Subapical Cell Research Articles

A fibrous scaffold for in vitro culture and experimental studies of Physcomitrium patens

AbstractThe model moss, Physcomitrium patens, is routinely cultured on cellophane placed over a solid nutrient medium. While this culture method is convenient for moss propagation, it is not suitable for studying how topographical features and mechanical cues from the environment influence the growth and development of moss. Here, we show that P. patens can be grown on fibrous scaffolds consisting of nanoscale, randomly oriented fibers composed of polyvinylidene tri‐fluoroethylene (NRP). The moss adheres tightly to NRP in contrast to the lack of adhesion to cellophane. Adhesion to the scaffold is associated with slower tip growth of moss protonema for some time, followed by an increase in tip growth rate that is equivalent to that on cellophane. In addition, the orientation of the first subapical cell division plane differs between NRP‐grown and cellophane‐grown protonema. Nonetheless, moss colonies grown on NRP did not show signs of nutrient or photosynthetic stress and developed normal gametophores. Together, these data establish NRP as a suitable substrate for the culture of P. patens and to probe the influence of mechanical forces on tip growth and cell division of moss.

Longevity of Fungal Mycelia and Nuclear Quality Checks: a New Hypothesis for the Role of Clamp Connections in Dikaryons.

This paper addresses the stability of mycelial growth in fungi and differences between ascomycetes and basidiomycetes. Starting with general evolutionary theories of multicellularity and the role of sex, we then discuss individuality in fungi. Recent research has demonstrated the deleterious consequences of nucleus-level selection in fungal mycelia, favoring cheaters with a nucleus-level benefit during spore formation but a negative effect on mycelium-level fitness. Cheaters appear to generally be loss-of-fusion (LOF) mutants, with a higher propensity to form aerial hyphae developing into asexual spores. Since LOF mutants rely on heterokaryosis with wild-type nuclei, we argue that regular single-spore bottlenecks can efficiently select against such cheater mutants. We then zoom in on ecological differences between ascomycetes being typically fast-growing but short-lived with frequent asexual-spore bottlenecks and basidiomycetes being generally slow-growing but long-lived and usually without asexual-spore bottlenecks. We argue that these life history differences have coevolved with stricter nuclear quality checks in basidiomycetes. Specifically, we propose a new function for clamp connections, structures formed during the sexual stage in ascomycetes and basidiomycetes but during somatic growth only in basidiomycete dikaryons. During dikaryon cell division, the two haploid nuclei temporarily enter a monokaryotic phase, by alternatingly entering a retrograde-growing clamp cell, which subsequently fuses with the subapical cell to recover the dikaryotic cell. We hypothesize that clamp connections act as screening devices for nuclear quality, with both nuclei continuously testing each other for fusion ability, a test that LOF mutants will fail. By linking differences in longevity of the mycelial phase to ecology and stringency of nuclear quality checks, we propose that mycelia have a constant and low lifetime cheating risk, irrespective of their size and longevity.

L-WNK1 is required for BK channel activation in intercalated cells.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.

The Cbk1-Ace2 axis guides Candida albicans from yeast to hyphae and back again

Since its description in S. cerevisiae, the Regulation of Ace2 and Morphogenesis (RAM) pathway has been studied for nearly 20 years in multiple model and pathogenic fungi. In pathogenic fungi, the RAM pathway carries out many functions through mechanisms that remain to be defined in detail. Recently, we reported that Cbk1-mediated phosphorylation of the transcription factor Ace2 functions to repress the hyphae-to-yeast transition in Candida albicans. This transition is understudied relative to the yeast-to-hyphae transition. Subapical hyphal cell compartments are arrested in G1 until the point at which lateral yeast emerge. Here, we discuss this model and report new data indicating that a second G1 associated protein, the mitotic exit regulator Amn1. In S. cerevisiae diploid cells, Amn1 negatively regulates Ace2 at both the gene expression level through a negative feedback loop and at the protein level by targeting Ace2 for degradation. In C. albicans, Amn1 and Ace2 also form a feedback loop at the level of gene expression. Deletion of AMN1 decreases lateral yeast formation relative to wild type in maturing hyphae and is associated with decreased expression of PES1, a positive regulator of lateral yeast formation. These data indicate that the regulation of mitotic exit plays a role in determining the timing of lateral yeast emergence from hyphae in C. albicans. We also propose an integrated model for the interplay between the Cbk1-Ace2 axis and other hyphal stage regulators during the process of filamentation and transition back to yeast.

