| dc.description.abstract |
Plants are exposed to external mechanical stress, such as wind, rain, pathogens or the soil through which the root has to grow. Mechanical stress can also originate from the plant itself, for example from cellular growth differences. These mechanical cues can change the morphology through an act of stress adjustment. For example, polarized wind can shape trees to become flagged trees and plants which are frequently touched are shorter but more robust. On the cellular level, polar mechanical cues can contribute to the cell shape, even in the complex multipolar jigsaw shape of pavement cells. This process is accompanied by local cell wall (CW) modifications, defining a delicate spatial pattern of restricted and promoted cell expansion. During polar expansion, mechanical stress is transferred from the CW to the plasma membrane (PM), as the PM follows the CW and can thus be stretched. To resist this mechanical stress, it is suggested that more membrane material is delivered to the stress bearing PM area. As mechanical stress displays a polarizing character, it is possible that the stress bearing area on the PM is polarized as well, which then requires polarized trafficking events. Mechanical stress induced polarized trafficking, that eventually affects cell morphology and cell identity. However, so far, no clear link between polar mechanical stress and polarized trafficking exists, as most insight was mostly gained from model systems with deficit mechanical properties. Thus, to test if polar mechanical stress is associated with polar trafficking events, the ablation (ABL) technique was used on meristematic root epidermal cells, as a polar mechanical stress pattern can be assumed on the ABL adjacent cells. To further strengthen the polar acting tension in ABL adjacent cells, PM dynamic was measured by using the fluorescence recovery after photobleach experiments. The ABL adjacent cells displayed polar PM dynamic differences, with the ABL facing side showing a higher dynamic than the opposite cell side, which displayed a control like PM dynamic. In addition, ABL adjacent cells showed smaller/less increasing endocytosis associated membrane aggregates, induced by the recycling and partial secretory inhibitor Brefeldin-A than control cells. This suggests that ABL induced tension affects trafficking pathways. However, this effect was decreased in cells more distant from the ABL, parallel to the potential tension gradient of the ABL. To test if the alternating trafficking is biologically relevant, the ABL adjacent cell expansion was measured, which also reflected the trafficking efficiency. A severely reduced ABL adjacent cell expansion was observed by blocking the ER exit sites, indicating the ER being an essential membrane source under mechanical stress. BFA induced inhibition of the recycling associated GNOM and secretion associated BIG1, BIG2 and BIG4, resulted in a significant reduction of the ABL induced polar cell expansion. Despite the role of GNOM in polar relocalization of PIN1, genetically modified BFA-resistant GNOM could not rescue the BFA-induced significant ABL induced cell expansion, indicating a minor role of GNOM mediated recycling in tension induced polar membrane delivery. Additional depletion of BFA-resistant secretion associated BIG3, did not lead to a more pronounced reduction of the ABL induced cell expansion. As BFA already inhibits three quarters of the secretion associated BIG’s, an essential role of the secretory pathway in polar trafficking upon polar tension can still be assumed. In contrast, the absence of BIG3 alone increased the ABL induced cell expansion, which was returned to WT level in big3big5 double mutant. As BIG5 plays a role in the endocytotic pathway, it is possible that endocytosed membrane represents an additional membrane source, that can be exhausted during locally acting tension.
To further test the link between polar mechanical stress and molecular responses, it was attempted to optimize the already existing psiTrap (Thorand, 2019), a microfluidic device capable of the controlled induction of mechanical stress on single immobilized cells, generating a more reliable and less bubble sensitive modified psiTrap (collaboration with Gregor Schmidt). Overall, this work strengthens the connection of polar mechanical stress and polar membrane delivery. The identification of potential key mediator as well as the gradual affected trafficking upon mechanical stress can provide valuable information to investigate how morphology can be influenced by mechanical stress. |
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