Vesicle Supply Center Research Articles

A Myosin Light Chain Is Critical for Fungal Growth Robustness in Candida albicans.

ABSTRACTIn a number of elongated cells, such as fungal hyphae, a vesicle cluster is observed at the growing tip. This cluster, called a Spitzenkörper, has been suggested to act as a vesicle supply center, yet analysis of its function is challenging, as a majority of components identified thus far are essential for growth. Here, we probe the function of the Spitzenkörper in the human fungal pathogen Candida albicans, using genetics and synthetic physical interactions (SPI). We show that the C. albicans Spitzenkörper is comprised principally of secretory vesicles. Mutant strains lacking the Spitzenkörper component myosin light chain 1 (Mlc1) or having a SPI between Mlc1 and either another Spitzenkörper component, the Rab GTPase Sec4, or prenylated green fluorescent protein (GFP), are viable and still exhibit a Spitzenkörper during filamentous growth. Strikingly, all of these mutants formed filaments with increased diameters and extension rates, indicating that Mlc1 negatively regulates myosin V, Myo2, activity. The results of our quantitative studies reveal a strong correlation between filament diameter and extension rate, which is consistent with the vesicle supply center model for fungal tip growth. Together, our results indicate that the Spitzenkörper protein Mlc1 is important for growth robustness and reveal a critical link between filament morphology and extension rate.

The evolution of fungal morphogenesis, a personal account

This article describes the evolution of the field of fungal morphogenesis, its beginning at the end of the 19th century and its exponential growth during the second half of the 20th century, continuing until the present day. The main theme correlates biological progress with the advent of new technologies. Accordingly the article describes the discovery of apical growth, the fibrillar nature of the fungal wall, the chemistry of the cell wall, the search for biochemical pathways in morphogenesis, the discovery of the Spitzenkörper, the apical gradient of wall synthesis, key highlights in ultrastructural research, the development of mathematical models particularly the vesicle supply center (VSC) model, the revolution brought about by molecular biology and unique discoveries such as the hydrophobins and γ-tubulin and some the latest triumphs of the marriage between molecular genetics and confocal microscopy. Credit is given to the investigators responsible for all the advances.

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center.

Secretion and filamentation are mediated by the Candida albicans t-SNAREs Sso2p and Sec9p.

To study the role of late secretion in Candida albicans pathogenesis, we created conditional mutant C.albicans strains in which the t-SNARE-encoding genes SSO2 or SEC9 were placed under the control of a tetracycline-regulated promoter. In repressing conditions, C.albicans tetR-SSO2 and tetR-SEC9 mutant strains were defective in cytokinesis and secretion of aspartyl proteases and lipases. The mutant strains also exhibited a defect in filamentation compared with controls, and thus, we followed the fate of the C.albicans Spitzenkörper, an assembly of secretory vesicles thought to act as a vesicle supply center for the growing hyphae. In the absence of Ca Sso2p, the Spitzenkörper dissipated within 5h and thin-section electron microscopy revealed an accumulation of secretory vesicles. Moreover, the hyphal tip developed into a globular yeast-like structure rather than maintaining a typical narrow hyphae. These studies indicate that late secretory t-SNARE proteins in C.albicans are required for fundamental cellular processes and contribute to virulence-related attributes of C.albicans pathogenesis. Moreover, these results provide direct evidence for a key role of SNARE proteins in vesicle-mediated polarized hyphal growth of C.albicans.

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth.

The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane-associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

Existence and linear stability of solutions of the ballistic VSC model

An equation for the dynamics of the vesicle supply center model of tip growth infungal hyphae is derived. For this we analytically prove the existence anduniqueness of a traveling wave solution which exhibits the experimentallyobserved behavior. The linearized dynamics around this solution is analyzed andwe conclude that all eigenmodes decay in time. Numerical calculation of thefirst eigenvalue gives a timescale in which small perturbations will die out.

