Microtubule organization during zoosporogenesis inAllomyces macrogynus

Summary The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamino-2-phenylindole - dH₂O deionized water - DMSO dimethyl sulfoxide - DS dilute salts solution - G/5 0.1% glucose medium - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOC microtubule-organizing center - PBS phosphate buffered saline - PCM pericentriolar matrix - TEM transmission electron microscopy - VELM videoenhanced light microscopy     

Meiotic prophase in diploid and tetraploid strains ofAllomyces macrogynus

Summary Three-dimensional serial section reconstructions of meiotic prophase nuclei ofAllomyces macrogynus (Chytridiomycetes, Blastocladiales) have been carried out. Serial section reconstructions of pachytene nuclei have revealed that the fungus when grown at 23 °C is an autotetraploid and is a diploid when grown at 35 °C for at least 6 months or on a substrate containing para-fluorophenylalanine for 1–2 weeks.Studies of the duplication and migration of the centrioles during the first stages of prophase revealed the existence of four centrioles in the 23 °C strain after centriole duplication and two centrioles in the 35 °C strain after duplication. It is observed that a bivalent attaches to the nuclear envelope at a site where a centriole is situated. It is proposed that the presence of the four centrioles in the 23 °C strain is due to the fact that each bivalent is represented twice in the strain.     

Analysis of cytoplasmic microtubules and flagellar roots in the zoospores ofAllomyces macrogynus

Summary Cytoskeletal and flagellar microtubules in the zoospores of the aquatic fungusAllomyces macrogynus are resistant to microtubule depolymerizing drugs. Consequently, we have analyzed the partial composition and organization of microtubules (Mts) in the cytoplasm and flagellar apparatus in the zoospores ofA. macrogynus. Evidence from two-dimensional gel electrophoresis demonstrated the presence of two -tubulin isoforms in axonemal and cytoplasmic Mts. In addition, a monoclonal antibody specific for acetylated -tubulin was used on one-dimensional protein blots to show that acetylated -tubulins are present in isolated zoospore cell bodies and axonemes. Immunofluorescence microscopy observations using this monoclonal antibody demonstrated that flagellar, kinetosomal, and cytoplasmic Mts were labeled. The nature of Mts in the flagellar apparatus was studied ultrastructurally. InA. macrogynus, the flagellar apparatus consists of the kinetosome, rhizopolast (striated flagellar rootlet), axoneme, and 9 sets of triplet Mts which radiate anteriorly from the proximal end of the kinetosome (microtubular rootlet), Analysis of the rhizoplast indicated that this structure does not contain Mts. The rhizoplast, which connects the functional kinetosome with a single, large basal mitochrondrion, consists of four electron-opaque bands. Serial-sectioning indicated that the rhizoplast is always adjacent to kinetosome triplets 1, 2, and 9, and thus lies perpendicular to the plane of flagellar beat. These results suggest that the primary function of the rhizoplast is to organize the kinetosome and mitochondrion with respect to one another and to bias flagellar beat in the appropriate orientation for cell motility.Abbreviations BSA bovine serum albumin - BCA bicinchoninic acid - DS dilute salts - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N-tetracetic acid - EM electron microscopy - Mes 2-(N-morpholinomethane sulfonic acid - Mt microtubule - NP-40 Nonidet P-40 - 1-D PAGE one-dimensional polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - 2-D PAGE two-dimensional polyacrylamide gel electrophoresis - Tween-20 polyoxyethylenesorbitan monolaurate     

The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

Gametangial development in Allomyces macrogynus

The possible role of mitochondria in determining the sex of the gametangia of Allomyces macrogynus was investigated. Quantitative studies of mitochondrial distribution in vegetative hyphae confirmed previous reports of apical mitochondrial clustering. However, by the time the male and female gametangia were partitioned off, no significant difference in mitochondrial distribution between the two sexes was present. Possible mechanisms for the redistribution of mitochondria during early differentiation are discussed. In addition, cytochrome oxidase activity was demonstrated in all mitochondria of both male and female gametangia by the use of diaminobenzidine. It is concluded that neither mitochondrial distribution nor differential mitochondrial activity plays a determining role in the differentiation of the sexual cells in Euallomyces.Abbreviations %M percent area occupied by mitochondria - DAB diaminobenzidine     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

