Hyphal tip growth inPhytophthora

Germinating cysts and isolated walls from germinating cysts incorporated¹⁴C-UDPG into wall material of which 22.5 and 15% respectively were insoluble in boiling 1 N HCl, indicating that part of the synthetase activity is located in the wall itself. A combination of Urografin and Ficol density gradients was used to separate various intracellular fractions. A consistent separation of -glucanase and UDPG-transferase enriched fractions was achieved. The -glucanase fraction contained dictyosome vesicles and fragments along with some plasma membranes. The UDPG-transferase fraction was relatively rich in membranes resembling rough and smooth ER. The results suggest the two enzymes are transported to the wall by different intracellular routes, and two types of vesicle may be involved. Alkaline phosphatase, -glucosidase and acid phosphatase were found extracellularly and their distribution in density gradients determined. The results of histochemical staining for acid phosphatase, alkaline phosphatase and polysaccharide are described and compared with the biochemical data. -1,3-glucanase, found intra- and extracellularly, induced distorted growth of germ tubes and also removed most of the apical wall when added to the incubation medium. None of these responses were observed with cellulase. Determinations of the osmotic pressure of germinating cysts and incubation medium revealed that the turgor of germinating cysts amounts to about 1.8 at under the conditions used.     

Polarized cell growth: double grip by CDK1

Precise coupling of cell growth and cell-cycle progression is crucial for achieving cell homeostasis. A recent study sheds light on two distinct roles of cyclin-dependent kinase 1 (CDK1) in promoting polarized cell growth in budding yeast.     

Rapid tip growth: insights from pollen tubes

Pollen tubes extend rapidly in an oscillatory manner by the extreme form of polarized growth, tip growth, and provide an exciting system for studying the spatiotemporal control of polarized cell growth. The Rho-family ROP GTPase is a key signaling molecule in this growth control and is periodically activated at the apical plasma membrane to spatially define the apical growth region and temporally precede the burst of growth. The spatiotemporal dynamics of ROP GTPase is interconnected with actin dynamics and polar exocytosis that is required for tip-targeted membrane and wall expansion. Recent advances in the study of the mechanistic interlinks between ROP-centered signaling and spatiotemporal dynamics of cell membrane and wall remodeling will be discussed.     

Signaling tip growth in plants

Tip growth is an extreme form of polar growth modulated by both intrinsic and extrinsic spatial cues. Pollen tubes and root hairs have been used as model systems to investigate tip growth signaling in higher plants. Recent studies have focused on tip-localized Ca2+ gradients and Rho GTPases in pollen tubes and a series of mutants affecting root hair tip growth. These molecular and genetic markers will serve as stepping stones towards uncovering tip growth pathways in plants.     

Polarized growth in fungi: symmetry breaking and hyphal formation

Cell shape is a critical determinant for function. The baker's yeast Saccharomyces cerevisiae changes shape in response to its environment, growing by budding in rich nutrients, forming invasive pseudohyphal filaments in nutrient poor conditions and pear shaped shmoos for growth towards a partner during mating. The human opportunistic pathogen Candida albicans can switch from budding to hyphal growth, in response to numerous environmental stimuli to colonize and invade its host. Hyphal growth, typical of filamentous fungi, is not observed in S. cerevisiae. A number of internal cues regulate when and where yeast cells break symmetry leading to polarized growth and ultimately distinct cell shapes. This review discusses how cells break symmetry using the yeast S. cerevisiae paradigm and how polarized growth is initiated and maintained to result in dramatic morphological changes during C. albicans hyphal growth.     

At the poles across kingdoms: phosphoinositides and polar tip growth

Phosphoinositides (PIs) are minor, but essential phospholipid constituents of eukaryotic membranes, and are involved in the regulation of various physiological processes. Recent genetic and cell biological advances indicate that PIs play important roles in the control of polar tip growth in plant cells. In root hairs and pollen tubes, PIs control directional membrane trafficking required for the delivery of cell wall material and membrane area to the growing tip. So far, the exact mechanisms by which PIs control polarity and tip growth are unresolved. However, data gained from the analysis of plant, fungal and animal systems implicate PIs in the control of cytoskeletal dynamics, ion channel activity as well as vesicle trafficking. The present review aims at giving an overview of PI roles in eukaryotic cells with a special focus on functions pertaining to the control of cell polarity. Comparative screening of plant and fungal genomes suggests diversification of the PI system with increasing organismic complexity. The evolutionary conservation of the PI system among eukaryotic cells suggests a role for PIs in tip growing cells in models where PIs so far have not been a focus of attention, such as fungal hyphae.     

Establishing and maintaining axial growth: wall mechanical properties and the cytoskeleton

Organ morphology depends on cell placement and directional cell expansion. Microtubules are involved in both of these processes so genetic approaches to understand the role microtubules play in organ expansion are not straightforward. Our use of the temperature-sensitive mor1-1 mutants led to the surprising discovery that Arabidopsis thaliana (L.) Heynh. root cells can establish and maintain transverse cellulose texture without well organized microtubule arrays. This work also demonstrated that cells can lose the ability to expand anisotropically without losing transversely oriented cellulose microfibrils. We suggest that microtubule disruption affects the cells ability to generate long cellulose microfibrils, which may be essential for achieving growth anisotropy. Thus organ shape may depend not only on the orientation but also on the relative length of cellulose microfibrils during axis establishment and growth. More recent work has shown an important correlation between microtubule organization and the deposition patterns of the glycosylphosphatidylinositol (GPI)-anchored wall protein COBRA. Loss of microtubule organization is associated with the dissipation of transverse banding patterns of COBRA, suggesting that COBRAs function in maintaining anisotropic expansion may be microtubule-dependent.     

