Embedded Files
Will a coated surface affect the expression of myosin and alpha actin in C2C12 mouse myoblasts as the cells differentiate?
Emily Wong (EW) and Ian Parsons (IP), hard at work in the lab to bring you this data.
Laminin-111 complex by Gad Armony - Own work, CC BY-SA 4.0, via Wikimedia Commons
The ECM protein laminin (LM) is a major component of basement membranes which, through integrins and the dystrophin-associated glycoprotein complex, connects to skeletal muscle fibers (Holmberg & Durbeej, 2013). LM has been shown to have a role in muscle cell signaling (Homberg & Durbeej, 2013) and the promotion of myoblast motility (Sanes, 2003), fusion, and differentiation (Csapo, 2020).
Laminin, pictured left, interacts with integrins embedded in the cell membrane via the C-terminal LM globular (LG) domain of the α arm (Hohenester, 2019). The protein additionally binds to other ECM components and proteins, such as collagen, with the arms of its β and γ chains. LM is important for transmitting extracellular signals through the cell membrane. Myogenic cells have critical interactions with LM at every stage of differentiation (Bajanca et al., 2006).
This structure and role in mind, we examined what effect a coating of laminin would have on developing C2C12 muscle cells. Our evidence, combined with other literature, backs the utility of LM coatings in in vitro muscle cell growth and proliferation.
Graphical Hypothesis, made in Clip Studio Paint, 2022, EW.
RESULTS
Day 12 Cells on LM Coating
Phase Contrast Microscopy, EW, 2022
Cells grown on laminin coatings grew in more organized aligned patterns and with more consistent size than those grown on uncoated dishes.
Day 12 Cells on Control Plate
Phase Contrast Microscopy, EW, 2022
Myoblast
Day 0
Day 4
Day 7
Day 11
Morphology throughout myogenesis of C2C12 cells grown on laminin coated or uncoated coverglasses, Myoblast to Day 11.
Control (Top, 2021, IP), LM (Bottom, 2021, EW)
Differential interference contrast (DIC) and DAPI fluorescent microscopy using Olympus BX53 at 400x magnification. Images taken using Olympus XM10; merged and coloured in CellSens. Timeline of C2C12 morphology course. Cells pictured in the bottom row were plated onto coverglasses coated in laminin solution, control group (top row) was plated onto uncoated glass coverslips. Cells were fixed at the myoblast stage two days post-plating, day 0 at 100% cell confluency three days post plating, days 4, 6, and 11.
Cells in both groups displayed appropriate maturation markers for each day - star shaped at MB, touching at D0, fusing between D4-D7, and multinucleation at D11-12.
Myoblast
Day 0
Day 4
Day 7
Day 11
Immuno-cytochemistry imaging of sarcomeric α-actin and myosin distribution during myogenesis of C2C12 cells grown on laminin coated coverglasses, from Myoblast to Day 11.
DNA (Blue), Myosin (Green), α-Actin (Red), Control (Top, 2021, IP), LM (Bottom, 2022, EW)
Primary antibody labelled sarcomeric α-actin on TritC at 175ms (red), primary antibody labelled myosin on FitC at 100ms (green), and DAPI at automatic exposure (blue). Imaged using Olympus BX53 at 600x magnification, 10.2 gain, with Olympus XM10 camera, and merged and coloured using CellSens. C2C12 cells grown on glass coverslips with laminin coating (bottom) and without (top). ICC negative control samples incubated without primary antibody.
Myosin and Actin increase in visibility and distribution as days progress. On day 11 the LM cells show individual myosin molecules visible in alignment with actin.
Myosin mRNA fold change measured by qPCR, throughout myogenesis for laminin coated and uncoated plates from MB to Day 12. (EW)
RNA extracted from C2C12 cells at MB, day 0 at 100% confluency, days 5, 7, and 12. qPCR performed on 1uL of 3.3ng/uL cDNA created from 100ng RNA. Relative gene expression calculated using Pfaffl method, data normalized to GAPDH mRNA, calibrated to control MB = 1. Each sample performed in triplicate shown as mean +/- SEM, (n=3). ANOVA Two-factor, P<0.05.
Myosin mRNA expression hit its peak at day 7 for both control and laminin groups. Laminin coating resulted in significant increase in myosin mRNA expression.
Myosin mRNA fold change measured by qPCR, throughout myogenesis for laminin coated and uncoated plates from MB to Day 12, y axis max set to 350 fold.
Figure 4. Gene expression of Skeletal α-Actin with qPCR
Conclusions
Conclusions
LM Enhanced Cell Organization
In Laminin the C2C12 cells had an apparent polarized alignment compared to the control with a distribution that curved in the petri dish.
LM Enhanced Myosin and Actin Localization
LM Enhanced Myosin mRNA Expression
Consistent with literature which shows laminin coatings increase myosin mRNA expression (van Dijk et al., 2008). Likely due to laminin's role in in vivo cell maturation.
Skeletal α-Actin mRNA expression was not significantly impacted by LM
Potentially due to other sources of actin, such as remodeling of isoforms, as the ICC imaging showed differences in actin appearance.
What Comes Next?
What Comes Next?
Further investigation into actin isoforms as antibodies used were specialized for one isoform of alpha-actin
Analysis of protein levels, mRNA of other actin isoforms, or use of more specific antibodies in ICC.
Longer maturation
Growing cells until they form proper sarcomeres to see a fuller picture of muscle development under LM.
Comparison to in vivo conditions
Analysis of animal cell vs. cultures to see if ECM coatings created a more physiologically relevant environment.
References
References
Bajanca, F., Luz, M., Raymond, K., Martins, G. G., Sonnenberg, A., Tajbakhsh, S., Buckingham, M., & Thorsteinsdóttir, S. (2006). Integrin α6β1-laminin interactions regulate early myotome formation in the mouse embryo. Development, 133(9), 1635–1644. https://doi.org/10.1242/dev.02336
Csapo, R., Gumpenberger, M., & Wessner, B. (2020). Skeletal muscle extracellular matrix–what do we know about its composition, regulation, and physiological roles? A narrative review. Frontiers in physiology, 11, 253. https://doi.org/10.3389/fphys.2020.00253
Hohenester E. (2019). Structural biology of laminins. Essays in biochemistry, 63(3), 285–295. https://doi.org/10.1042/EBC20180075
Holmberg, J., & Durbeej, M. (2013). Laminin-211 in skeletal muscle function. Cell adhesion & migration, 7(1), 111–121. https://doi.org/10.4161/cam.22618
Sanes, J. R. (2003). The basement membrane/basal lamina of skeletal muscle. Journal of Biological Chemistry, 278(15), 12601-12604. https://doi.org/10.1074/jbc.R200027200
van Dijk, A., Niessen, H. W. M., Zandieh Doulabi, B., Visser, F. C., & van Milligen, F. J. (2008). Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin. Cell and Tissue Research, 334(3), 457–467. https://doi.org/10.1007/s00441-008-0713-6
Page updated
Report abuse