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Tip Growth in Walled Cells: Cellular Expansion and Invasion Mechanisms

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... Since turgor is a scalar, for nonspherical cell shapes to develop during differentiation, the cell wall mechanical behavior must differ between subcellular regions. This can be achieved through the variation of wall thickness or heterogenous distribution of the material properties (Green, 1962;Sanati Nezhad and Geitmann, 2015). The mechanical aspects of shaping or deformation processes can be explored using a variety of mathematical approaches (Dyson and Jensen, 2010). ...
... Tip-growing cells such as pollen tubes, root hairs, and fungal hyphae feature a spatially confined expansion zone allowing these cells to perform invasive behavior (Sanati Nezhad et al., 2013;Sanati Nezhad and Geitmann, 2015;Bascom et al., 2018). The profile of the growing tip is radially symmetrical and remains self-similar when moving forward. ...
Article
(Full-text Open Access! www.plantphysiol.org/content/176/1/41): Plant cells come in a striking variety of different shapes. Shape formation in plant cells is controlled through modulation of the cell wall polymers and propelled by the turgor pressure. Understanding the shaping aspects of plant cells requires knowledge of the molecular players and the biophysical conditions under which they operate. Mechanical modeling has emerged as a useful tool to correlate cell wall structure, composition, and mechanics with cell and organ shape. The finite element method is a powerful numerical approach employed to solve problems in continuum mechanics. This Update critically analyzes studies that have used finite element analysis for the mechanical modeling of plant cells. Focus is on models involving single cell morphogenesis or motion. Model design, validation, and predictive power are analyzed in detail to open future avenues in the field.
... The local modification of cell wall properties determines whether cell-surface expansion is uniformly distributed over the cell envelope or whether it occurs at specific locations creating a non-uniform distribution of the expansion pattern 60 (Figure 4). It also regulates whether the expansion of a given unit element in the cell envelope occurs equally in all directions (isotropically) or preferentially along one axis (anisotropically) 61 . This cell wall-focused concept of plant cell growth is crucial since the driving force for cell expansion, the turgor, is virtually uniform within the cell volume and, as pressure is a scalar, it is non-directional. ...
Article
The plant cell wall is an extracellular matrix that envelopes cells, gives them structure and shape, constitutes the interface with symbionts, and defends plants against external biotic and abiotic stress factors. The assembly of this matrix is regulated and mediated by the cytoskeleton. Cytoskeletal elements define where new cell wall material is added and how fibrillar macromolecules are oriented in the wall. Inversely, the cytoskeleton is also key in the perception of mechanical cues generated by structural changes in the cell wall as well as the mediation of intracellular responses. We review the delivery processes of the cell wall precursors that are required for the cell wall assembly process and the structural continuity between the inside and the outside of the cell. We provide an overview of the different morphogenetic processes for which cell wall assembly is a crucial element and elaborate on relevant feedback mechanisms.
... Thus, the force must be dramatically reduced to maintain cell wall integrity upon the pollen tube's emergence from the restricted growth region by monitoring and responding to the increased membrane tensile stress. The penetrative force could result from an increase in the internal turgor pressure and/or the softening of the apical cell wall (Nezhad and Geitmann, 2015;. Modeling and experimental data favor the notion that the enhanced softening of pectin at the apical cell wall at least partly provides the penetrative force (Sanati . ...
Article
Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1’s ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.
... Cellular differentiation in plants involves dramatic alterations in cell shape leading to highly specialized cell types, such as trichomes, fibers, or guard cells. Cell growth and shaping involve a turgor-driven deformation of the cell wall and are assumed to be governed by at least two crucial parameters related to the material properties of the primary cell wall (Altartouri and Geitmann, 2015;Sanati Nezhad and Geitmann, 2015). The first parameter is the degree of spatial uniformity of the wall stiffness that is controlled by the extensibility of the cell wall material and the dimensions (thickness) of the wall. ...
Article
Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.
... In walled cells, anisotropy is an essential feature on the other hand, especially in elongating cells. Combined with subcellular heterogeneity, anisotropy determines the growth pattern of the individual cell (Baskin, 2005;Cosgrove, 2005;Sanati Nezhad and Geitmann, 2015;Bidhendi and Geitmann, 2016). Both parameters, therefore, need to be considered when evaluating plant cell mechanics, and the methods need to be tailored for these features. ...