Developmental control of cell morphogenesis: a focus on membrane growth

To date, the role of transport and insertion of membrane in the control of membrane remodelling during cell and tissue morphogenesis has received little attention. In contrast, the contributions of cytoskeletal rearrangements and both intercellular and cell-substrate attachments have been the focus of many studies. Here, we review work from many developmental systems that highlights the importance of polarized membrane growth and suggests a general model for the role of endocytic recycling during cell morphogenesis. We also address how the spatio-temporal control of membrane insertion during development can account for various classes of tissue rearrangements. We suggest that tubulogenesis, tissue spreading and cell intercalation stem mostly from a remarkably small number of cell intrinsic surface remodelling events that confer on cells different modes of migratory behaviours.     

酵母交配过程的极性生长及其调控机制

酿酒酵母单倍体细胞能够与相反交配型的单倍体细胞发生交配。交配时酿酒酵母放弃原有出芽位点,根据信息素的浓度梯度,重新选择生长位点,向相反交配型细胞伸出突起进行极性生长。交配因子受体指导选择交配突起的位点,通过G蛋白激活Ste20p,将信号经由Ste11p、Ste7p和Fus3p组成的MAPK模块传递到Far1p和Ste12p等因子,调控相关基因的转录,抑制原有的出芽位点,选择新的生长位点,并使细胞周期停止在G1期,G蛋白与Cdc24p、Cdc42p和Bem1p等蛋白作用,聚集在细胞,使得肌协蛋白细胞骨架在交配突起处聚集,呈极性化分布,使细胞发生极性生长。     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Microtubules guide root hair tip growth

The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip-growing fission yeast cells have been shown to play a particularly important role in regulating cell polarity. By placing proteins that serve as spatial cues in the cell cortex of the expanding tip, microtubules determine the site where exocytosis, and therefore growth, takes place. Transport and the targeting of exocytotic vesicles to the very tip depend on the actin cytoskeleton. Recently, endoplasmic microtubules have been identified in tip-growing root hairs, which are an experimental system for plant cell growth. Here, we review the data that demonstrate involvement of microtubules in hair elongation and polarity of the model plants Medicago truncatula and Arabidopsis thaliana. Differences and similarities between the microtubule organization and function in these two species are discussed and we compare the observations in root hairs with the microtubule-based polarity mechanism in fission yeast.     

Mechanisms of hyphal tip growth: tube dwelling amebae revisited

Over 100 years ago, Reinhardt suggested that hyphal tip growth is comparable to ameboid movement inside a tube; the apical cytoplasm being protruded like a pseudopodium with the wall assembled on its surface. There are increasing data from hyphae which are explicable by this model. Fungi produce pseudopodia-like structures and their cytoplasm contains all of the major components implicated in pseudopodium production in animal cells. Most of these components are concentrated in hyphal tips and tip growth involves actin, a major component of pseudopodia. Together these data indicate that the essence of the ameboid model is still tenable. However, detailed mechanisms of tip growth remain too poorly known to provide definitive proof of the model and the behavior of the trailing cytoplasm indicates differences which are probably a response to the walled lifestyle.     

Critical evaluation of the VSC model for tip growth

The vesicle supply centre (VSC) model (Bartnicki-Garcia et al., 1989) for hyphal tip growth is powerful because it can model diverse developmental morphologies and predicts cellular organization based in current cell biology. It predicts that tip growth results from the random distribution of cell surface synthesizing vesicles from a point in the tip, the VSC, which determines their pattern of impact and fusion at the plasma membrane. We derive equations for tip-high gradients of vesicle fusions, generated by mechanisms not related to a supply centre, which create typical hyphal morphologies. These equations direct the conceptual basis for tip growth to vesicle fusion gradients, presumably mediated by a putative membrane skeleton associated with the plasma membrane. We also show that the organization and behaviour of motile organelles in growing hyphal tips of the oomycete,Saprolegnia ferax, argue against the presence of an apparatus capable of generating the distribution of vesicles postulated by the VSC model. We conclude that the VSC model is unlikely to describe the mechanistic basis of tip growth inS. ferax, and therefore, at best, it is not universally applicable.     

The tip growth apparatus of Aspergillus nidulans

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.     

Calcium regulation of tip growth: new genes for old mechanisms

We review the recent advances on Ca(2+) in tip-growing cells, with a special focus on pollen tubes. New genes for Ca(2+) pumps, channels and sensing proteins have been recently described, with special emphasis on cyclic nucleotide gated channels (CNGCs) and glutamate receptor-like channels (GLRs). We also review the current state of knowledge in what concerns Ca(2+) sensor and relay proteins, where the knowledge of the cell models is less advanced. While these newly described genes offer promise to a better understanding of the spatial and temporal patterns of Ca(2+) signalling that may be relevant for the formation of the phenotype, we discuss the necessity to investigate further links in the network downstream of the Ca(2+) signature, with a special need for mechanisms of feed-back that might render functional feed-back loops approachable by modelling and genetics. Given the available literature, we conclude on the need to investigate more on the role of two specific classes of proteins, the calcium binding protein kinases (CPKs) and the Calcineurin B-like proteins (CBLs) and their regulatory relationships to ion channels (summarized in Figure 3b).     

Building a hair: tip growth in Arabidopsis thaliana root hairs

The Arabidopsis thaliana root hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip of Arabidopsis root hairs has been identified by patch-clamp measurements. Actin has been visualized in vivo in Arabidopsis root hairs by using a green-fluorescent-protein-talin reporter and shown to form a dense mesh in the apex of the growing tip. The kojak gene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role for LRX1, a leucine-rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.