Apical Branching in a Temperature Sensitive Mutant ofAspergillus niger

An apical branching, temperature-sensitive, mutant ofAspergillus niger(ramosa-1) was isolated by UV mutagenesis.Ramosa-1has a wild type morphology at 23°C, but branches apically when shifted to 34°C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenkörper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210–240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenkörper two new Spitzenkörper appeared, each giving rise to an apical branch. The second branch Spitzenkörper appeared with a 60- to 100-s delay. We did not observe the original Spitzenkörper dividing in two; instead, the new Spitzenkörper arosede novofrom vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenkörper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenkörper. Accordingly, we propose that the apical branching phenotype inramosa-1is triggered by a molecular event that induces a transient alteration in cytoskeleton organization.     

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with the subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenk?rper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289–298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenk?rper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropism in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton. Received: 14 September 1997 / Accepted: 16 October 1997     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi

Summary. The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell–substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.Correspondence and reprints: Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.Received October 25, 2002; accepted February 26, 2003; published online August 26, 2003     

Rhizoids and protonemata of characean algae: model cells for research on polarized growth and plant gravity sensing

Gravitropically tip-growing rhizoids and protonemata of characean algae are well-established unicellular plant model systems for research on gravitropism. In recent years, considerable progress has been made in the understanding of the cellular and molecular mechanisms underlying gravity sensing and gravity-oriented growth. While in higher-plant statocytes the role of cytoskeletal elements, especially the actin cytoskeleton, in the mechanisms of gravity sensing is still enigmatic, there is clear evidence that in the characean cells actin is intimately involved in polarized growth, gravity sensing, and the gravitropic response mechanisms. The multiple functions of actin are orchestrated by a variety of actin-binding proteins which control actin polymerisation, regulate the dynamic remodelling of the actin filament architecture, and mediate the transport of vesicles and organelles. Actin and a steep gradient of cytoplasmic free calcium are crucial components of a feedback mechanism that controls polarized growth. Experiments performed in microgravity provided evidence that actomyosin is a key player for gravity sensing: it coordinates the position of statoliths and, upon a change in the cell's orientation, directs sedimenting statoliths to specific areas of the plasma membrane, where contact with membrane-bound gravisensor molecules elicits short gravitropic pathways. In rhizoids, gravitropic signalling leads to a local reduction of cytoplasmic free calcium and results in differential growth of the opposite subapical cell flanks. The negative gravitropic response of protonemata involves actin-dependent relocation of the calcium gradient and displacement of the centre of maximal growth towards the upper flank. On the basis of the results obtained from the gravitropic model cells, a similar fine-tuning function of the actomyosin system is discussed for the early steps of gravity sensing in higher-plant statocytes.     

Diversity and dynamics of the Spitzenkörper in growing hyphal tips of higher fungi

Summary The Spitzenkörper, located in the apex of growing hyphae of septate fungi, has been portrayed previously as a spheroid complex containing a cluster of apical (secretory) vesicles which sometimes encloses a differentiated core area. With the aid of computer-enhanced video microscopy and phase-contrast optics, we studied 32 fungi in the Ascomycetes, Deuteromycetes, Hyphomycetes, Basidiomycetes, and Agonomycetes. The Spitzenkörper appeared as a highly dynamic and pleomorphic multicomponent complex capable of changing shape, size, and position within the hyphal apex during growth. The main theme of this study is to demonstrate two kinds of morphological diversity/variation in Spitzenkörper from diverse fungi: (a) inherent diversity — Spitzenkörper features characteristic of particular fungi, and (b) dynamic pleomorphism — gradual or rapid changes in size, shape, and position of the Spitzenkörper within a single hyphal tip. Several components associated with the Spitzenkörper were identified: (a) vesicle cluster, (b) vesicle cloud, (c) differentiated core region(s) within the Spitzenkörper, (d) apical granules, (e) cytoplasmic filaments. Eight morphological patterns of Spitzenkörper organization are described in the higher fungi based on the shape and distribution of their components. An additional (ninth) pattern was recognized in the chytridiomyceteAllomyces macrogynous from recent work by others. All these patterns appeared to be conserved at the genus level. In all patterns but one, a core region was observed by light microscopy. The Spitzenkörper not only exhibited spontaneous dynamic pleomorphism but also reacted to stress conditions (light, mechanical, and electrical fields). These reactions include migration of the Spitzenkörper back into the subapical zone and/or disassembly of its components. The understanding and conceptualization of this dynamic complex is problematic and should remain flexible enough to encompass the diversity of Spitzenkörper patterns and the dynamic pleomorphism of this specialized apical apparatus which appears to drive hyphal tip growth in the higher fungi.Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

The Neurospora crassa exocyst complex tethers Spitzenk?rper 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.     

How hyphae grow: Morphogenesis explained?

Summary 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 Spitzenkörper (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.Abbrevations VSC vesicle supply center - SPK Spitzenkörper     

Association of spectrin-like proteins with the actin-organized aggregate of endoplasmic reticulum in the Spitzenkörper of gravitropically tip-growing plant cells

Spectrin-like epitopes were immunochemically detected and immunofluorescently localized in gravitropically tip-growing rhizoids and protonemata of characean algae. Antiserum against spectrin from chicken erythrocytes showed cross-reactivity with rhizoid proteins at molecular masses of about 170 and 195 kD. Confocal microscopy revealed a distinct spherical labeling of spectrin-like proteins in the apices of both cell types tightly associated with an apical actin array and a specific subdomain of endoplasmic reticulum (ER), the ER aggregate. The presence of spectrin-like epitopes, the ER aggregate, and the actin cytoskeleton are strictly correlated with active tip growth. Application of cytochalasin D and A23187 has shown that interfering with actin or with the calcium gradient, which cause the disintegration of the ER aggregate and abolish tip growth, inhibits labeling of spectrin-like proteins. At the beginning of the graviresponse in rhizoids the labeling of spectrin-like proteins remained in its symmetrical position at the cell tip, but was clearly displaced to the upper flank in gravistimulated protonemata. These findings support the hypothesis that a displacement of the Spitzenk?rper is required for the negative gravitropic response in protonemata, but not for the positive gravitropic response in rhizoids. It is evident that the actin/spectrin system plays a role in maintaining the organization of the ER aggregate and represents an essential part in the mechanism of gravitropic tip growth.     

Gravity perception requires statoliths settled on specific plasma membrane areas in characean rhizoids and protonemata

Summary. The noninvasive infrared laser micromanipulation technique (optical tweezers, optical trapping) and centrifugation were used to study susception and perception, the early events in the gravitropic pathway of tip-growing characean rhizoids and protonemata. Reorientation of the growth direction in both cell types was only initiated when at least 2–3 statoliths settled on specific areas of the plasma membrane. This statolith-sensitive plasma membrane area is confined to the statolith region (10–35 μm behind the tip) in positively gravitropic rhizoids, whereas in negatively gravitropic protonemata, this area is limited to the apical plasma membrane (0–10 μm). Statolith sedimentation towards the sensitive plasma membrane areas is mediated by the concerted action of actin and gravity. The process of sedimentation, the pure physical movement, of statoliths is not sufficient to initiate graviresponses in both cell types. It is concluded that specific statolith-sensitive plasma membrane areas play a crucial role in the signal transduction pathway of gravitropism. These areas may represent the primary sites for gravity perception and may transform the information derived from the gravity-induced statolith sedimentation into physiological signals which trigger the molecular mechanisms of the opposite graviresponses in characean rhizoids and protonemata. Received September 10, 2001 Accepted November 16, 2001