Cell walls are composed of cellulose microfibrils, embedded in a polysaccharide matrix. The cellulose microfibrils are produced by cellulose synthase (CESA) complexes, visible in the electron microscope as particle rosettes in the plasma membrane.
The synthetic part of the complex consists of glycosyl transferases, which bind UDP-glucose and process it into a linear polymer of ß-(1,4) glucose residues. The 18-36 polymers produced by one complex crystallize and form a microfibril in the cell wall. During the polymerization/crystallization process, the CESA complex moves inside the plasma membrane and its route determines the orientation of its product, the cellulose microfibril. Electron microscopical studies have revealed that cellulose microfibrils in the cell wall are highly organized in a limited number of cell wall textures.
The texture type determines the strength and other mechanical properties of the cell wall and in elongating cells cellulose microfibrils are perpendicular to the elongation axis. How this orientation is created is not completely clear. In elongating etiolated Arabidopsis hypocotyl cells live cell imaging of XFP-labeled CESA’s has shown that the CESA complexes follow the cortical microtubules. However, in the total absence of cortical microtubules CESA complexes move in ordered patterns in the plasma membrane as well and a highly ordered cell wall texture is being formed.We conclude that cortical microtubules are:
· not needed for production of cellulose microfibrils,
· not needed for ordering of cellulose microfibrils,
· not needed for determining velocity of CESA complex movement,
BUT: are needed for specific ordering of cellulose microfibrils in elongating cells
Therefore, we have asked the question: ‘what is the default ordering mechanism in the absence of cortical microtubules?’ In other words: ‘can a complex ordered texture, such as that of a helicoidal cell wall be formed in the absence of direct cellular guidance mechanisms for the CESA complexes?’ We have proposed that cellulose microfibril textures can be formed by a mechanism that is based on geometrical considerations in which is the angle g between every single cellulose microfibril and the transverse axis is determined by:
- the density of cellulose microfibrils (N) and thus on the density of active cellulose synthase complexes , which itself relies on insertion rate, life time and velocity of CESA complexes,
- the distance between the microfibrils (d) and thus the cellulose to wall matrix ratio,
- the radius of the cell (R).
This geometrical model explains the genesis of the most complicated helicoidal texture in the absence of a guiding structure, and shows that the cell has intrinsic, versatile tools for creating a variety of textures. If the three parameters are known, the model is able to produce all possible cell wall textures, axial, helical, helicoidal, longitudinal. A compelling feature of the model is that local rules generate global order, a typical phenomenon of life. The mathematical part of this work is being done by Prof. Dr. B. M. Mulder.
Since CESA density is the most important parameter of the model and depends on its insertion into the plasma membrane, its lifetime and velocity, we measured CESA insertion frequency and location. This latest work in collaboration with the group of David Ehrhardt from Carnegie Institution at Stanford University has shown that in elongating etiolated arabidopsis hypocotyl cells microtubules not only guide the cellulose synthase complexes but also serve as signposts to locate where the CESA complexes are inserted into the membrane (Nature Cell Biology 11, 797–806 Ryan Gutierrez , Jelmer J. Lindeboom , Alex R. Paredez , Anne Mie C. Emons & David W. Ehrhardt (2009)
The movie shows a FRAP experiment (Fluorescence recovery after photobleaching). Shown is a part of the plasma membrane with cell cortex of a living growing hypocotyl cell of Arabidopsis thaliana, in red the microtubules (labeled met RFP::TUA5: red fluorescent protein coupled to tubulin, and in green de cellulose synthase complexes (labeled met GFP::CESA3: green fluorescent protein coupled to cellulose synthase. The bigger red dots are Golgi bodies in the cortex of the cell. SmaCCS (small CESA compartments) lie just under the plasma membrane and can be discriminated from the synthases inside the membrane by their behavior; their movement is erratic, while the movement in the plasma membrane of synthesizing synthases is slow and steady, 350 nm/min. Jelmer Lindeboom used the photobleaching technique to visualize the actual insertion of the cellulose synthase complexes into the plasma membrane. Figure left before bleaching, figure middle after bleaching of GFP::CESA3, figure right recovery 90 sec; Arrow heads are insertion events.
Actin filament bundles transport organelles including Golgi bodies. CESA-complexes insert into the plasma membrane in area’s where Golgi bodies are. The above mentioned research has also shown that Golgi bodies can be tethered to Small CESA containing compartments (SmaCCS), which move on depolymerizing microtubule ends, especially observed under stress conditions, and deliver the complexes to the plasma membrane. In one of our projects we study Golgi body movement on actin filament (bundles)s and their behavior at the plasma membrane. Many questions about the role of the actin and microtubule cytoskeleton in the process of cellulose microfibril deposition remain to be solved.
Plant cell biological systems
As a system we use Arabidopsis thaliana hypocotyl cells and root epidermal cells (Jelmer Lindeboom) including root hairs, growing as well as fully grown (Ying Zhang). In addition to the study of the trajectories of the cellulose synthase complexes, we study the behavior of the origin from which they are delivered, the Golgi system (Miriam Akkerman) , and their product, the cellulose microfibrils, at the ultrastructural level in the electron microscope. For ordering of cortical microtubules (Jelmer Lindeboom) and in a microgravity project on cortical microtubules and CESA complexes tobacco BY-2 suspension cultured cells are being used (Jan Vos) In another project we observe and analyze the polymerization of cellulose in vitro from CESA enzymes isolated from BY-2 suspension cultured cells with a liquid crystal polarized light microscope (LC-Polscope), which quantitatively measures birefringence of oriented filaments arrays. This microscope is also being used to quantify cellulose production in thickening bands of individual tracheray element cells of a Zinnia elegans suspension cultured cells (Carolina Cifuentes).Research funding:
EU NEST Adventure project: CASPIC