Publication date: 25th July 2016
Introduction: Actin fibers are essential for cellular survival, development and many physiological process. They can be present as thick and stable structures in non-migrating cells or thinner and dynamic filaments in migrating ones1. Understanding how these fibers influence endothelial cells’ dynamics will help to improve the knowledge of blood vessels' formation and the maintenance of their integrity. The goal is to correlate cellular dynamics with an intact or disrupted actin cytoskeleton.
Methodology: Hydrogels of polyethylene glycol (PEG; 4-arms, 20 kDa, vinyl sulfone terminus) conjugated with RGD (200 µM, Ac-GCGYGRGDSPG-NH2) and crosslinked by DL-Dithiothreitol were produced with or without fluorescent beads dispersed in the PEG matrix. The hydrogels had a Young’s Modulus of 1 and 2.5 kPa. Human Umbilical Vein Endothelial Cells (HUVECs) transduced by LifeAct adenoviral vectors were grown in endothelial cell growth medium and seeded on the hydrogels. Experiments started after the cells were attached and spread by incubating the cells with or without 0.1 µM Cytochalasin D (CytoD), an inhibitor of actin polymerization, for up to 6 more hours. Confocal laser-scanning microscopy was used to track migrating cells and visualize the beads in their stressed and relaxed state. Obtained data were analyzed by MatLab.
Results: Both with and without CytoD, HUVECs on 2.5 kPa hydrogels presented a higher fluorescent signal, suggesting more or thicker actin fibers were present, and higher traction magnitudes mostly located at the termination of ventral and dorsal fibers. CytoD was able to disrupt the actin cytoskeleton with a faster effect on cells on the 1 kPa hydrogels. After 60 minutes of treatment, these cells started to reorganize actin-rich zones on the membrane borders and above the nucleus, which allowed the emission of filopodia with an energy cost about 20 times smaller than before. Comparing cells treated with CytoD to control cells, the speed during migration was increased 3 and 2 times and the persistence 16 and 3 times for 1 and 2.5 kPa hydrogels, respectively.
Conclusions: The present work identified and tracked different types of actin stress fibers along with cellular behavior, such as migration speed and persistence. Future work will quantify the tractions produced by these different fibers in the same cell, their resistance to disruption, their influence on cell morphology during migration and the force exerted under non-favorable conditions of polymerization (CytoD effect).
Acknowledgements: The European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ ERC Grant Agreement n° 308223) and the Brazilian Agency CNPq for financial support.
References: [1] Pellegrin S. and Mellor H.J. Cell. Sci. 120, 3491, 2007.
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