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Meet the team
Helena Vadnais
Marissa Nelson
Christian Donnelly
Research Question
Research Question
What is the expression and localization of alpha-actin and beta-actin during the days of differentiation of C2C12 myoblasts?
Rationale
Rationale
Previous research has shown that there are relative expression differences between actin isoforms in C2C12 myoblasts during myogenesis. As myotubes continue to mature and nucleation increases throughout differentiation, alpha-actin expression is expected to increase and help facilitate cytoskeletal modifications required for contractile movement (Medline, 2020). In contrast, beta-actin is expected to have a higher expression during the early stages of myogenesis since it is involved in the motility and structure of the myoblast (Dugina et al., 2009). In this study, we aim to identify the expression and localization in alpha- and beta-actin throughout myogenesis by common techniques of immunocytochemistry (ICC) and immunofluorescence (IF) microscopy. Alpha-actin, along with myosin, will form the contractile units within cells known as sarcomeres, which develop at later time points of differentiation. In contrast, beta-actin is a housekeeping protein that is abundant prior to differentiation and is used in the motility and structural determination of the precursor cells. What remains less clear are the patterns of expression at different time points throughout myogenesis. We aim to use ICC and IF techniques at various time points throughout cell differentiation (from before differentiation at the myoblast (MB) phase to Day 11 of cell-to-cell contact). We can identify cell fusion through DIC (Differential Interference Contrast) imaging with DAPI overlay to characterize morphology and multinucleation of the myotubes at each time point. By using qPCR techniques we will gather relative gene expression data on the isoforms of alpha-actin, beta-actin, and myosin. We will be able to then identify the points of interest to where the genes of interest are most expressed during the differentiation stages of cell development. This research may provide important information about how these proteins may impact differentiation and could inform future experiments investigating sarcomere formation.
Image found https://drive.google.com/drive/folders/1929GohZXPEY03Gps2J3P0AtIYRkLM5-X
Background
Background
Myogenesis is the development and differentiation of skeletal muscle cells from the myoblast into myotubes. This differentiation from mononucleated to multinucleated cells displays a morphological change from star-shaped to a long, rectangular shape. There are MRFs (myogenic regulatory factors) that are proteins responsible for inducing the expression of proteins such as alpha-actin (Burattini et al., 2004, p. 223). Beta-actin is highly expressed in myoblasts and decreases in the early stages of differentiation. In contrast, we see a higher expression of alpha-actin, after cell-to-cell contact, during the later stages of myotube maturation.
Actin is one of the most abundant proteins that are found within the majority of cells (Lee, 2013, p. 3). The cytoskeletal structures provide stability and motility will vary in shape and size, but overall, they play a significant role in structural determination (Lee, 2013, p. 3). Actin filaments have bound myosin proteins that allow the contractile movement of the muscle cells, providing they are more expressed in myotubes (Lee, 2013; Medline, 2020). The cooperation of actin and myosin is facilitated by the use of sarcomeres, a basic unit of muscle fibre that has the role of providing the muscle with contractions (Mansfield & Neumann, 2019). This relationship with actin and myosin creates the necessary mechanics for the differentiation of cell shape and migration (Lee, 2013, p. 17).
Image found:https://experiment.com/u/qlgq5A
Beta-actin plays a role with cytoskeletal actin filaments involving itself in mobility and structure (Dugina et al., 2009), allowing it to be visualized more so in the myoblast stage. The mechanisms that are involved with actin polymerization demonstrate an influence on cell migration, and where overexpression of actin proteins can affect cells (Artman, et al., 2014). For example, the upregulation of B-actin may increase cell movement (Artman, et al., 2014). If beta-actin has this effect on the motility of cells, this is a good reason why we would expect to see more beta-actin during early myogenesis. This expression allows the myoblast cells to perform cell division and move closer together to reach cell-to-cell contact, thus, initiating the next phase into myotubes.
