- M. Akkerman (1)
- L. Bouwman-Smits (1)
- Bandan Chakrabortty (1)
- E.E. Deinum (1)
- H.S. Honing van der (1)
- J. Jonge de (1)
- Aniek Jongerius (1)
- R. Ramaker (1)
- D. Weijers (1)
- J.R. Wendrich (1)
- Ying Ying Zhang (1)
Control mechanisms of microtubule overlap regions
Jongerius, Aniek - \ 2017
University. Promotor(en): Marcel Janson. - Wageningen : Wageningen University - ISBN 9789463436113 - 133 p.
microtubuli - celbiologie - plantencelbiologie - modellen - cellen - microtubules - cellular biology - plant cell biology - models - cells
Microtubule organization in cells is an important process. An example of careful microtubule organization is the mitotic spindle. The spindle is a bipolar structure with microtubules emanating from the poles at both sides. These microtubules form antiparallel overlaps in the centre of the spindle where they are bundled by bundling proteins. The overlaps are centred in the spindle and their constant length is regulated. The overlaps are important for the stability of the microtubule network, without bundling proteins the overlaps are lost and the spindle collapses. The antiparallel overlaps are also the site where microtubules slide apart to induce spindle elongation. Sliding is induced by tetrameric motor proteins that can bind to two bundled microtubules. Spindle elongation also requires microtubule growth at the overlaps. All these different functions at the overlap, sliding, growth/shrinkage and bundling, have to cooperate to maintain overlap length. While sliding reduces overlap length, growth will increase overlap length. These activities have to be coordinated for the maintenance of a constant overlap length. We propose that a feedback mechanism is present where growth of the microtubules is limiting the sliding in the overlap. This would prevent sliding when the overlap decreases and helps to maintain the overlap. We designed in vitro experiments to make antiparallel overlaps in vitro. In these experiments we use purified proteins from S. pombe. We combine ase1 and klp9 in a relative sliding assay to mimic the sliding in the midzone. In our experiments we combine relative sliding with dynamic microtubules for the first time. This allows us to test how these activities are coordinated. In other experiments we combine ase1 and cls1 with dynamic microtubules to see if the rescue activity of cls1 can be confined to the overlaps. Furthermore, interactions between motor proteins and diffusive proteins are investigated on single microtubules.
Plant cortical microtubule dynamics and cell division plane orientation
Chakrabortty, Bandan - \ 2017
University. Promotor(en): Ben Scheres; Bela Mulder. - Wageningen : Wageningen University - ISBN 9789463431828 - 124
microtubules - plant cell biology - cell division - plant development - molecular biology - morphogenesis - simulation - microtubuli - plantencelbiologie - celdeling - plantenontwikkeling - moleculaire biologie - morfogenese - simulatie
This thesis work aimed at a better understanding of the molecular basis of oriented cell division in plant cell. As, the efficiency of plant morphogenesis depends on oriented cell division, this work should contribute towards a fundamental understanding of the molecular basis of efficient plant morphogenesis. We describe a modelling framework that allows us to simulate microtubule dynamics on the surface of arbitrary shapes. We further explored the generic role of microtubule regulatory effects such as shape anisotropy, edge-catastrophe and enhanced microtubule stabilization on the orientation of the microtubule array. Through a combined approach of experimental observations of cell division patterns and simulation of microtubule dynamics, we describe a possible molecular basis of oriented cell division during Arabidopsis early embryogenesis. We also infer the necessity of incorporating anisotropic growth/stress response of microtubules towards understanding division plane orientation in the growing epidermal root cells of Arabidopsis.
Stem cell organization in Arabidopsis : from embryos to roots
Wendrich, J.R. - \ 2016
University. Promotor(en): Dolf Weijers, co-promotor(en): Bert de Rybel. - Wageningen : Wageningen University - ISBN 9789462577350 - 192 p.
arabidopsis - stem cells - roots - plant embryos - morphogenesis - biological development - cellular biology - plant cell biology - stamcellen - wortels - plantenembryo's - morfogenese - biologische ontwikkeling - celbiologie - plantencelbiologie
Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a highly predictable and relatively simple model to study several developmental processes. Chapter 1 discusses the Arabidopsis embryo as a model for development and morphogenesis and describes the currently known factors involved in these processes.
Molecular cloning is a vital technique of today’s plant biological research. The ability to quickly produce reliable constructs for follow-up analyses can greatly accelerate biological research. In Chapter 2, we describe the optimization of a highly efficient Ligation Independent Cloning method. This method makes use of sticky overhangs that enable in vivo ligation of cloning products. We present a step-by-step protocol that enables generating plant transformation-ready constructs in a semi-high-throughput manner, within two to three days. This method can for example facilitate follow-up analysis of genome-wide approaches.
