The Organization of mitochondria in growing hyphae of Neurospora crassa

Details of mitochondrial system organization and behavior in the tip of growing N. crassa hyphae and hyphal fragments were studied by means of cell-permeable mitochondria-selective probes in glucose- and sorbitol-containing media. It was shown that filamentous mitochondria concentrate in the 30-μm apical zone of growing hyphae and hyphal fragments independently of the carbon source. The mitochondrial assemblies propagate forward with elongation of hypha, split upon apical branching, and are formed de novo when branches are formed away from the growing tip. These activities resemble microtubule behavior. Co-staining with Mitotracker Red and Mitotracker Green revealed possible functional heterogeneity in subpopulations of mitochondria. It was proposed that the observed features are governed by the microtubular network, while the primary function of mitochondria was to sustain the growth rate. The organizing role of electrical gradients on the growing tip and their influence on mitochondrial and microtubular networks are discussed.     

Patterns of cortical and perinuclear microtubule organization in meristematic root cells ofAdiantum capillus veneris

Summary The interphase meristematic root cells ofAdiantum capillus venerispossess a well developed cytoskeleton of cortical microtubules (Mts), which disappear at prophase. The preprophase-prophase cells display a well organized preprophase microtubule band (PMB) and a perinuclear Mt system. The observations favour the suggestion that the cell edges included in the PMB cortical zone possess a Mt organizing capacity and thus play an important role in PMB formation. The perinuclear Mts are probably organized on the nuclear surface. The preprophase-prophase nuclei often form protrusions towards the PMB cortical zone and the spindle poles, assuming a conical or rhomboid shape. Mts may be involved in this nuclear shaping.Reinstallation of cortical Mts in dividing cells begins about the middle of cytokinesis with the reappearance of short Mts on the cell surface. When cytokinesis terminates, numerous Mts line the postcytokinetic daughter wall. Many of them converge or form clusters in the cytoplasm occupying the junctions of the new and the old walls. In the examined fern, the cortical Mt arrays seem to be initiated in the cortex of post-cytokinetic root cells. A transitory radial perinuclear Mt array, comparable to that found in post-telophase root cells of flowering plants, was not observed inA. capillus veneris.     

Aggregation and collapse in a mechanical model of fungal tip growth

 Interconnected hyphal tubes form the mycelia of a fungal colony. The growth of the colony results from the elongation and branching of these single hyphae. The material being incorporated into the extending hyphal wall is supplied by vesicles which are formed further back in the hyphal tip. Such wall-destined vesicles appear conspicuously concentrated in the interior of the hypha, just before the hyphal apex, in the form of an apical body or Spitzenk?rper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenk?rper and the transport of vesicles, but as yet there is no postulated mechanism for this. We propose a mechanism by which forces generated by the cytoskeleton are responsible for biasing the movement of vesicles. A mathematical model is derived where the cytoskeleton is described as a viscoelastic fluid. Viscoelastic forces are coupled to the conservation equation governing the vesicle dynamics, by weighting the diffusion of vesicles via the strain tensor. The model displays collapse and aggregation patterns in one and two dimensions. These are interpreted in terms of the formation of the Spitzenk?rper and the initiation of apical branching. Received: 16 September 1996 / Revised version: 20 July 1998     

Key differences between lateral and apical branching in hyphae of Neurospora crassa

We examined in fine detail growth kinetics and intracellular events during lateral and apical branching in hyphae of Neurospora crassa. By high-resolution video-enhanced light microscopy, we found remarkable differences in the events preceding lateral vs apical branching. While apical branching involved a significant disturbance in the apical growth of the parental hypha, lateral branching occurred without any detectable alterations in the growth of the parental hypha. Prior to the emergence of a lateral branch, an incipient Spitzenk?rper was formed about 12-29 microm behind the apex. Lateral branch formation did not interfere with the elongation rate of the primary hypha, the shape of its apex or the behavior of its Spitzenk?rper. In sharp contrast, apical branching was preceded by marked changes in physiology and morphology of the parental hypha and by a sharp drop in elongation rate. The sequence involved a cytoplasmic contraction, followed by a retraction, dislocation, and disappearance of the Spitzenk?rper; hyphal elongation decreased sharply and a transient phase of isotropic growth caused the hyphal apex to round up. Growth resumed with the formation of two or more apical branches, each one with a Spitzenk?rper formed by gradual condensation of phase-dark material (vesicles) around an invisible nucleation site. The observed dissimilarities between lateral and apical branching suggest that these morphogenetic pathways are triggered differently. Whereas apical branching may be traced to a sudden discrete disruption in cytoplasmic organization (cytoplasmic contraction), the trigger of lateral branching probably stems from the subapical accumulation of wall precursors (presumably vesicles) reaching a critical concentration.     