Image found:https://www.sinobiological.com/resource/beta-actin/proteins#pid=1
Alpha-actin comes in two forms; cardiac and skeletal. These actin isoforms are of great importance to the study of myogenesis because alpha-actin has the crucial role of forming thin filaments that interreact with the myosin based thick filaments to form sarcomeres. Sarcomeres are the basic contractile unit of muscles cells. Sarcomeres contract by the myosin thick filament pulling on actin filaments running in opposite direction as illustrated in the diagram to the right. muscle contraction is made possible by synchronized contraction of numerous sarcomeres. The two alpha-actin isoforms are each the predominate actin isoform in cardiac and skeletal muscle tissue respectively.
Image found:https://opentextbc.ca/biology/chapter/19-4-muscle-contraction-and-locomotion/
Experimental Design
Experimental Design
Our project consists of culturing C2C12 myoblasts within a 10% Fetal Bovine Serum (FBS) until 80% (for our myoblast fixation stage) and 100% confluency is achieved (full coverage of the growth surface). Once the desired confluency is met, the culturing media is changed and upkeep to maintain cell growth for the individual 2 week trial periods (a total of three trials). The methodologies of fixation and permeabilization were completed on different differentiation phases (myoblast, Day 0, 4, 7, 11). By using the techniques of immunocytochemistry and microscopy, the targets of alpha- and beta-actin within the C2C12 cells are to be identified during differentiation time points of myogenesis. The sub-targets of myosin and phalloidin are used as placeholders to better understand the localization of the isoforms of actin when looking at them separately.
Performing qPCR we will be able to gather the relative gene expression for our target groups of a-skeletal, a-cardiac, beta-actin and myosin. The relative gene expression data collected from sample day myoblast, and days 0, 5, 7, and 12 (for a total of 3 trials) will present the overall expression of the target genes within the stages of myogenesis. Preparation techniques for qPCR include reverse transcriptase for cDNA synthesis and primer efficiency to test target gene primers before qPCR. RNA analysis techniques included Nanodrop, Qubit BR, Qubit IQ, and gel electrophoresis.
Relative Gene expression
Relative Gene expression
qPCR data present the relative gene expression of the target genes of alpha-actin (cardiac and skeletal), beta-actin and myosin.
The relative gene expression between the genes of interest and the reference gene will provide data on the physiological changes within the gene expression levels within the sampling days of C2C12 cells.
The standard deviation for each is calculated according to the total trials (n=3) and the specific sample days (MB, Day 0, 5, 7, 12).
Using the Pfaffl method the data was normalized to the MB day, as it is the initial day of sample collection and the cells have not reached cell-to-cell contact (~70-80% confluency).
Genes of interest:
Skeletal alpha-actin
Cardiac alpha-actin
Beta-actin
Myosin - myh4
Reference gene: GAPDH
Calibrator is the technical triplicate and the test is the reference (GAPDH) triplicate Cq mean values.
Primer Efficiency (E) = (10 ^ ( -1 / slope ) ) - 1
Primer efficiency was done for all the genes of interest to test the quality of the primers used. Expected efficiency is 90-110%.
Relative mRNA gene expression of myosin (myh4) marker over the course of myogenesis determined by qPCR. The myoblast (MB) expression level is normalized to 1, while all other sampling days (D0, D5, D7, and D12)(n=3) are reported as fold changes relative to the MB sample (n=2). There is a rising increase of myh4 from MB to Day 12, expression of myosin is largely expressed in the final days of myogenesis (Day 12).
SD' (+/-): MB (1.48E+01), Day 0 (1.01E+01), 5 (5.49E+00), 7 (5.87E+00), 12 (6.55E-01)
Relative mRNA gene expression of β-actin markers over the course of myogenesis determined by qPCR. Each graph shows relative mRNA gene expression of the gene of interest β actin. The myoblast (MB) expression level is normalized to 1, while all other sampling days (D0, D5, D7, and D12) are reported as fold changes relative to the MB sample (n=2).
