|Title||Exocytosis and polarity in plant cells: insights by studying cellulose synthase complexes and the exocyst|
|Author(s)||Ying Zhang, Ying|
|Source||Wageningen University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar; C.M. Liu. - S.l. : s.n. - ISBN 9789461734075 - 132|
Laboratory of Cell Biology
|Publication type||Dissertation, internally prepared|
|Keyword(s)||plantencelbiologie - cellen - exocytose - cellulose - polariteit - microtubuli - celwanden - celwandstoffen - plant cell biology - cells - exocytosis - cellulose - polarity - microtubules - cell walls - cell wall components|
|Categories||Plant Cell Biology|
The work presented in this thesis covers aspects of exocytosis, plant cell growth and cell wall formation. These processes are strongly linked as cell growth and cell wall formation occur simultaneously and exocytosis is the process that delivers cell wall components to the existing cell wall and integral membrane proteins to the plasma membrane. The chapters in this thesis describe work on the exocyst, a group of proteins thought to be involved in polarized secretion, the regulation of CESA complex mediated cellulose microfibril deposition by cortical microtubules, and the organization of cortical microtubules. Chapter 1 is a review in which research on the plant exocyst is discussed. We compare the literature about the plant exocyst with knowledge about well-studied yeast and mammalian exocysts and explore which aspects of exocyst functioning are conserved in plants and which aspects are not. We propose that the plant exocyst has acquired distinct functions and mechanisms in exocytosis for plant cell growth, based on the fact that each subunit of the exocyst in yeast and mammals is encoded by one gene, whereas some exocyst subunits in plants, particularly EXO70, are encoded by multiple genes. In Chapter 2, we presented experimental data on the exocyst. Using a yeast two hybrid based approach we present novel interactions between different exocyst subunits. We continue by focusing on the exocyst subunit SEC3, which functions as a landmark protein in yeast and mammalian cells. We show that both SEC3 genes in Arabidopsis are essential for plant development; A T-DNA insertion in the SEC3A gene causes embryo development to arrest at the globular stage and a T-DNA insertion in the SEC3B gene causes gametophytic lethality. We were able to complement the sec3a mutant by introducing a pSEC3A::SEC3A:GFP construct and used the resulting lines to study the subcellular localization of SEC3A. The fusion protein shows a similar localization to cytokinetic cell plates as has been shown for other exocyst subunits. In interphase cells SEC3A:GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctuate structures with discrete average lifetimes of 6-12 seconds. These puncta are equally distributed over the cell surface of root epidermal cells and tip growing root hairs and the density of puncta does not decrease after growth termination of these cells. Either SEC3a puncta may not participate in exocytosis for polarized cell expansion, or the plasma membrane recruitment of SEC3 is a default process that requires other, polarly localized factors to mediate exocytotic events. Chapter 3 focused on the role of cortical microtubules in the insertion, guidance and occurrence in the plasma membrane of cellulose microfibril producing CESA complexes. We characterized a wide range of parameters that give insight in CESA complex behavior, such as velocity, density and movement angles in the expanding tip and non-expanding tube of growing root hairs and the same areas in fully-grown root hairs. Then we performed co-localization studies of CESA complexes with cortical microtubules. In tubes of both growing and fully-grown root hair cells CESA complex insertion occurred preferentially on cortical microtubules. Part of the population of CESA complexes that were moving in the plasma membrane was tracking cortical microtubules, whereas others were moving in between cortical microtubules. CESA complexes tracking cortical microtubules had a slightly different movement direction, but also a much lower variation in movement direction than the CESA complexes that were moving in between cortical microtubules. When microtubules are absent, all CESA complexes move in the same direction as those that do not track cortical microtubules in the presence of microtubules, and the variation in the movement direction is similar to that of CESA complexes moving in between cortical microtubules. This shows that CMTs in root hairs focus CESA complex movement, by which they order cellulose microfibrils into a tighter helix. In the absence of microtubules, the average lifetime of CESA complexes increases from 12.8 minutes to 22.3 minutes, showing that there is a feedback mechanism between CESA complex insertion and CESA complex lifetime. Since their velocity of movement in the plasma membrane does not change, they produce longer cellulose microfibrils in the absence of cortical microtubules. In Chapter 4, we addressed the question how CESA complexes that are guided by widely spaced cortical microtubules can produce a uniform layer of cellulose microfibrils with a much smaller spacing in axially growing root epidermal cells. We studied the orientation, density, alignment and movement of cortical microtubules and CESA complexes using immunocytochemistry and live cell imaging of root epidermal cells. The CMTs, the rows of CESA complexes and the innermost CMFs lay in the same orientation, approximately transverse to the elongation axis in both the inner and outer periclinal cell face in the elongation zone and root hair zone. CESA complexes predominantly move in rows along CMTs in both directions. Analysis of timelapse movies of CMTs revealed that the position shifting of cortical microtubules accounts for how the uniform layer of cellulose microfibrils can be formed. Chapter 5 is the general discussion of the thesis, where we provide a framework in which the results presented in the previous chapters fall.