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The morphogenesis of sessile plants is mainly driven by directional cell growth and cell division. The organization of their cytoskeleton and the mechanical properties of the cell wall greatly influence morphogenetic events in plants. It is well known that cortical microtubules (CMTs) contribute to directional growth by regulating the deposition of the cellulose microfibrils, as major cell wall fortifying elements. More recent findings demonstrate that mechanical stresses existing in cells and tissues influence microtubule organization. Also, in dividing cells, mechanical stress directions contribute to the orientation of the new cell wall. In comparison to the microtubule cytoskeleton, the role of the actin cytoskeleton in regulating shoot meristem morphogenesis has not been extensively studied.
This thesis focuses on the functional relevance of the actin cytoskeleton during cell and tissue scale morphogenesis in the shoot apical meristem (SAM) of Arabidopsis thaliana. Visualization of transcriptional reporters indicates that ACTIN2 and ACTIN7 are two highly expressed actin genes in the SAM. A link between the actin cytoskeleton and SAM development derives from the observation that the act2-1 act7-1 double mutant has abnormal cell shape and perturbed phyllotactic patterns. Live-cell imaging of the actin cytoskeleton further shows that its organization correlates with cell shape, which indicates a potential role of actin in influencing cellular morphogenesis.
In this thesis, a detailed characterization of the act2-1 act7-1 mutant reveals that perturbation of actin leads to more rectangular cellular geometries with more 90° cell internal angles, and higher incidences of four-way junctions (four cell boundaries intersecting together). This observation deviates from the conventional tricellular junctions found in epidermal cells. Quantitative cellular-level growth data indicates that such differences in the act2-1 act7-1 mutant arise due to the reduced accuracy in the placement of the new cell wall, as well as its mechanical maturation. Changes in cellular morphology observed in the act2-1 act7-1 mutant result in cell packing defects that subsequently compromise the flow of information among cells in the SAM.
Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells.
Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands
(2013)
Secretory cells in blowfly salivary gland are specialized via morphological and physiological attributes in order to serve their main function, i.e. the transport of solutes at a high rate in response to a hormonal stimulus, namely serotonin (5-HT). This study examines the way that 5-HT-insensitive precursor cells differentiate into morphologically complex 5-HT-responsive secretory cells. By means of immunofluorescence microscopy, immunoblotting and measurements of the transepithelial potential changes, we show the following. (1) The apical membrane of the secretory cells becomes organized into an elaborate system of canaliculi and is folded into pleats during the last pupal day and the first day of adulthood. (2) The structural reorganization of the apical membrane is accompanied by an enrichment of actin filaments and phosphorylated ERM protein (phospho-moesin) at this membrane domain and by the deployment of the membrane-integral part of vacuolar-type H+-ATPase (V-ATPase). These findings suggest a role for phospho-moesin, a linker between actin filaments and membrane components, in apical membrane morphogenesis. (3) The assembly and activation of V-ATPase can be induced immediately after eclosion by way of 8-CPT-cAMP, a membrane-permeant cAMP analogue. (4) 5-HT, however, produces the assembly and activation of V-ATPase only in flies aged for at least 2 h after eclosion, indicating that, at eclosion, the 5-HT receptor/adenylyl cyclase/cAMP signalling pathway is inoperative upstream of cAMP. (5) 5-HT activates both the Ca2+ signalling pathway and the cAMP signalling cascade in fully differentiated secretory cells. However, the functionality of these signalling cascades does not seem to be established in a tightly coordinated manner during cell differentation.
Adherent cells constantly collect information about the mechanical properties of their extracellular environment by actively pulling on it through cell-matrix contacts, which act as mechanosensors. In recent years, the sophisticated use of elastic substrates has shown that cells respond very sensitively to changes in effective stiffness in their environment, which results in a reorganization of the cytoskeleton in response to mechanical input. We develop a theoretical model to predict cellular self-organization in soft materials on a coarse grained level. Although cell organization in principle results from complex regulatory events inside the cell, the typical response to mechanical input seems to be a simple preference for large effective stiffness, possibly because force is more efficiently generated in a stiffer environment. The term effective stiffness comprises effects of both rigidity and prestrain in the environment. This observation can be turned into an optimization principle in elasticity theory. By specifying the cellular probing force pattern and by modeling the environment as a linear elastic medium, one can predict preferred cell orientation and position. Various examples for cell organization, which are of large practical interest, are considered theoretically: cells in external strain fields and cells close to boundaries or interfaces for different sample geometries and boundary conditions. For this purpose the elastic equations are solved exactly for an infinite space, an elastic half space and the elastic sphere. The predictions of the model are in excellent agreement with experiments for fibroblast cells, both on elastic substrates and in hydrogels. Mechanically active cells like fibroblasts could also interact elastically with each other. We calculate the optimal structures on elastic substrates as a function of material properties, cell density and the geometry of cell positioning, respectively, that allows each cell to maximize the effective stiffness in its environment due to the traction of all the other cells. Finally, we apply Monte Carlo simulations to study the effect of noise on cellular structure formation. The model not only contributes to a better understanding of many physiological situations. In the future it could also be used for biomedical applications to optimize protocols for artificial tissues with respect to sample geometry, boundary condition, material properties or cell density.