SD' (+/-): MB (1.25E+00), Day 0 (1.10E+00), 5 (1.75E+00), 7 (1.57E+00), 12 (9.61E+00)
Relative mRNA gene expression of ɑ-cardiac actin and ɑ-skeletal actin over the course of myogenesis is determined by qPCR. Each graph shows relative mRNA gene expression of the gene of interest: A) ɑ-cardiac and B) ɑ skeletal actin. The myoblast (MB: n=1) expression level is normalized to 1, while all other sampling days D0, D5, D7, and D12 are reported as fold changes relative to the MB sample (n=3 for cardiac and n=1 for skeletal).
ɑ-Cardiac actin standard deviation. SD' (+/-): MB (9.48E+00), Day 0 (6.25E+00), Day 5 (4.89E+00), Day 7 (3.28E+00), and Day 12 (1.51E+00)
ɑ-Skeletal actin standard deviation. SD' (+/-): MB (7.27E-01), Day 0 (1.03E+00), Day 7 (4.52E-01), and Day 12 (7.78E-1).
Target groups
Target groups
We are focusing on 3 target groups within our experiment: alpha-actin (cardiac and skeletal), beta-actin, and myosin.
Alpha-actin and myosin form contractile sarcomeric units, so we theorize that these markers will present themselves in the later-stage differentiation. Interestingly, the data collected from ICC imaging indicated that there are two types of alpha-actin present within the differentiating cells. A recent development suggested that the alpha-actin antibodies used were not specific to either subtype. This data supports preliminary evidence that both cardiac-alpha actin and skeletal-alpha actin localize within the c2c12 cells, thus, alpha-actin is evident at stages of differentiation (Bains et al., 1984). Alpha-actin and beta-actin are our second group. The colocalization of alpha- and beta-actin allows us to visualize these targeted proteins at each stage of n differentiation. Looking at the actin isoforms together over a two-week growth period we can identify at what point the two targets present themselves during cell differentiation. It is theorized that beta-actin will appear within the earlier stages and the alpha-actin will follow in the ladder once the myoblasts reach 100% confluence and transition to myotubes.
Finally, beta-actin and phalloidin are used together to allow us to confirm expression patterns of filamentous actin throughout myogenesis as shown in prior student research, and allow us to gain insight into how much of that signal is due to beta-actin. Phalloidin is used because it is to stain all forms of actin filaments present within the cell (ie. F-actin) (ThemoFisher, 2006). Along with the staining of all actin forms, beta-actin would be included, thus presenting in the data an overlapping is the antibody targeting and phalloidin staining.
So, by putting both together in our immunofluorescence staining protocol we should expect to see the two types at their designated stages. Interestingly, it was found that alpha-actin was localizing throughout all differentiating days. Indicating that there was more than one type of alpha-actin present within the immunofluorescence microscopy imaging. With further investigation, two forms of alpha-actin were determined present: cardiac-alpha and skeletal-alpha actin.
Conclusion
Conclusion
Expression patterns of myosin are consistent with known literature. However, as myosin is present within all differentiation stages the data suggest a higher expression of myh4 is in the later stages of myogenesis, more specifically, day 12.
Expression patterns of ɑ cardiac and ɑ skeletal actin are indistinguishable due to the type of antibody used, but relative gene expression shows different levels at various stages of myogenesis. Cardiac actin was more so expressed on sampling days 5-12, whereas skeletal actin was more expressed on days 7 and 12.
Preliminary data of beta-actin expression was profoundly expressed within the earlier stages of development.
Preliminary data provided that the target genes are pronounced within different developmental stages of myogenesis, aligning with our initial theory.
The identification of expression and localization of the actin filaments can provide further knowledge of where sarcomeric formation occurs at which time point in myogenesis.
Image found: https://www.researchgate.net/figure/Myogenic-lineage-progression-and-expression-profile-of-key-myogenic-regulators-a_fig1_332352650
Future Directions
Future Directions
Further investigation into the localization and relative gene expression of the actin isoforms and myosin, can lead to a better comprehension of sarcomere development during myogenesis.
Image found: https://www.britannica.com/science/sarcomere
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