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.

Analysis of Statoliths Displacement in Chara Rhizoids for Validating the Microgravity-Simulation Quality of Clinorotation Modes

In single-celled rhizoids of the green algae Chara, positively gravitropic growth is governed by statoliths kept in a dynamically stable position 10–25 μ m above the cell tip by a complex interaction of gravity and actomyosin forces. Any deviation of the tube-like cells from the tip-downward orientation causes statoliths to sediment onto the gravisensitive subapical cell flank which initiates a gravitropic curvature response. Microgravity experiments have shown that abolishing the net tip-directed gravity force results in an actomyosin-mediated axial displacement of statoliths away from the cell tip. The present study was performed to critically assess the quality of microgravity simulation provided by different operational modes of a Random Positioning Machine (RPM) running with one axis (2D mode) or two axes (3D mode) and different rotational speeds (2D), speed ranges and directions (3D). The effects of 2D and 3D rotation were compared with data from experiments in real microgravity conditions (MAXUS sounding rocket missions). Rotational speeds in the range of 60–85 rpm in 2D and 3D modes resulted in a similar kinetics of statolith displacement as compared to real microgravity data, while slower clinorotation (2–11 rpm) caused a reduced axial displacement and a more dispersed arrangement of statoliths closer to the cell tip. Increasing the complexity of rotation by adding a second rotation axis in case of 3D clinorotation did not increase the quality of microgravity simulation, however, increased side effects such as the level of vibrations resulting in a more dispersed arrangement of statoliths. In conclusion, fast 2D clinorotation provides the most appropriate microgravity simulation for investigating the graviperception mechanism in Chara rhizoids, whereas slower clinorotation speeds and rotating samples around two axes do not improve the quality of microgravity simulation.

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs.

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs.

Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells.

BackgroundIn the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells.ResultsWe used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles.ConclusionsTo our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles from different tip growing plant cells, thus enhancing our understanding of the mechanisms of plant polarized cell growth.

Sexual structures in Ptilothamnion sphaericum and Pterosiphonia complanata (Ceramiales, Rhodophyta) from the Atlantic Iberian Peninsula

Sexual structures of Ptilothamnion sphaericum and Pterosiphonia complanata are described for the first time. Spermatangial heads of P. sphaericum are densely clustered in short lateral branchlets, each cell of which bears up to three spermatangial heads. They consist of a stalk cell and 3-4 axial cells bearing a cluster of spermatangial mother cells that cut-off 1–2 spermatangia. The last two cells in female axes of P. sphaericum are relatively short, with the hypogenous cell longer and more similar to vegetative cells. The subapical cell bears three periaxial cells: two sterile and the supporting cell, which bears a four-celled carpogonial branch and one sterile cell. The hypogenous cell bears 1–2 involucral filaments prior to fertilization. These features support the placement of P. sphaericum in the genus Ptilothamnion. Spermatangial branches of Pterosiphonia complanata are borne on unbranched modified trichoblasts. They consist of an elongate suprabasal cell that bears a fertile central axis from which four periaxial cells arise, bearing quadrangular spermatangial mother cells that bear the spermatangia. Spermatangial branches have 1 (-3) apical sterile cells. Procarps of P. complanata consist of a four-celled carpogonial branch and two sterile cells borne on the supporting cell. These features are similar to all Pterosiphonia species for which they have been described.

Ultrastructure of glandular hairs of <i>Sigesbeckia jorullensis</i> Kunth (Asteraceae)

Multicellular long-stalked glandular hairs of the outer involucral bracts of Sigesbeckia jorullensis Kunth were investigated with transmission electron micros-copy. The apical layer of head cells was characterized by many irregular leucoplasts, a well developed and in the secretory phase mostly agranular endoplasmic reticu-lum, few dictyosomes, and one calcium oxalate crystal per cell. The prevalent part of the secretion seems to originate from leucoplasts of apical head cells and is secreted into a subcuticular space after crossing cytoplasm and the plasma membrane. The sheath of this sub-cuticular space was formed exclusively by a thin cuticle, which was detached from the cell wall in the secretory phase. Some small wall protuberances were found between the sub-apical and apical cell layer. Whether the three to six layers of sub-apical head cells, containing many chloroplasts, contribute to the biogenesis of the lipophilic secretion or not, remains unclear. Light microscopy demonstrated that there was a...

