Tip growth is a ubiquitous process throughout the plant kingdom in which a single cell elongates in one direction in a self-similar manner. To sustain tip growth in plants, the cell must regulate the extensibility of the wall to promote growth and avoid turgor-induced rupture. This process is heavily dependent on the cytoskeleton, which is thought to coordinate the delivery and recycling of vesicles containing cell wall materials at the cell tip. Although significant work has been done to elucidate the various molecular players in this process, there remains a need for a more mechanistic understanding of the cytoskeletonÂ’s role in tip growth. For this reason, specific emphasis should be placed on understanding the dynamics of the cytoskeleton, its associated motors, and their cargo. Since the advent of fluorescence fusion technology, various quantitative fluorescence dynamics techniques have emerged. Among the most prominent of these techniques is fluorescence recovery after photobleaching (FRAP). Despite its prominence, it is unclear how to interpret fluorescence recoveries in confined cellular geometries such as tip-growing cells. Here we developed a digital confocal microscope simulation of FRAP in tip-growing cells. With this simulation, we determined that fluorescence recoveries are significantly influenced by cell boundaries. With this FRAP simulation, we then measured the diffusion of VAMP72-labeled vesicles in the moss Physcomitrella patens. Using finite element modeling of polarized cell growth, and the measured VAMP72-labeled vesicle diffusion coefficient, we were able to show that diffusion alone cannot support the required transport of wall materials to the cell tip. This indicates that an actin-based active transport system is necessary for vesicle clustering at the cell tip to support growth. This provides one essential function of the actin cytoskeleton in polarized cell growth. After establishing the requirement for actin-based transport, we then sought to characterize the in vivo binding interactions of myosin XI, vesicles, and filamentous actin. Particle tracking evidence from P. patens protoplasts suggests that myosin XI and VAMP72-labeled vesicles exhibit fast transient interactions. Hidden Markov modeling of particle tracking indicates that myosin XI and VAMP72- labeled vesicles move along actin filaments in short-lived linear trajectories. These fast transient interactions may be necessary to achieve the rapid dynamics of the apical actin, important for growth. This work advances the fieldÂ’s understanding of fluorescence dynamics, elucidates a necessary function of the actin cytoskeleton, and provides insight into how the components of the cytoskeleton interact in vivo.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-dissertations-1539 |
| Date | 19 February 2018 |
| Creators | Bibeau, Jeffrey Philippe |
| Contributors | Luis Vidali, Advisor, Erkan Tuzel, Committee Member, Jagan Srinivasan, Committee Member, Elizabeth F. Ryder, Committee Chair |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Doctoral Dissertations (All Dissertations, All Years) |
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