Cloning and characterization of a novel gene, striamin, that interacts with the tumor suppressor protein p53

Expression analysis of a novel cDNA isolated from immortal murine fibroblasts revealed a single transcript of 3.0 kilobase pairs that was highly expressed in mouse and human striated muscle and in mouse heart. The gene has therefore been named striamin. Its expression was confined to skeletal muscle types with a fast glycolytic (2B) contractile phenotype. It was also detected in C2C12 mouse myoblasts and was down-regulated during in vitro myogenesis. The cDNA has a single open reading frame encoding a predicted 16.8-kDa protein of 149 amino acids with no homology to known proteins. Microinjection and transfection of green fluorescence protein-tagged striamin demonstrated that it localizes to the nucleus. Coimmunoprecipitations revealed that it can interact with p53 (a positive marker for myoblast differentiation) in vivo and in vitro. Furthermore, it repressed p53 activity in p53-mediated reporter assays. Fluorescence in situ hybridization with a mouse P1 genomic clone localized the gene to chromosome 12C3, which is syntenic to human chromosome 14q21-22

Skeletal muscle repair in a mouse model of nemaline myopathy

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.Despina Sanoudou, Mark A. Corbett, Mei Han, Majid Ghoddusi, Mai-Anh T. Nguyen, Nicole Vlahovich, Edna C. Hardeman, and Alan H. Begg

Reappearance of the minor alpha-sarcomeric actins in postnatal muscle.

The postnatal expression profiles of alpha-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. alpha-Cardiac actin (alpha-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching approximately 90% that found in the adult mouse heart. Although alpha-CA is the alpha-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. alpha-Skeletal actin (alpha-SA) transcripts accumulate to approximately 32% of the total alpha-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of alpha-CA and alpha-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor alpha-sarcomeric actins

Skeletal muscle of mice with a mutation in slow ¿-tropomyosin is weaker at lower lengths

Skeletal muscle function was measured in anaesthetised transgenic mice having a mutation in the TPM3 gene (slow α-tropomyosin), a similar mutation as found in some patients with nemaline myopathy, and was compared with control muscles. Measurements of isometric and dynamic muscle performance were done with electrical nerve stimulation at physiological temperatures. No muscle weakness was found in the transgenic muscles when performance was measured at muscle optimum length. This was true not only with full activation but also at lower activation levels, indicating that calcium sensitivity was not affected at this length. Also, fatigability was not affected in these conditions. However, isometric force of the muscles with the mutation in TPM3 was lower at lengths below optimum, with more impairment at decreasing length. As the muscles are active over a large range of different muscle lengths during daily activities, this finding may explain, at least in part, the muscle weakness experienced by patients with nemaline myopathy. © 2002 Elsevier Science B.V. All rights reserved

Epidermal YAP2-5SA-DeltaC drives beta-catenin activation to promote keratinocyte proliferation in mouse skin in vivo

The epidermis is a highly regenerative tissue. YAP is a pivotal regulator of stem/progenitor cells in tissue regeneration, including in the epidermis. The molecular mechanisms downstream of YAP that activate epidermal cell proliferation remain largely unknown. We found that YAP and β-catenin co-localize in the nuclei of keratinocytes in the regenerating epidermis in vivo and in proliferating HaCaT keratinocytes in vitro. Inactivation of YAP in HaCaT keratinocytes resulted in reduced activated β-catenin and reduced keratinocyte numbers in vitro. In addition, we found that in the hyperplastic epidermis of YAP2-5SA-ΔC mice, the mutant YAP2-5SA-ΔC protein was predominantly localized in the keratinocyte nuclei and caused increased expression of activated nuclear β-catenin. Accordingly, β-catenin transcriptional activity was elevated in the skin of live YAP2-5SA-ΔC/TOPFLASH mice. Lastly, loss of β-catenin in basal keratinocytes of YAP2-5SA-ΔC/K14-creERT/CtnnB1(-/-) mice resulted in reduced proliferation of basal keratinocytes and a striking rescue of the hyperplastic abnormalities. Taken together, our work shows that YAP2-5SA-ΔC drives β-catenin activity to promote basal keratinocyte proliferation in the mouse skin in vivo. Our data shine new light on the etiology of regenerative dermatological disorders and other human diseases that display increased YAP and β-catenin activity.Bassem Akladios, Veronica Mendoza-Reinoso, Michael S. Samuel, Edna C. Hardeman, Kiarash Khosrotehrani, Brian Key and Annemiek Beverda

Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice

Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct–RFP and of Tpm3.1–mNeonGreen on secretory granules in LifeAct–RFP transgenic, Tpm3.1–mNeonGreen and myosin IIA–GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment.Andrius Masedunskas, Mark A. Appaduray, Christine A. Lucas, María Lastra Cagigas, Marco Heydecker, Mira Holliday, Joyce C.M. Meiring, Jeff Hook, Anthony Kee, Melissa White, Paul Thomas, Yingfan Zhang, Robert S. Adelstein, Tobias Meckel, Till Böcking, Roberto Weigert, Nicole S. Bryce, Peter W. Gunning, and Edna C. Hardema

Visuomotor control of walking in Parkinson’s disease: Exploring possible links between conscious movement processing and freezing of gait

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordIntroduction Changes in visual attention have been argued to influence freezing of gait (FOG) in people with Parkinson’s Disease (PD). However, the specific visual search patterns of people with FOG pathology (PD + FOG) and potential underlying mechanisms are not well understood. The current study explored visual search behavior in PD + FOG while walking on a pathway featuring environmental features known to exacerbate FOG (e.g., narrow doorway and tripping hazards). Potential attentional underpinning mechanisms were also assessed, such as conscious movement processing. Methods Visual search behavior of twelve people with PD + FOG tested in ON-state (Mage = 74.3) and twelve age-matched healthy controls (Mage = 72.5) were analysed during a complex walking task. The task required participants to step over an obstacle and navigate through a narrow doorway, surrounded by clutter. Results People with PD + FOG more frequently directed visual attention to ongoing and imminent steps compared to healthy controls (Mdn = 26% vs Mdn = 14%, respectively; p = 0.042). Self-reported conscious movement processing was also significantly higher in people with PD+FOG. The one participant who froze during the walking task fixated the future trip hazard (obstacle, approximately 6 steps ahead) almost exclusively during freezing trials (i.e., 60-100% of the trial). In contrast, during ‘non-freeze’ trials, this participant increased the duration of fixations towards ongoing and imminent steps. Conclusion Results suggest that people with PD + FOG strongly monitor/control ongoing and immediately upcoming stepping movements. However, prolonged fixations towards threats to future movements might prevent people with PD + FOG from processing the visual information needed to do this, thereby provoke freezing episodes