Immunofluorescence Study of Microtubule Organization in Some Polarized Cell Types of Selected Brown Algae

The microtubule (Mt) organization in apical cells of Sphacelaria rigidula. as well as in branch initials of S. rigidula and Ectocarpus siliculosus, was studied by immunofluorescence. The apical interphase cells of S. rigidula show an impressive cytoskeleton of Mts, converging on the centrosome(s). A number of Mt bundles are perinuclear, but most of them run in axial orientation from the centrosomes to the cell cortex. The anterior Mt system consists of numerous thin Mt bundles, whereas the posterior system contains fewer and thicker bundles. In cells entering prophase, the cytoplasmic Mts gradually disappear. This process is somewhat faster at the posterior than at the anterior pole of the premitotic nucleus. After mitosis, the cytoplasmic Mts of the apical region appear to be re-organized more rapidly than those of the basal part of the cell. The apical daughter nucleus retains a lobed shape and condensed chromatin for a longer time, and increases considerably in size between telophase and cytokinesis, compared to the basal one. Duplication of the centrosomes proceeds more rapidly in the anterior region of apical cells than in the basal part. During branch formation in S. rigidula and E. siliculosus, a new polarity axis is established, and the Mts extend towards the protrusion into which the nucleus migrates before mitosis. After nuclear division, one of the daughter nuclei is positioned at the tip of the branch, where the apical Mt focussing point is localized.     

Microtubules Are required for motility and positioning of vesicles and mitochondria in hyphal tip cells of Allomyces macrogynus

We have used video-enhanced light microscopy and digital image processing to characterize the intracellular motility and positioning of vesicles ( approximately 1-microm diameter) and mitochondria in growing hyphal tip cells of Allomyces macrogynus. These observations were coupled with cytoskeletal inhibitory experiments to define the roles of the microtubule and actin cytoskeletons in organelle translocation and positioning. Vesicles and mitochondria were abundant in apical and subapical hypha regions. Vesicles traveled along paths that were parallel to the longitudinal axis of the cell. Anterograde (i.e., toward the hyphal apex) and retrograde (i.e., away from the hyphal apex) movements of vesicles occurred at average rates of 4.0 and 2.2 microm/s, respectively. Bidirectional travel of vesicles along common paths was noted in the cortical cytoplasm. Mitochondria were aligned mostly parallel to the long axis of the hypha, except those extending into the hyphal apex, which were oriented toward the Spitzenk?rper. In regions of the subapical hypha mitochondria were often restricted to the cortical cytoplasm and nuclei occupied the central cytoplasmic region. Mitochondria displayed rapid anterograde movements reaching speeds of 3.0 microm/s, but primarily maintained a constant position relative to either the advancing cytoplasm or the lateral cell wall. Cytoskeletal disruption experiments showed that the positioning of mitochondria and motility of vesicles and mitochondria were microtubule-based and suggested that the actin cytoskeleton played uncertain roles.     

Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Aggregation and collapse of fungal wall vesicles in hyphal tips: a model for the origin of the Spitzenk?rper

The intracellular origins of polarity and branch initiation in fungi centre upon a localization in the supply of fungal wall constituents to specific regions on the hyphal wall. Polarity is achieved and maintained by accumulating secretory vesicles, prior to incorporation into the wall, in the form of an apical body or Spitzenkörper. However, neither the mechanisms leading to this accumulation nor the initiation of branching, are as yet understood. We propose a mechanism, based on experimental evidence, which considers the mechanical properties of the cytoskeleton in order to explain these phenomena. Cytoskeletal viscoelastic forces are hypothesized to be responsible for biasing vesicles in their motion, and a mathematical model is derived to take these considerations into account. We find that, as a natural consequence of the assumed interactions between vesicles and cytoskeleton, wall vesicles aggregate in a localized region close to the tip apex. These results are used to interpret the origin of the Spitzenkörper. The model also shows that an aggregation peak can collapse and give rise to two new centres of aggregation coexisting near the tip. We interpret this as a mechanism for apical branching, in agreement with published observations. We also investigate the consequences and presumptive role of vesicle–cytoskeleton interactions in the migration of satellite Spitzenkörper. The results of this work strongly suggest that the formation of the Spitzenkörper and the series of dynamical events leading to hyphal branching arise as a consequence of the bias in vesicle motion resulting from interactions with the cytoskeleton.     

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans

Filamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. Until now, there have been no tools to visualize actin or the actin-binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in Aspergillus nidulans . We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch-depleted zone occupied the apical 0.5 μm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.     

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.     

Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle ofDictyota dichotoma (Phaeophyta,Dictyotales)

Summary Interphase cells ofDictyota dichotoma (Hudson) Lamour. lack cortical microtubules (Mts) but display an impressive network of cytoplasmic microtubules (c-Mts). These are focussed on two opposed perinuclear centriolar sites where centrin or a centrin-homologue is localized. Some of the Mts surround the nucleus, but the majority traverse the cytoplasm as bundles variously directed towards the plasmalemma. In apical cells, and to a lesser extent in the square or slightly elongated meristematic cells, Mts are more or less evenly arranged. In elongated cells they form thick bundles longitudinally traversing the cytoplasm; a pattern maintained in differentiated cells. In early prophase the non-perinuclear Mts disappear but by late prophase a bi-astral arrangement of short Mts is observed. They enter polar nuclear depressions and attach to differentiated regions of the nuclear envelope where polar gaps open. By metaphase the spindle Mts converge on the centrioles at the polar gaps. At anaphase, interzonal Mts are evident and the asters start to reassemble. After telophase disruption of the interzonal Mts, the daughter nuclei approach each other, but move apart again before cytokinesis. The latter movement keeps pace with the development of two interdigitating Mt systems, ensheathing both daughter nuclei. The partition membrane bisects this Mt cage. Between telophase and cytokinesis the centrosomes separate, finally occupying opposed perinuclear sites. New Mts arise at the new centrosomes, some terminating on the consolidating partition membrane. Our data show thatD. dichotoma vegetative cells display a prominent cytoplasmic Mt cytoskeleton, which undergoes continual, but definite, change in organization during the cell cycle.     

NUCLEAR BEHAVIOR,SEPTATION, AND HYPHAL GROWTH OF ALTERNARIA SOLANI

Nuclei of multinucleate hyphal tip cells divided almost synchronously. Nuclear division was followed by multiple septation during which the elongated hyphal tip cell was divided into several, generally multinucleate, cells. Within the same hypha a distinct growth pattern was not observed with respect to number, length, and nuclear number of cells formed with each successive nuclear division-septation cycle. Hyphal branches originated and received nuclei from hyphal tip cells.     

Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.     

Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

A screen for Neurospora knockout mutants displaying growth rate dependent branch density

Branch density (the spatial distribution of branch initiation points along a growing hypha) in wild-type Neurospora has been shown to remain constant at different growth rates due to a hypothesized system which compensates for hyphal growth rate. Here we report the results of a survey of the Neurospora knockout library for mutants affecting this proposed growth rate compensation system. The mutants identified fail to maintain branching homeostasis?at different growth rates, thus showing growth rate-dependent branch density. The gene functions highlighted by this screen are diverse with several emerging themes including: ubiquitin-binding proteins, kinases, metal binding/metal metabolism proteins, reactive oxygen species (ROS) control proteins, and clock-associated/clock-controlled proteins. Other than their common influence on branch density homeostasis, the relationships between these gene functions and how they interact to influence branching are unclear.     

On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

Microtubular fir-trees in mitotic spindles of onion roots

Summary The organization of kinetochore fibers was examined inAllium root cells processed for tubulin immunocytochemistry. Metaphase fibers consist of a core or trunk of Mts to which are attached numerous branches, yielding a bottle-brush of fir-tree pattern similar to that reported inHaemanthus endosperm cells. Many of the branches cross the midzone and extend into the opposite half-spindle. In addition, branch Mts associate with more than one kinetochore fiber. During anaphase, branch Mts elongate while the trunks shorten and fuse into polar caps. Our results are discussed in terms of spindle fiber organization and Mt polarity.Abbreviations K Kinetochore - Mt microtubule     

THE CORTICAL MICROTUBULAR CYTOSKELETON OF OXYRRHIS MARINA (DINOPHYCEAE) OBSERVED WITH IMMUNOFLUORESCENCE AND ELECTRON MICROSCOPY1

The cortical microtubular cytoskeleton of Oxyrrhis marina Dujardin was investigated using indirect immunofluorescence and transmission electron microscopy. The cortical microtubular cytoskeleton is unlike that of other previously examined dinoflagellates because all cortical microtubules are oriented longitudinally and do not attach or abut tranverse microtubular arrays. This difference is considered along with other morphological and cytological variables as indicative of Oxyrrhis's phylogenetic position relative to the Dinophyceae.     

Analysis of morphological relationship between micro- and macromorphology of Mortierella species using a flow-through chamber coupled with image analysis

Using a flow-through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro- and macromorphology was investigated. On potato-dextrose-agar plates, 5 species formed rose petal-like colonies, 3 formed large round colonies, and 3 formed donut-like colonies. By observing micromorphology in a flow-through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (q(b): tip/microm/h) and a low tip extension rate (q(tip): microm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet-like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow-specific hyphal growth rate (mu(l)) range (0.35-0.45 h(-1)), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet-like morphology and a pellet core. When the branch formation rate was lower than 2 x 10(-3) tips/microm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h(-1)), low tip extension rate (20 tips/microm/h), and high branch formation rate (8 x 10(-3) tips/microm/h) forms the suitable macromorphology for arachidonic acid production.