Project properties
Title Do cytoplasmic strands contain microtubules?
Group Plant Cell Biology, Laboratory of
Project type thesis
Credits 18-39
Supervisor(s) Hannie van der Honing, Tijs Ketelaar
Examiner(s) Prof. dr. Marcel Janson
Contact info tijs.ketelaar@wur.nl, 0317-482453
Begin date 2008/04/01
End date 2010/04/01
Description Proper intracellular organization is of vital importance for cellular functioning and plant development. The cytoplasm of plant cells is organized such that a pool of cytoplasm, present around the nucleus and a pool of cytoplasm in the periphery of the cell are connected by strands of cytoplasm. These cytoplasmic strands penetrate the vacuole that separates the two pools of cytoplasm. Cytoplasmic strands are essential for the transport of transcripts, proteins, and signals from the nucleus to the periphery of the cell and vice versa. All cytoplasmic strands of interphase plant cells contain actin filaments, and when actin filaments are depolymerized, the strands disappear. Thus, actin filaments are the structural basis of cytoplasmic strands. Microtubules are thought to be absent from interphase cytoplasmic strands, and when microtubules are depolymerized, the strands remain intact. There is some evidence that suggests that cytoplasmic strands do contain microtubules just prior to and after cell division. The function of these microtubules is not clear. Microtubules in cytoplasmic strands might, for instance, play a role in the positioning of the nucleus in the cell center prior to cell division. In this project, you will determine when exactly microtubules are present in cytoplasmic strands and determine the function of these microtubules. The approach will be as follows:
In our lab, we have a tobacco BY-2 suspension culture expressing the GFP:α-tubulin fusion protein, which is, like normal α-tubulin, incorporated into microtubules. Fluorescence Recovery After Photobleaching (FRAP) is a technique that discriminates between GFP:α-tubulin that has been incorporated into microtubules and GFP:α-tubulin that is present as a free dimmer. FRAP will reveal the presence or absence of microtubules in cytoplasmic strands at specific moments of the cell cycle. The use of specific drugs interfering with the cytoskeleton will show whether microtubules and/or actin filaments are important for the stability and dynamics of cytoplasmic strands that contain microtubules. This work will finally solve the question IF, and WHEN, cytoplasmic strands contain microtubules and what microtubules are doing in cytoplasmic strands.
Used skills Culturing of tobacco BY-2 suspension cells
Cell cycle synchronization
Treatments with cytoskeleton drugs
Microscopy techniques (DIC, CLSM, FRAP)
Quantitative analysis of FRAP results
Requirements PCB-30306