Project properties
Title The role of specific actin binding proteins in the organization of the cytoplasm
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. The amount and locations of actin polymerisation, and thus the formation of cytoplasmic strands, are regulated by actin interacting proteins. Although many actin interacting proteins have been characterized in vitro, it is not known which actin interacting proteins regulate cytoplasmic organization. The work in this MSc project will reveal which actin interacting proteins play a role in the organization of the cytoplasm.

Experimental setup of the MSc project:
1) Generate constructs that enable simultaneous visualization of actin filaments and the vacuolar membrane (tonoplast). Introduce these constructs into several Arabidopsis lines in which actin interacting proteins have been knocked out.
2) Collect 3D time lapse image sequences of the organization of cytoplasmic strands and actin filaments in several cell types or cell suspension cultures of the Arabidopsis mutants, and analyse these image sequences to quantitatively asses parameters, such as the number, width, dynamics and locations of strands.
3) Apply actin depolymerizing drugs to the lines generated in (2) to investigate whether knocking out actin interacting proteins increases the sensitivity of actin filaments in cytoplasmic strands to these drugs. These experiments give additional information about the influence of specific actin binding proteins on the stability of actin filaments and cytoplasmic strands.
Used skills Molecular cloning and Agrobacterium mediated transformation of plant cells
Crossing of Arabidopsis
Generation of suspension cultures; cell culturing
Application of cytoskeleton drugs
Microscopy techniques (DIC, CLSM)
Digital image analysis
Requirements PCB-30306