Article
The primary plant cell wall is a dynamically regulated composite material of multiple biopolymers that forms a scaffold enclosing the plant cells. The mechanochemical make-up of this polymer network regulates growth, morphogenesis, and stability at the cell and tissue scales. To understand the dynamics of cell wall mechanics, and how it correlates with cellular activities, several experimental frameworks have been deployed in recent years to quantify the mechanical properties of plant cells and tissues. Here we critically review the application of biomechanical tool sets pertinent to plant cell mechanics and outline some of their findings, relevance, and limitations. We also discuss methods that are less explored but hold great potential for the field, including multiscale in silico mechanical modeling that will enable a unified understanding of the mechanical behavior across the scales. Our overview reveals significant differences between the results of different mechanical testing techniques on plant material. Specifically, indentation techniques seem to consistently report lower values compared with tensile tests. Such differences may in part be due to inherent differences among the technical approaches and consequently the wall properties that they measure, and partly due to differences between experimental conditions.
... One of the principal motivations for monitoring cell wall polysaccharides is the fact that they exert spatial and temporal control on cellular morphogenesis and hence plant developmental processes. To be specific, expansive growth of walled cells under the effect of turgor pressure is influenced by at least two parameters characterizing the mechanical behavior of the wall [5]. The first parameter is the degree of uniformity of the wall properties, which determines whether the cellular surface expands entirely or only partially, that is, some cellular regions expand rapidly while others resist yielding. ...
Article
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Sexual reproduction in flowering plants is unique in multiple ways. Distinct multicellular gametophytes contain either a pair of immotile, haploid male gametes (sperm cells) or a pair of female gametes (haploid egg cell and homodiploid central cell). After pollination, the pollen tube, a cellular extension of the male gametophyte, transports both male gametes at its growing tip and delivers them to the female gametes to affect double fertilization. The pollen tube travels a long path and sustains its growth over a considerable amount of time in the female reproductive organ (pistil) before it reaches the ovule, which houses the female gametophyte. The pistil facilitates the pollen tube's journey by providing multiple, stage-specific, nutritional, and guidance cues along its path. The pollen tube interacts with seven different pistil cell types prior to completing its journey. Consequently, the pollen tube has a dynamic gene expression program allowing it to continuously reset and be receptive to multiple pistil signals as it migrates through the pistil. Here, we review the studies, including several significant recent advances, that led to a better understanding of the multitude of cues generated by the pistil tissues to assist the pollen tube in delivering the sperm cells to the female gametophyte. We also highlight the outstanding questions, draw attention to opportunities created by recent advances and point to approaches that could be undertaken to unravel the molecular mechanisms underlying pollen tube-pistil interactions. WIREs Dev Biol 2012, 1:96-113. doi: 10.1002/wdev.6 For further resources related to this article, please visit the WIREs website. Additional Supporting Information may be found in the online version of this article. Movie1-Palanivelu-Tsukamoto.mov Movie1-Palanivelu-Tsukamoto.mov.
Chapter
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The pollen tube wall differs in both structure and function from walls of vegetative plant cells. Cellulose represents only asmall portion of the cell wall polymers, so an organized microfibrillar system has not been identified yet. The initial wall, formed by secretion at the growing tip, is mostly composed of methyl esterified pectins. During cell wall maturation, concomitant with its translocation from apex to shank, these are demethylated by pectin methylesterase to yield carboxyl groups which have the potential to bind calcium ions, adding mechanical strength to the gel. Callose synthase activity is established close to the growing tip, and builds acallose layer beneath the fibrous pectic layer. The mature wall also contains proteins, arabinogalactan proteins and pollen extensin-like proteins. The mature wall is acylinder that resists turgor expansion, but is stronger at the base than the tip due to the presence of the callose layer and the gelation of pectin polymers in the shank. Permeability of the wall is essential, to allow passage of both ions and sporophytic proteins that determine compatibility in many species. Influx of calcium ions affects the tip cytoplasm, especially the cytoskeleton, and oscillatory changes in these fluxes are involved in the “pulsatile” mode of growth. This process deposits extra wall material during the “slow” growth phase, which generates rings of increased density in the walls that can be readily seen with appropriate antibodies.
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The frequency and amplitude of oscillatory pollen tube growth can be altered by changing the osmotic value of the surrounding medium. This has motivated the proposition that the periodic change in growth velocity is caused by changes in turgor pressure. Using mathematical modeling we recently demonstrated that the oscillatory pollen tube growth does not require turgor to change but that this behavior can be explained with a mechanism that relies on changes in the mechanical properties of the cell wall which in turn are caused by temporal variations in the secretion of cell wall precursors. The model also explains why turgor and growth rate are correlated for oscillatory growth with long growth cycles while they seem uncorrelated for oscillatory growth with short growth cycles. The predictions made by the model are testifiable by experimental data and therefore represent an important step towards understanding the dynamics of the growth behavior in walled cells.