Existence of Solutions to the Diffusive VSC Model

We prove existence of classical solutions to the so-called diffusive vesicle supply center (VSC) model describing the growth of fungal hyphae. It is supposed in this model that the local expansion of the cell wall is caused by a flux of vesicles into the wall and that the cell wall particles move orthogonally to the cell surface. The vesicles are assumed to emerge from a single point inside the cell (the VSC) and to move by diffusion. For this model, we derive a nonlinear, nonlocal evolution equation and show the existence of solutions relevant to our application context, namely, axially symmetric surfaces of fixed shape, traveling along with the VSC at constant speed. Technically, the proof is based on the Schauder fixed point theorem applied to Hölder spaces of functions. The necessary estimates rely on comparison and regularity arguments from elliptic PDE theory.

Self-regulation in tip-growth: The role of cell wall ageing

We develop a model to describe the effect of cell wall ageing on the local expansion rate of tip-growing cells. Starting from an exact equation for the stationary age-distribution of the wall material, we propose a generic measure for the local expansion propensity of the wall if the ageing process is described by a constant rate Poissonian decay process. This ageing process may be either interpreted as biochemical in nature describing the finite lifetime of regulatory proteins, or as mechanical in nature describing the gradual “hardening” of the wall through cross-linking or gelation of the wall polymers. In this way we can construct models for tip-growth in which material deposition, evolving wall properties and surface expansion are self-consistently intertwined. As a proof of principle, we implement our ageing approach in two different idealised models of tip-growth, obtaining the stationary tip shapes as a function of the ageing parameter. In the first, the spatial distribution of delivery of growth material is determined by the local curvature of the cell and the growth mode is orthogonal. In the second, the growth material originates from a Vesicle Supply Center, a point-like representation of the Spitzenkörper as found in fungal hyphae, and the growth mode is isometric.

Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB(G2A)::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.

The diffusive vesicle supply center model for tip growth in fungal hyphae

We propose the diffusive vesicle supply center model for tip growth in fungal hyphae. The model is based on the three-dimensional vesicle supply center (VSC) model [Gierz, G., Bartnicki-García, S., 2001. A three-dimensional model of fungal morphogenesis based on the vesicle supply center concept: J. Theor. Biol. 208, 151–164], but incorporates two aspects of a more realistic vesicle delivery mechanism: vesicle diffusion from the VSC and a finite rate constant for vesicle fusion with the cell membrane. We develop a framework to describe tip growth for a general class of models based on the vesicle supply center concept. Combining this with a method for calculating the steady state distribution of diffusive vesicles we iteratively solve for stationary cell shapes. These show a blunter tip than predicted by the original VSC model, which we attribute to increased forward-directed vesicle delivery via diffusion. The predicted distance between the VSC and the utmost tip of the cell is set by the ratio between the diffusion constant and the rate constant for vesicle exocytosis. Combined with the cell radius, these define the only dimensionless parameter for our model.

The Role of the Kinesin Motor KipA in Microtubule Organization and Polarized Growth ofAspergillus nidulans

Polarized growth in filamentous fungi requires the integrity of the microtubule (MT) cytoskeleton. We found that growing MTs in Aspergillus nidulans merge at the center of fast growing tips and discovered that a kinesin motor protein, KipA, related to Tea2p of Schizosaccharomyces pombe, is required for this process. In a DeltakipA strain, MT plus ends reach the tip but show continuous lateral movement. Hyphae lose directionality and grow in curves, apparently due to mislocalization of the vesicle supply center (Spitzenkörper) in the apex. Green fluorescent protein (GFP)-KipA accumulates at MT plus ends, whereas a KipA rigor mutant protein, GFP-KipA(G223E), coated MTs evenly. These findings suggest that KipA requires its intrinsic motor activity to reach the MT plus end. Using KipA as an MT plus-end marker, we found bidirectional organization of MTs and determined the locations of microtubule organizing centers at nuclei, in the cytoplasm, and at septa.