pH gradients and cell polarity inPelvetia embryos

Summary Endogenous pH profiles were measured around single fertilized eggs of the brown algaPelvetia during the earliest stages of development. Profiles were constructed by measuring the pH near the cell surface at several positions using a pH sensitive microelectrode. Transcellular pH differences in the medium surrounding zygotes were detected soon after fertilization, as the developmental axis was being formed. The future rhizoid end of the cell was relatively alkaline and the presumptive thallus was acidic. At germination and throughout the first 5 d of embryogenesis, the apex of the elongating rhizoid was alkaline with respect to more distal regions. However, conditions that dissipated or reversed this extracellular pH gradient had little or no effect on polarization or growth, indicating that the gradient was not essential for early development.Inhibition of respiratory electron transport by cyanide and antimycin A eliminated the pH gradient, while uncouplers of oxidative phosphorylation [2,4-dinitrophenol (DNP) and carbonylcyanide m-chlorophenylhydrazone (CCCP)] stimulated acidification of the thallus regions. Proton ATPase inhibitors had no effect. Acidification, therefore, is not generated by ATP-dependent proton pumps in the plasma membrane, and instead probably reflects secretion of metabolic acids. Localized metabolism may establish an internal pH gradient that controls regional differentiation, and we are presently investigating this possibility.Abbreviations ASW artificial seawater - CCCP carbonylcyanide m-chlorophenylhydrazone - CD cytochalasin D - DNP 2,4-dinitrophenol     

The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus

Summary The tubulin cytoskeleton in hyphal tip cells ofAllomyces macrogynus was detected with an -tubulin monoclonal antibody and analyzed with microscopic and immunoblot techniques. The -tubulin antibody identified a 52 kilodalton polypeptide band on immunoblots. Immunfluorescence data were collected from formaldehyde-and cryofixed hyphae. Both methods provided similar images of tubulin localization. However, cryofixation yielded more consistent labeling and did not require detergent extraction or cell-wall lytic treatments. Tubulin was primarily localized as microtubules observed in the peripheral and central cytoplasmic regions and in mitotic spindles. Cytoplasmic microtubules were oriented parallel to the cells' longitudinal axis, with central microtubules more often varied in their alignment, and emanated from a region in the hyphal apex resulting in an apical zone of bright fluorescence. A thin layer of microtubules appearing as bands of fluorescence encircled many nuclei. Discrete spots of fluorescence were also associated with nuclei. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins that may be involved in the regulation of microtubule nucleation, stained centrosomes but not apical regions of hyphae. Nocodazole was used to depolymerize the microtubule network and reveal its regions of origin. A hocodazole concentration of 0.01 g/ ml (3.3× 10^–8M) provided a 70 to 75% inhibition of hyphal tip growth and was used throughout this study. The number of cells having an apical zone of fluorescence declined by 15 min of exposure. This zone was present in only a few cells after 60 min. After 30 min, the central cytoplasm consisted of small microtubule fragments and nuclear-associated spots. A small number of peripheral microtubules and nuclear-associated spots persisted throughout nocodazole treatments. Spindle microtubules were restored by 30 min after removal of nocodazole. This was followed by the reappearance of the apical zone of fluorescence and then by central and peripheral cytoplasmic microtubules. Apical fluorescence coincided with the presence of a Spitzenkörper. The results suggest that the Spitzenkörper and centrosome function as centers of microtubule nucleation and organization during hyphal tip growth in this fungus.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IB incubation buffer - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate buffered saline - PIPES 1,4-piperazinedietha-nesulfonic acid - PFB PIPES fixation buffer - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SPB spindle pole body - TEM transmission electron microscopy - YpSs yeast extract-inorganic phosphate-soluble starch     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.Abbreviations CCO cytochrome-c oxidase - CCR KCN-insensitive NADH-dependent cytochrome-c reductase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IDPase inosine 5-diphosphatase - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA 1-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - UDPG uridine diphosphoglucose     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

Cytoskeletal polarity in mammalian lymphocytes in situ

Summary The distribution of vimentin and spectrin in lymphocytes within murine lymphoid tissues was studied by means of immunofluorescence. A polarized submembranous aggregate of intermediate filaments was observed to be characteristic of lymphocytes within the medulla of the thymus as well as in lymphocytes within specific areas of spleen and lymph-node. This aggregate was determined to be in close association with a similarly polarized aggregate of spectrin. Lymphocytes of both B and T surface phenotype comprise the population of cells that are naturally polarized in terms of these cytoskeletal proteins. Lymphocytes with such a naturally polarized cytoskeleton are not observed in the spleen until approximately 5 days after birth, but are observed in the thymus by day 19 of gestation. Incubating lymphocytes with cytochalasin D, but not colchicine, caused a rapid dispersal of the spectrin aggregate without altering the polar accumulation of intermediate filaments. When splenic B-cells were allowed to form uropods as a result of ligand binding, the uropod (as well as surface receptor cap) was positioned above the region containing the polar aggregate of spectrin and vimentin. The possible physiological significance of naturally occurring cytoskeletal polarity in lymphocytes is discussed.     

