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Meet the team
Meet the team
Brandee Tucker
Haley Leavitt
Nicholas R Pannell
Hazel Macaldo
Emily Howard
Emily Wong
Ian Parsons
Research Question
Research Question
What effect will extracellular matrix protein-coated growth surfaces have on differentiation in a cell culture model of myogenesis?
What effect will extracellular matrix protein-coated growth surfaces have on differentiation in a cell culture model of myogenesis?
Rationale
Rationale
C2C12 cells cultured in vitro deviate from their in vivo counterparts. For example, myotubes form in random arrangements rather than aligned bundles. We hypothesize that this is in part due to the lack of the extracellular matrix (ECM), which cells in vivo naturally grow within. The ECM provides structural support and facilitates cell signaling. By coating our growth plates with various ECM proteins - laminin, gelatin, and fibronectin - we hope to produce a better model of the physiological system.
C2C12 myoblasts proliferate easily and differentiate quickly, and thus are used as a model to study myogenesis progression. Our goal is to observe the progression of myogenesis by noting changes in cell morphology, nucleation and mRNA expression.
Alpha-actin and myosin are proteins which form the contractile unit known as sarcomeres and are thus late-stage markers of myogenesis. Using immunocytochemistry we will visualize the localization of alpha-actin and myosin. The quality and alignment of myotubes will be compared between cells grown on slides coated with laminin, gelatin, fibronectin, and no substrate. Real time qPCR comparisons will be used to observe if ECM proteins contribute to changes in alpha- actin and myosin mRNA expression.
Background
Background
Myogenesis is the process of muscle development. Skeletal muscle development is divided into multiple events starting from the migration and proliferation of precursor muscle cells known as myoblasts. Once the cells reach 100% confluency, the cell cycle is terminated and the cells begin differentiating into mature muscle fibers known as myotubes. Along with morphological changes, differentiation of muscle cells changes the gene expression from a non-contractile protein, beta-actin, to major contractile proteins called ɑ-actin and myosin (Cox et al., 1990). Physiologically, muscle cells grow in a parallel manner due to extracellular matrix proteins (ECM) such as collagen, laminin and fibronectin surrounding the muscle fibers (Grzelkowska-Kowalczyk, 2015). The ECM provides a mechanical structure by regulating the formation of fibrillar network and increasing muscle tissue elastic properties during myofibril contraction (Goncalves et al., 2019). Typically, muscle cells are cultured in vitro on smooth pyrex plates, which generally results in a random distribution of myotubes. Therefore, the results produced by these models may not be fully representative of physiological myogenesis. In an attempt to simulate in vivo myogenesis conditions, ECM coatings are added during cell plating to provide an adhesive substrate for each cell to bind to (Goncalves et al., 2019).
Sarcomeres
Sarcomeres
In mature muscle cells, alpha-actin and myosin form sarcomeres which are responsible for muscle contraction. The mechanism of contraction follows a sliding-filament model where actin filaments slide past myosin filaments towards the middle of the sarcomere, shortening the sarcomere without any change in filament length (Cooper, 2000).
https://www.researchgate.net/profile/Siobhan-Loughna/publication/230810761/figure/fig1/AS:202680053047298@1425334098050/Schematic-representation-of-a-sarcomere-The-thick-and-thin-filaments-overlap-in-the.png
Actin Filaments
Actin Filaments
Actin filaments have versatile applicability to many different ECM-related structures and associated functions. Of particular relevance to the alpha-actin isoforms discussed in the current work, actin filament bundling and stabilization (by tropomyosin) is the inherent process preceding the structural assembly of the actin macromolecular substituents, depicted in alpha-actin microscopy.
In the central portion of the actin image panel below, the process of treadmilling, whereby a net disassembly and assembly, from plus and minus ends of the actin filament (respectively) are observed. That is, although a given actin microfilament may appear to have a constant length, the continuous flux of polymerizing and depolymerizing subunits leads to a relative net change which can be deemed to be negligible (i.e. 0).
Actin filaments participate in many dynamic functional assemblies within the context of even just a single cell (Alberts, B., et al., 2014, pp.905).
Common actin arrays in the intracellular distribution of actin microfilaments is illustrated above (Alberts, B., et al., 2014, pp.911). Filipodium and lamellipodium actin-rich extensions of the cell membrane are frequently visible across all culture conditions, regardless or experimental and control conditions (albeit, in healthy C2C12 myocytes).
SARCOMERIC ALPHA-ACTIN
Localization of alpha-actin and nuclei of Day 7 C2C12 myotubes. Image captured using OLYMPUS IX51 IF microscopy at 600x magnification. Images combined and captured using cellSense 1.16 software. Cells stained with Sarcomeric alpha Actin Monoclonal (red) and DAPI (blue). Large multinucleated myotubes are visible with alpha-actin localized outside of the nucleus revealing the elongated morphology.
