Year: 2019
Type: dissertation
Abstract: Embryos repair epithelial wounds rapidly, in a process driven by collective cell behaviors. Upon wounding, filamentous actin and the motor protein non-muscle myosin II polarize in the cells adjacent to the wound, forming a supracellular cable around the lesion. Contraction at the wound margin coordinates cell movements and drives rapid wound closure. Using FRAP in Drosophila embryos, we found that myosin turnover at the wound margin was slower than in actomyosin networks with reduced contractility. Using laser ablation we showed that tension in the wound margin cable increased as wound closure progressed, which was associated with reduced myosin turnover. Reducing tension resulted in increased turnover and loss of myosin from the wound edge. Finally, myosin motor activity was necessary for its stabilization around the wound and for efficient wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair, however we could not discount regulation of myosin through its binding partner, actin. To investigate a role for tension in regulating actin during wound repair, we used FRAP to show that actin was stabilized around embryonic wounds. Loss of tension through laser ablation led to loss of actin fluorescence, however, photobleaching experiments after laser severing showed no change in actin turnover. We found no changes in actin dynamics between early and late wound closure, when tension is higher. Together these data suggest that tension may be partially necessary for actin localization, but it is neither necessary nor sufficient to regulate actin turnover. To begin to pick apart the relationship between actin and myosin at the wound margin, we inhibited myosin activity and measured actin turnover. Actin stabilization was independent of myosin activity, suggesting actin may be regulated independently. To understand whether myosin turnover is independent of actin, we stabilized actin networks pharmacologically and monitored for changes in myosin dynamics. Myosin turnover was unaffected by actin stabilization. These results suggest that actin and myosin are independently regulated, and that these networks are more complex than expected. This work provides insights into the mechanisms used by cells to coordinate their behaviour and could have implications for development and disease.
Source: TSpace (University of Toronto)
Author: Anna B. Kobb
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3013947214