Lipid function in plant cell polarity

The establishment and maintenance of cell polarity play pivotal roles during plant development. During the past five years, proteins that are required for different aspects of plant cell polarity have been identified. However, the functions of lipids and their interactions with proteins that mediate polarity remained largely unaddressed. Recent genetic studies have discovered cell and tissue polarity mutants that have defects in sterol composition, glycosylphosphatidylinositol-anchored proteins, glycosylphosphatidylinositol biosynthesis and phospholipid signalling. Analyses of the affected gene products have provided a first glance at the roles of lipids in cell polarity signalling, as well as in the trafficking and anchoring of polar proteins.     

Bioelectric aspects of the hemipteran telotrophic ovariole (Dysdercus intermedius)

Summary Two systems of steady extra-cellular currents were found along the surface of the telotrophicDysdercus ovarioles by means of a vibrating probe. The first covers the subgerminal tropharium and all the previtellogenic follicles. The current leaves the 3 or 4 small follicles of early euplasmic growth stages laterally and enters the syncytial tropharium. We presume that a similar intracellular current flows between the trophoplasm and the ooplasm which are interconnected by narrow nurse strands. Preliminary intracellular measurements indicate a potential gradient within this continuous cytoplasm, the ooplasm being electropositive to that of the tropharium. This current system fits into a model of polarized intracytoplasmic transport by electrophoresis. It is possible to explain the well known directed and selective flow of RNA from the tropharium via the nurse strands into the oocytes by means of such a model. The second current system occurs around every one of the 2 to 8 vitellogenic follicles. The pattern is completely different from that described for the first system. In the vitellogenic stages the current enters the follicle laterally all along the now much extended surface. It is balanced by a strong peak current which leaves the interfollicular region. As data on intracellular currents are not yet avialable, it is only a matter of speculation whether the circuit is closed through the ooplasm or only by a tangential loop through the follicle epithelium. The possible significance of this second current system for vitellogenin accumulation and uptake by the vitellogenic oocytes is also uncertain as yet.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Effect of nutrient availability on hyphal maturation and topographical sensing in Aspergillus niger

Topographical sensing (thigmotropism) is an essential component of efficient fungal growth. It is an important element in the complex pathway of sensory and mechanical elements that drive and control the growing hyphal tip, a fuller understanding of which will bring the mycological community a step closer to complete comprehension of the hyphal growth mode. Previous work has led us to hypothesize that the stress induced by nutrient deficiency causes structural changes in the hyphal tip that induces a thigmotropic response in Aspergillus niger, a soil fungus that does not display thigmotropism under normal conditions. In this study, we have sought to identify some of the factors that influence this induction of thigmotropism using a novel combination of microengineered substrates and imaging and analysis techniques to quantify thigmotropic behavior in complex hyphal systems. We have shown that the sensitivity of fungal contour sensing appears to be directly linked to nutrient availability and hypothesize that this may be caused by a stress-induced flattening of the tip and increased immaturity of the hyphal apex. Parts of this paper were presented at the Mycological Society of Japan (MSJ)/British Mycological Society (BMS) Joint Symposium, “The new generation of mycologists in Japan and the UK” held in Chiba, Japan, on June 3, 2006.     

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     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Depletion of Aspergillus nidulans cotA causes a severe polarity defect which is not suppressed by the nuclear migration mutation nudA2

The Aspergillus nidulans homologue of Neurospora crassa cot-1, cotA, encoding a member of the NDR protein kinase family, has been cloned and expressed under the control of the conditional alcA promoter. Depletion of CotA by repression of the alcA promoter led to a severe growth defect accompanied by loss of polarity. Germlings show greatly enlarged volume of the spores and hyphae, accompanied by an increase in number of nuclei per compartment, though the nucleus/volume ratio is not significantly altered. The depleted CotA phenotype was not suppressed by a nuclear migration mutation nudA2. Double mutants showed an additive, defective phenotype, unlike the suppression of the cot-1 ts mutation by ropy mutations seen in N. crassa, suggesting a different relationship between nuclear migration and the cot signalling pathway in A. nidulans. A functional CotA–GFP fusion protein was found in punctate regions of fluorescence similar to the distribution reported for human NDR2, and as a cap at the hyphal tip.