaki ◽

...

Keyword(s):

Muscle Development ◽

Striated Muscle ◽

Anchoring Protein

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Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1002430107 ◽

2010 ◽

Vol 107 (17) ◽

pp. 7746-7750 ◽

Cited By ~ 37

Author(s):

Alexander R. Dunn ◽

Peiying Chuan ◽

Zev Bryant ◽

James A. Spudich

Keyword(s):

Myosin Vi ◽

Tail Domain ◽

Processive Stepping

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Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion

Cells ◽

10.3390/cells9071673 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1673

Author(s):

Lilya Lehka ◽

Małgorzata Topolewska ◽

Dominika Wojton ◽

Olena Karatsai ◽

Paloma Alvarez-Suarez ◽

...

Keyword(s):

Focal Adhesion ◽

Cellular Localization ◽

Fold Increase ◽

Time Dependent ◽

Myoblast Differentiation ◽

Focal Contact ◽

Myosin Vi ◽

Cytoskeleton Organization ◽

Hindlimb Muscles ◽

Primary Myoblast

We have previously postulated that unconventional myosin VI (MVI) could be involved in myoblast differentiation. Here, we addressed the mechanism(s) of its involvement using primary myoblast culture derived from the hindlimb muscles of Snell’s waltzer mice, the natural MVI knockouts (MVI-KO). We observed that MVI-KO myotubes were formed faster than control heterozygous myoblasts (MVI-WT), with a three-fold increase in the number of myosac-like myotubes with centrally positioned nuclei. There were also changes in the levels of the myogenic transcription factors Pax7, MyoD and myogenin. This was accompanied by changes in the actin cytoskeleton and adhesive structure organization. We observed significant decreases in the levels of proteins involved in focal contact formation, such as talin and focal adhesion kinase (FAK). Interestingly, the levels of proteins involved in intercellular communication, M-cadherin and drebrin, were also affected. Furthermore, time-dependent alterations in the levels of the key proteins for myoblast membrane fusion, myomaker and myomerger, without effect on their cellular localization, were observed. Our data indicate that in the absence of MVI, the mechanisms controlling cytoskeleton organization, as well as myoblast adhesion and fusion, are dysregulated, leading to the formation of aberrant myotubes.

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Single-Molecule and Molecular Dynamics Study of the Dimerization of Myosin VI Medial Tail Domain

Biophysical Journal ◽

10.1016/j.bpj.2009.12.3969 ◽

2010 ◽

Vol 98 (3) ◽

pp. 724a

Author(s):

HyeongJun Kim ◽

Jen Hsin ◽

Yanxin Liu ◽

Monalisa Mukherjea ◽

Daniel Safer ◽

...

Keyword(s):

Molecular Dynamics ◽

Single Molecule ◽

Myosin Vi ◽

Tail Domain

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A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle

10.1101/600841 ◽

2019 ◽

Author(s):

Glenda Comai ◽

Églantine Heude ◽

Sebastien Mella ◽

Sylvain Paisant ◽

Francesca Pala ◽

...

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Striated Muscle ◽

Striated Muscles ◽

Loss Of Function ◽

Pharmacological Studies ◽

Posterior Migration ◽

Functional Relevance ◽

Genetic Signatures ◽

Genetic Hierarchy

SUMMARYIn most vertebrates, the upper digestive tract is composed of muscularised jaws linked to the esophagus that permit food uptake and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to Met/HGF signalling for antero-posterior migration of esophagus muscle progenitors, where HGF ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulate esophagus myogenesis and identify distinct genetic signatures that can be used as a framework to interpret pathologies arising within CPM derivatives.

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