Proteins regularly function as part of larger protein-complexes and their interaction partners can often be indicative of functionality. Unbiased, in vivo analysis of protein complexes can therefore be very informative for the functional characterization of a protein of interest. In Chapter 3, we describe an optimized method for immunoprecipitation followed by tandem mass-spectrometry. By performing mass-spectrometry measurements on at least three biological replicates, relative abundance of proteins in GFP-tagged sample compared to background controls can be statistically evaluated to identify high-confidence interactors. In this step-by-step protocol we detail the entire procedure from plant material to data analysis and visualization.
The establishment of distinct cellular identities is of critical importance for multicellular organisms. The first step that leads to cell identity is the activation of a unique set of transcripts and this often exploited in order to infer cell identity. In Chapter 4, we have generated 12 gene expression marker lines and describe their expression domain in the Arabidopsis embryo. We divided them into four different categories based on their expression domain: (I) ground tissue; (II) root stem cell; (III) shoot apical meristem; and (IV) post-embryonic. In addition, we used two stem cell markers to show their use as marker lines in genetic studies.
A central player in development of the Arabidopsis root meristem is the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP). Several downstream targets of this transcription factor have been characterized, but the main focus has been on targets that were themselves transcription factors. An open question remains, therefore, how MP can orchestrate cellular responses during development. Chapter 5 describes the in-depth functional and biochemical characterization of a group of IQ-domain proteins. We show that their expression is regulated by the hormone auxin and that they bind microtubules and Calmodulins, in vivo. In addition, we show that the subcellular localization of IQD18 is cell cycle dependent. Loss- and gain-of-function analysis resulted in differential auxin- and calcium-signaling output, suggesting these proteins may form a bridge between these two major signaling pathways. Furthermore, this indicates a mode for how MP may be affecting cellular responses, during root development.
In Chapter 6, we take a step back and re-evaluate the currently prevailing model for stem cell organization in the Arabidopsis (embryonic) root. Using different gene expression markers, we were able to generate non-cell type specific and cell type specific transcriptomic datasets from systematically obtained ontogenetic cell populations in the root meristem. Follow-up analyses give support for an extended model for stem cell organization in the root.
Finally, in Chapter 7, we discuss the novel findings of this thesis and suggestions are made for future research directions.
Plant krijgt vorm door de regels te breken (interview met Dolf Weijers)
Ramaker, R. ; Weijers, D. - \ 2014
Resource: weekblad voor Wageningen UR 8 (2014)16. - ISSN 1874-3625 - p. 8 - 8.
plantencelbiologie - plantenontwikkeling - celdeling - ruimtelijke modellen - 3d visualisatie - auxinen - plantengroeiregulatoren - plant cell biology - plant development - cell division - spatial models - 3d visualization - auxins - plant growth regulators
Plantencellen blijken zich volgens een simpele regel in tweeën te delen; dit gebeurt in het midden, maar wel met een zo klein mogelijk deelvlak. Dit inzicht helpt te verklaren hoe planten hun definitieve vorm krijgen. Opvallend genoeg blijken cellen deze regel ook te overtreden.
Shoot apical meristem arrest in brassica and tomato
Jonge, J. de - \ 2013
University. Promotor(en): Gerco Angenent, co-promotor(en): Steven Groot. - Wageningen : Wageningen UR - ISBN 9789461738417 - 137
brassica oleracea - solanum lycopersicum - meristemen voor scheuten - apicale meristemen - plantenontwikkeling - celdeling - plantencelbiologie - shoot meristems - apical meristems - plant development - cell division - plant cell biology
A pool of cells known as stem cells located in the center of the shoot apical meristem (SAM) are responsible to maintain meristematic activity throughout a plants life in order to produce organs. The maintenance of these stem cells is tightly controlled by a complex genetic and hormonal network. Any disruption that leads to the loss of stem cells will end the formation of new plant organs and therefore the plants life-cycle.
The balance between leaf initiation and meristem maintenance is controlled by internal and external factors, although our knowledge about the nature of these factors is very limited.
This thesis reports the results of a study on SAM loss in tomato and brassica and the genetic and environmental factors causing this arrest. The aim was to study which environmental conditions could lead to so-called blind tomato and brassica plants and to develop a method that could induce this phenomenon. Furthermore, a genomic region responsible for blindness in brassica was identified.