Image analysis of hyphal morphogenesis in Saprolegniaceae (Oomycetes)

Because of their wide range of apical morphology, several members of saprolegniaceous fungi (Oomycetes) were chosen to examine concordance with the vesicle supply center (VSC) model of hyphal morphogenesis. Two computer routines were devised to measure diameter changes over long stretches of hyphae and to test compatibility with the theoretical hyphoid shape, y= xcot( xV/ N). In all four genera examined, the apex followed closely the contour described by the hyphoid equation; divergences became evident in the subapex. The hyphae of Saprolegnia parasitica showed maximum concordance with the VSC model, i.e., their profile matched a hyphoid curve from the apex to the entire length of the mature hyphal tube. In Aphanomyces and Leptolegnia, growth in the subapical region subsided becoming less than that specified by the hyphoid equation. In Achlya bisexualis, the reverse was true, the subapical region expanded beyond that specified by the hyphoid equation. The two divergent subapical tendencies gave the hyphal tips a cylindroid or conoid appearance, respectively. Since the hyphal apex of all four species conformed to the curvature dictated by the hyphoid equation, we concluded that a basic VSC mechanism operates in all of these oomycetous fungi. Accordingly, we suggest that the shape of an oomycetous hypha is generated by a VSC-driven gradient of wall formation, which is subject to additional modification in the subapex to produce a range of hyphal tip morphologies. The mathematical basis for generating a conoid hyphal tip by elongating the VSC is described in Appendix A. Index descriptors: Conoid tips; Growth; Hyphae; Hyphal diameter; Hyphoid model; Morphogenesis; Morphology; Oomycetes; VSC

A Three-Dimensional Model of Fungal Morphogenesis Based on the Vesicle Supply Center Concept

We developed a three-dimensional model of hyphal morphogenesis under the same basic assumption used for the construction of a two-dimensional model. Namely, that the polarized growth of tubular cells (hyphoids) arises from a gradient of wall-building vesicles generated by a vesicle supply center (VSC). Contrary to the 2-D mathematical formulation, the three-dimensional derivation led to an indetermination whose solution required defining a priori the pattern of expansion of the wall, i.e. defining the overall spatial movement of the wall as the newly inserted wall elements displace the existing wall fabric. The patterns of wall expansion can be described by tracing the movement of marker points on the cell surface (point trajectories). Point trajectories were computed for three different modes of wall expansion of the VSC-generated hyphoids: orthogonal, isometric, and rotational. The 3-D VSC models allowed us to either stipulate or calculate the degree of anisotropy for each type of wall expansion. Wireframe models were built to visualize growth anisotropy in each model. Although the overall shape of the three hyphoid models is similar, they differ substantially in point trajectories and anisotropy. Point trajectories are experimentally testable and were the basis for the conclusion that hyphae grow in orthogonal fashion. (Bartnicki-Garcia et al., 2000. Biophys. J.79, 2382–2390.)

Mapping the Growth of Fungal Hyphae: Orthogonal Cell Wall Expansion during Tip Growth and the Role of Turgor

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.

What Determines Growth Direction in Fungal Hyphae?

We used high-resolution video microscopy and image analysis to map the trajectory of the Spitzenkörper in growing hyphae ofNeurospora crassaand to correlate it with growth directionality. The Spitzenkörper followed a tortuous trajectory produced by a dominant forward motion accompanied by frequent, transverse oscillations. In hyphae with a fixed growth direction, the regression line of the Spitzenkörper trajectory corresponded to the longitudinal axis of the hypha. A permanent change in growth direction, i.e., the establishment of a new growth axis, was correlated with a sustained shift in Spitzenkörper trajectory away from the existing cell axis. In meandering hyphae, changes in growth directionality occurred somewhat erratically but there was a strong compensatory tendency reversing directional shifts and maintaining an overall fixed direction of growth. Although external factors greatly affect hyphal growth direction (tropisms), they are probably not the primary determinants of growth directionality. Inhibitors of microtubules, but not of actin microfilaments, caused hyphae to lose their growth directionality—providing support for the idea that Spitzenkörper trajectory is determined internally by a growing scaffolding of cytoplasmic microtubules. The meandering morphology ofN. crassahyphae was duplicated by computer simulation in support of the idea that hyphal morphogenesis is controlled by the position of the Spitzenkörper functioning as a vesicle supply center.

How hyphae grow: Morphogenesis explained?