Actin is an important protein for many various cellular processes, for example, beta-actin is abundant in the cytoskeleton and functions in cell motility, whereas alpha-actin forms the contractile sarcomeres. As myoblasts begin to fuse and differentiate into myotubes beta-actin transcription is ceased while alpha-actin transcription begins, thus making alpha-actin a useful marker in late myotube maturation (Cox et al., 1990)
MYOSIN
Myosin and nuclei of C2C12 myotubes aged 7 days. Image created using with OLYMPUS IX51 at x600 magnification. coloured overlay was made using cellsense 1.16 with (Green) being the nuclei stained with DAPI and (Pink) being myosin stained with Alexa 488 anti-rabbit.
Myosin is a motor protein that binds onto the actin filament through the use of ATP making it a mechanochemical enzyme. There are 13 different forms of myosin with myosin 1 and 2 being the most abundant and are found in all eukaryotic cells. Myosin 2 is responsible for the budding of splitting daughters cells and muscle contraction. Myosin 1 and 5 are involved in the transportation of vesicles using the cell membrane and cytoskeletal filaments. (H.L, 2000)
By analyzing the localization of expression of myosin and alpha-actin during the various stages of myogenesis, it is possible to infer whether extracellular protein coatings have an impact on the maturation of C2C12 skeletal muscle cells. Analyzing the morphological changes during cell proliferation, migration, fusion, and maturation will allow us to determine if the specific extracellular coatings provide a more physiologically relevant model.
Experimental design
Experimental design
In the first half of our project, each partnership studied the effect of a different extra-cellular matrix protein (fibronectin, gelatin, or laminin) on cell morphology and multinucleation. Relative expression and localization of alpha-actin & myosin was observed using fluorescence microscopy and morphology was observed with DIC. This diagram explains the cell culturing, fixation, immunocytochemistry, and microscopy processes involved. The second half of our project was to determine the relative gene expression of both late stage markers during myogenesis on uncoated and ECM coated plates. Using GAPDH as a reference gene, the relative gene expression was calculated via the Pfaffl method, and compared among groups. This diagram explains the cell culturing, RNA extraction, purification, quality, validation, creation of the cDNA library and real time qPCR processes involved.
Target Subgroups
Target Subgroups
Diagram of ECM proteins and the cell membrane, made in Clip Studio Paint, Emily Wong, 2022
References
References
Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2014). Molecular Biology of the Cell (Sixth ed.). W. W. Norton & Company.Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Actin, Myosin, and Cell Movement. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9961/Cox, R. D., Garner, I., & Buckingham, M. E. (1990). Transcriptional regulation of actin and myosin genes during differentiation of a mouse muscle cell line. Differentiation: Ontogeny and Neoplasia.Gonçalves, T., Boutillon, F., Lefebvre, S., Goffin, V., Iwatsubo, T., Wakabayashi, T., Oury, F., & Armand, A. S. (2019). Collagen XXV promotes myoblast fusion during myogenic differentiation and muscle formation. Scientific reports, 9(1), 5878. https://doi.org/10.1038/s41598-019-42296-6
Grzelkowska-Kowalczyk, K. (2015). The importance of extracellular matrix in skeletal muscle development and function. In F. Travascio (Ed.), Composition and function of the extracellular matrix in the human body. IntechOpen. https://doi.org/10.5772/62230 H, L., & SL, Z. (2000). Section 18.3 Myosin: The Actin Motor Protein. In B. A (Ed.), Molecular Cell Biology (4th ed.). essay, NCBI.Rebuzzini, P., Cebral, E., Fassina, L., Alberto Redi, C., Zuccotti, M., & Garagna, S. (2015). Arsenic trioxide alters the differentiation of mouse embryonic stem cell into cardiomyocytes. Scientific Reports, 5(1). https://doi.org/10.1038/srep14993
Grzelkowska-Kowalczyk, K. (2015). The importance of extracellular matrix in skeletal muscle development and function. In F. Travascio (Ed.), Composition and function of the extracellular matrix in the human body. IntechOpen. https://doi.org/10.5772/62230 H, L., & SL, Z. (2000). Section 18.3 Myosin: The Actin Motor Protein. In B. A (Ed.), Molecular Cell Biology (4th ed.). essay, NCBI.Rebuzzini, P., Cebral, E., Fassina, L., Alberto Redi, C., Zuccotti, M., & Garagna, S. (2015). Arsenic trioxide alters the differentiation of mouse embryonic stem cell into cardiomyocytes. Scientific Reports, 5(1). https://doi.org/10.1038/srep14993
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