Simple models for complex questions on plant development
Deinum, E.E. - \ 2013
University. Promotor(en): Bela Mulder; Ton Bisseling. - S.l. : s.n. - ISBN 9789461736314 - 283
plantenontwikkeling - groei - modellen - plantenfysiologie - plantencelbiologie - plant development - growth - models - plant physiology - plant cell biology
This thesis combines several modelling studies on plant growth and development. The core interest is the nitrogen fixing legume-rhizobium symbiosis, specifically how different signals interact in specifying the location of the nodule primordium.
For one of them, the hormone cytokinin, little is known about its movement through the tissue. All sufficiently small molecules, however, can move by non-targeted symplastic transport. We therefore start with a study of the biophysical properties of this often overlooked mechanism.
The study of the nodule primordium proper starts with an investigation of different mechanisms for local auxin accumulation, because this hormone structurally accumulates at the site of the first cell divisions. Both studies are then combined to investigate how an epidermal cytokinin signal can induce auxin accumulation in the right -- species dependent -- cortical position.
Plant growth and development also has strong mechanical components: the differential expansion of cell walls due to their anisotropic structure and the orientation of cell division planes. Both are controlled by the interphase cortical microtubule array. We investigate the effects of several experimental observations on array organisation and their resulting developmental impact.
We conclude with a critical review of different ways of using models to address biological questions.
Exocytosis and polarity in plant cells: insights by studying cellulose synthase complexes and the exocyst
Ying Zhang, Ying - \ 2012
University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar; C.M. Liu. - S.l. : s.n. - ISBN 9789461734075 - 132
plantencelbiologie - cellen - exocytose - cellulose - polariteit - microtubuli - celwanden - celwandstoffen - plant cell biology - cells - exocytosis - polarity - microtubules - cell walls - cell wall components
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.
From Golgi body movement to cellulose microfibril alignment
Akkerman, M. - \ 2012
University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar. - S.l. : s.n. - ISBN 9789461733030 - 122
plantencelbiologie - golgiapparaat - organellen - cellulose - cellen - celwanden - microtubuli - arabidopsis thaliana - plant cell biology - golgi apparatus - organelles - cells - cell walls - microtubules
Chapter 1 is an introduction into cellulose deposition and an outline of this thesis.
In chapter 2 the movement and distribution of Golgi bodies is studied in the cortex of cells of different growth stages, early elongation zone compared to late elongation zone, in relation to the configuration of the actin cytoskeleton. Golgi bodies in the cortex of cells in the early elongation zone, where growth accelerates to rapid growth, show slow random oriented movement, called wiggling. In the cortex of cells in the late elongation zone, where cell elongation ceases, they also show a second kind of motility, fast directed movement with velocities of up to 7 µm.s-1, like in cytoplasmic strands in the same cells. The cortical areas where Golgi body movement is slow and random co-localize with fine F-actin, a configuration of single or thin bundles of filaments. On the other hand, areas where Golgi body movement is fast and directed co-localize with thick actin filament bundles. When Golgi bodies enter an area with a different actin cytoskeleton configuration they change their type of motility concomitantly. We conclude that Golgi body dynamics correlate with the actin cytoskeleton organization.
CESA complexes are known to run in rows along CMTs in Arabidopsis hypocotyl cells. In chapter 3 we studied the orientation, density, alignment and movement of CMTs and CESA complexes using immunocytochemistry and live cell imaging. Furthermore we studied the orientation and density of the product of the CESA complexes, the CMFs, in the innermost wall layer with Field Emission Scanning Electron Microscopy (FESEM). The CMTs, the tracks 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, where cell elongation ceases. CESA complexes predominantly move in rows along CMTs in both directions. While the CMFs form a uniform cell wall layer, CESA complexes run one after the other along CMTs that are wider spread from each other than the CMFs and only few CESA complexes move in between the CMTs. To understand how CESA complexes can produce a uniform layer of CMFs, instead of local CMF thickenings, we studied whether the CMTs change position during CMF production. Time lapse movies of CMTs show that CMTs reposition over time, so that CESA complexes produce an even CMF layer. In this way we can understand how the density of CMFs in the nascent cell wall can be higher than that of the CMTs and the moving rows of CMFs in the plasma membrane. CMFs are deposited consecutively next to earlier deposited ones in the same orientation.
In chapter 4 we used several different electron microscopy techniques to visualize CMF texture: transmission Electron Microscopy (TEM) of ultrathin sections after mild or complete matrix extraction, TEM of surface preparations and FESEM of surface preparations. We used root hairs of three different species; Arabidopsis thaliana, Medicago truncatula and Vicia sativa. We compare and discuss the results of the techniques for the capacity to measure orientation, density, length and width of the CMFs. In ultrathin sections and surface preparations we observed that the three species studied have root hairs with an axial/helical wall texture. Surface preparations are best suitable for density and orientation measurements of CMFs within the most inner cell wall layer. Ultrathin sections showed that the thickness of CMFs in Arabidopsis is approximately 3 nm. which indicates that these CMFs are produced by single CESA complexes.