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Expansive growth in plant cells is a formidable problem for biophysical studies, and the mechanical principles governing the generation of complex cellular geometries are still poorly understood. Pollen, the male gametophyte stage of the flowering plants, is an excellent model system for the investigation of the mechanics of complex growth processes. The initiation of pollen tube growth requires first of all, the spatially confined formation of a protuberance. This process must be controlled by the mechanical properties of the cell wall, since turgor is a non-vectorial force. In the elongating tube, cell wall expansion is confined to the apex of the cell, requiring the tubular region to be stabilized against turgor-induced tensile stress. Tip focused surface expansion must be coordinated with the supply of cell wall material to this region requiring the precise, logistical control of intracellular transport processes. The advantage of such a demanding mechanism is the high efficiency it confers on the pollen tube in leading an invasive way of life.
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The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.
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Large scale phenotyping of tip growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high throughput execution of more sophisticated assays, an experimental platform was developed based on microfluidic and MEMS (microelectromechanical systems) technology, the TipChip. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels harboring microscopic experimental setups. The tip growing cells, pollen tubes, filamentous yeast or fungal hyphae, can be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform we were able to answer several outstanding questions on pollen tube growth. We established that unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air raising the question how and where water is taken up by the cell. The platform opens new avenues for both, more efficient experimentation and large scale phenotyping of tip growing cells under precisely controlled, reproducible conditions. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
Article
Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening, and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used the Cellular Force Microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20-90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM in combination with a FEM-based model provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
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Fungi, in particular, basidiomycetous fungi, are very successful in colonizing microconfined mazelike networks (for example, soil, wood, leaf litter, plant and animal tissues), a fact suggesting that they may be efficient solving agents of geometrical problems. We therefore evaluated the growth behavior and optimality of fungal space-searching algorithms in microfluidic mazes and networks. First, we found that fungal growth behavior was indeed strongly modulated by the geometry of microconfinement. Second, the fungus used a complex growth and space-searching strategy comprising two algorithmic subsets: 1) long-range directional memory of individual hyphae and 2) inducement of branching by physical obstruction. Third, stochastic simulations using experimentally measured parameters showed that this strategy maximizes both survival and biomass homogeneity in microconfined networks and produces optimal results only when both algorithms are synergistically used. This study suggests that even simple microorganisms have developed adequate strategies to solve nontrivial geometrical problems.
Chapter
Filamentous fungi penetrate diverse solid substrates, including plant and animal tissues, by a process called invasive hyphal growth. Extending hyphae overcome the resistance of their food sources by the secretion of lytic enzymes and the exertion of mechanical force. The forces utilized for invasive growth are derived from turgor pressure and are regulated through loosening of the apical cell wall of the hypha. This chapter explains how hyphae are pressurized and how they apply this pressure during invasive growth. Recent experimental work is discussed, including the use of miniature strain gauges and laser tweezers to measure the forces exerted by hyphae, and information on hyphal mechanics obtained by atomic force microscopy. Other topics in this chapter include current thinking on the role of secreted enzymes and the cytoskeleton in the invasive process, and the remarkable mechanism of leaf penetration by melanized appressoria.
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In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood. We designed an experimental setup using a microscope-mounted galvanotropic chamber that allowed us to induce the redirection of in vitro pollen tube growth through a precisely timed and calibrated external signal. Actin destabilization, reduced calcium concentration in the growth medium and inhibition of calcium channel activity decreased the responsiveness of the pollen tube to a tropic trigger. An increased calcium concentration in the medium enhanced this response and was able to rescue the effect of actin depolymerization. Time-lapse imaging revealed that the motion pattern of vesicles and the dynamics of the subapical actin array undergo spatial reorientation prior to the onset of a tropic response. Together these results suggest that the precise targeting of the delivery of new wall material represents a key component in the growth machinery that determines directional elongation in pollen tubes.
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Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca2+]c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca2+]c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca2+]c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca2+]c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh, root hairs was found to be at a fixed angle from the root of 85 +/- 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca2+]c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface.
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In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.
Article
The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.
Conference Paper
Polymer microstructures were used to examine the manner in which fungal filaments negotiate obstacles in confined environments. When faced with an obstacle requiring a right-angle turn, two different responses were observed. In 21% of cases, hyphae turned around the corner and continued growth, while in the remaining 79% of cases, filaments continued apical growth into the corner, resulting in bending of the distal portions of the filament. The different reactions could not be linked to physical constraints (e.g., filament flexibility) since the filament deflection required to negotiate the obstacle was the same in all cases. Instead, the response appeared to be related to the original direction of growth at the time of filament formation (branching), with filaments turning only if the resultant growth vector was no more than 90° from their original branching vector. The results suggest that filaments are somehow able to retain a memory of their original branching direction, consistent with an overall survival strategy based on continued growth away from the colony center and into the surrounding environment.