Apical growth of fungal hyphae represents a relatively simple instance of cellular morphogenesis. Thanks to the polarized transport and exocytosis of precursor vesicles, new cell wall and plasma membrane are continuously deposited at the hyphal apex; the question is how the characteristic shape of tube and tapered tip comes about. Recent experiments lend support to a model whose central feature is a mobile vesicle supply center corresponding to the Spitzenkorper (apical body) visible in growing hyphae. Shapes predicted by the model agree remarkably well with those of actual hyphae. Nevertheless, critical examination of the model's premises suggests that it requires extension so as to incorporate both a driving force for expansion and a gradient of cell wall plasticity. I propose that a mobile vesicle supply center may be one, but only one, of a range of physiological devices employed by tip-growing organisms to localize the exocytosis of precursor vesicles. Apical growth should ensue whenever the loci of exocytosis advance vectorially, and nascent cell wall expands in a graded manner.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger.

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the FUNGUS SIMULATOR program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC-i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Determinants of fungal cell wall morphology: the vesicle supply center

Because fungal cell walls are shaped during construction, factors that regulate wall biogenesis must play a decisive role in morphogenesis. By delivering ingredients for wall formation in vesicles, fungi have a de facto mechanism to mold their own shape. The gradients in wall formation required for cell morphogenesis ensue from the pattern of vesicle discharge. A vesicle-based, mathematical model of fungal morphogenesis revealed that a relatively simple process could establish these gradients. The model and its underlying hyphoid equation assumes that wall-building vesicles emanate from a vesicle supply center (VSC). The VSC serves as an organizing center from which vesicles would move radially to the cell surface in all directions at random. VSC displacement is then an immediate cause of morphogenesis: a sustained linear displacement of the VSC would generate a polarized pattern of exocytosis required to make a hypha. The model predicts that the Spitzenkörper functions as a VSC. We have tested this prediction by analyzing instances where dislocations in Spitzenkörper position result in hyphal deformations. When the VSC was programmed to duplicate the movements of the Spitzenkörper before, during, and after a deformation, the resulting shapes mimicked closely the observed deformations. These correlations support the contention that the position and movement of the VSC determines the morphology of the fungal cell wall. The computer model has been refined to incorporate transverse random oscillations of the VSC to simulate more realistically the meandering shape of fungal hyphae. The model predicts that hyphal ring formation results from a sustained directional bias to the transverse oscillations of the Spitzenkörper. Key words: hyphal morphogenesis, Spitzenkörper, vesicle supply center, mathematical model, hyphoid equation, hyphal curvature.

Evidence That Spitzenkörper Behavior Determines the Shape of a Fungal Hypha: A Test of the Hyphoid Model

Bartnicki-Garcia, S. Bartnicki, D. D., Gierz, G., López-Franco, R., and Bracker, C. E. 1995. Evidence that Spitzenkörper behavior determines the shape of a fungal hypha; A test of the hyphoid model. Experimental Mycology 19, 153-159. Hyphae of the fungus Rhizoctonia solani have a characteristic Spitzenkörper in their growing tips and a cell shape described by the mathematical hyphoid equation. A mild disturbance of hyphae growing in a slide culture chamber on a microscope stage caused the Spitzenkörper to move away from its usual position next to the apical pole and wander briefly inside the apical dome. Hyphal elongation rate declined abruptly, and the apex became rounded and increased in diameter. As the Spitzenkörper migrated back to its polar position, rapid cell elongation resumed, and the contour of the growing hyphal tip returned to the typical hyphoid shape. The brief dislocation of the Spitzenkörper left a permanent bulge in the hyphal profile. This morphogenetic sequence was mimicked by computer simulation, based on the hyphoid equation which relates the generation of hyphal shape to the linear displacement of a vesicle supply center (VSC). The VSC was programmed to retrace the observed movements of the Spitzenkörper during the above sequence. The resulting similarity of shape between real and computer-simulated cells reinforces the mathematical prediction that the Spitzenkörper acts as a VSC and that its continuous linear advancement generates a typical hyphal tube with the characteristic hyphoid shape. Accordingly, the hyphoid model and its VSC concept provide a plausible hypothesis to explain the cellular basis of polarized growth of fungal hyphae.