Chapter 5 is a general discussion of our work in relation to the field. It describes the role of the actin cytoskeleton , Golgi body motility and CMTs in the deposition of an organized texture of CMFs.
Actin-mediated cytoplasmic organization of plant cells
Honing, H.S. van der - \ 2011
University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar. - [S.l.] : S.n. - ISBN 9789085859383 - 124
cytoplasma - plantencelbiologie - cellen - actine - celskelet - celstructuur - cytoplasm - plant cell biology - cells - actin - cytoskeleton - cell structure
In this thesis, I present results that give insight in the role of the actin cytoskeleton in the production of an organized cytoplasm in plant cells, which is, for instance, required for proper cell morphogenesis.
Chapter 1 is a review in which we discuss the possible role of actin-based force generation in the production of an organized cytoplasm in plant cells. We compare the functions of actin binding proteins of three well-studied mammalian model systems that depend on actin-based force generation with the possible functions of their homologues in plants, and predict how these proteins might determine the cytoplasmic architecture of plant cells.
In chapter 2, we describe the use a combined setup of optical tweezers with a confocal laser scanning microscope to study whether stiffness is an actin-related property of plant cytoplasm, and to study parameters involved in the reorganization of the actin cytoskeleton during physical manipulation of the cytoplasm. We used optical tweezers to produce cytoplasmic protrusions that resemble cytoplasmic strands, while imaging the behaviour of the actin cytoskeleton. We determined the trapping force needed to produce cytoplasmic protrusions, and show that the presence of actin filaments stiffens the cytoplasm. The deactivation of a 2,3-butanedione monoxime (BDM)-sensitive factor, probably the molecular motor myosin, stiffens the cytoplasm even more. The observation that actin filaments do not enter the tweezer-formed protrusions during this BDM treatment, suggests that the actin cytoskeleton can reorganize by a myosin-based relocation of actin filaments. Such a myosin-based reorganization of the actin cytoskeleton might be involved in the production of an organized cytoarchitecture in plant cells.
Lifeact:Venus, which consists of the first 17 amino acids from the yeast protein Abp140 fused to a yellow fluorescent protein, is a novel probe for actin filament visualization. In chapter 3, we compare the (re)organization of the actin cytoskeleton visualized with Lifeact:Venus with that of the actin cytoskeleton visualized with GFP:FABD2, a commonly used marker for filamentous actin in plants that consists of GFP fused to the second actin binding domain of Arabidopsis FIMBRIN1. We show that Lifeact:Venus reduces remodeling of the actin cytoskeleton inArabidopsis root epidermal cells, as well as concomitant reorganization of the cytoplasm. Nonetheless, expression of Lifact:Venus neither affects cytoplasmic organization, nor plant growth and development. The data imply that the organization of the actin cytoskeleton, but not its dynamic relocation over time, is a determining factor in plant cell growth, and show that Lifeact should be used with caution when studying reorganization of actin filaments.
In cytoplasmic strands, actin filaments are organized in thick bundles. The actin bundling protein villin is involved in maintaining these bundles. In chapter 4, we analyze the role of VLN2 and VLN3, two members of the villin protein family in Arabidopsis, and show that mutations in the genes encoding these villins result in a decrease in the number of thick actin filament bundles. Double mutant plants have abnormal leaves, stems, siliques and roots. The wavy, twisted appearance of these organs in the double mutant shows that the coordination of cell expansion is affected. Furthermore, the rotational movements (circumnutation) of vln2 vln3 inflorescences have larger amplitudes than those of wild type Col-0 inflorescences and are less regular.The data show that VLN2 and VLN3 are involved in the generation of thick actin filament bundles, and suggest that these bundles are important for coordinated cell expansion.
Chapter 5 is the general discussion of the thesis. We discuss research in which actin binding proteins that could play a role in cytoplasmic organization have been described. In this chapter, we have included our initial data about the role of the actin bundling protein fimbrin on actin organization. We further discuss how manipulation of cytoplasmic organization by optical tweezers can give insight into physical properties of actin filaments in the plant cytoplasm.
Docentencursus Cel- en ontwikkelingsbiologie van planten : theorie en praktijk in de klas
Bouwman-Smits, L. - \ 2011
docenten - agrarisch onderwijs - bijscholing - plantencelbiologie - plantenontwikkeling - teachers - agricultural education - continuing training - plant cell biology - plant development
Poster met informatie over de docentencursus Cel- en ontwikkelingsbiologie van planten.