Vacuole inheritance regulates cell size and branching frequency of Candida albicans hyphae.

Hyphal growth of Candida albicans is characterized by asymmetric cell divisions in which the subapical mother cell inherits most of the vacuolar space and becomes cell cycle arrested in G1, while the apical daughter cell acquires most of the cell cytoplasm and progresses through G1 into the next mitotic cell cycle. Consequently, branch formation in hyphal compartments is delayed until sufficient cytoplasm is synthesized to execute the G1 ‘START’ function. To test the hypothesis that this mode of vacuole inheritance determines cell cycle progression and therefore the branching of hyphae, eight tetracycline-regulated conditional mutants were constructed that were affected at different stages of the vacuole inheritance pathway. Under repressing conditions, vac7, vac8 and fab1 mutants generated mycelial compartments with more symmetrically distributed vacuoles and increased branching frequencies. Repression of VAC1, VAM2 and VAM3 resulted in sparsely branched hyphae, with large vacuoles and enlarged hyphal compartments. Therefore, during hyphal growth of C. albicans the cell cycle, growth and branch formation can be uncoupled, resulting in the investment of cytoplasm to support hyphal extension at the expense of hyphal branching.

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.

Choroid plexus epithelial monolayers – a cell culture model from porcine brain

BackgroundThe goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa.MethodsCells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR), were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1) and multidrug resistance protein 1 (Mrp1, Abcc1) was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments.ResultsChoroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing) membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed) membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123.ConclusionIt was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic transport across the blood-cerebrospinal fluid barrier (BCSFB).

Actin Organization During Eucalyptus Root Hair Development and Its Response to Fungal Hypaphorine

The fungus Pisolithus microcarpus establishes an ectomycorrhiza with Eucalyptus globulus. This symbiosis involves a fungal synthesis and secretion of hypaphorine, an indolic compound. Previous studies have shown that hypaphorine induces an alteration in the actin cytoskeleton of elongating root hairs and inhibits hair elongation. Using an alternative approved method, we analyzed the effects of hypaphorine on the E. globulus root hair cyto-architecture and actin configuration in more detail and provide new results. One mM hypaphorine stops root hair elongation within 20 min, and changes the hair cyto-architecture. Semi-quantitative analysis of the actin cytoskeleton before and after treatment with hypaphorine shows that hypaphorine induces a shift from fine F-actin to F-actin bundles in the sub-apex of the hair, which occurs first in the mid-plane of the cell. This creates a sub-apical cell centre free of filamentous actin, an actin configuration that differs from that during developmental growth arrest. The mechanism of action of hypaphorine is discussed.

Gravisensing in single-celled systems – update on characean rhizoids and protonemata

Single-celled and tip-growing rhizoids and protonemata of the characean algae have been intensively studied and there is considerable progress in the understanding of the molecular and cellular mechanisms underlying gravisensing and gravity-dependent growth. In higher plant statocytes, the role of actin in both processes is still a matter of intense debate, but there is clear evidence that actin coordinates both processes in characean rhizoids and protonemata. The multiple functions and dynamic nature of the actin cytoskeleton in these cells are based on the concerted action of a variety of actin-binding proteins. Profilin, actin-depolymerizing factor, a spectrin-like protein, villin and fimbrin have been detected which control apical actin polymerization and regulate the dynamic remodeling of the actin arrangement. An actomyosin-based system was shown to (i) mediate the transport of secretory vesicles to the growing tip, (ii) establish the incorporation of cell wall material and (iii) coordinate the tip-focussed distribution of calcium channels which establish the tip-high calcium gradient for local exocytosis. Experiments performed in microgravity have shown that the actomyosin system precisely coordinates the position of statoliths in rhizoids and protonemata and, upon a change in orientation, directs sedimenting statoliths to specific areas at the plasma membrane where physical contact with gravisensor molecules initiates growth reorientation. The upward growth response of protonemata was shown to be preceded by a statolith-induced and actin-dependent relocalization of the Ca 2+-gradient to the upper flank that does not occur in positively gravitropic rhizoids, in which sedimented statoliths cause differential growth of the opposite subapical cell flank. Thus, constant actin polymerization in the growing tip and the spatiotemporal control of actin remodeling by numerous actin-binding proteins are essential for gravity sensing and polarized growth of characean rhizoids and protonemata.

Role of peg formation in clamp cell fusion of homobasidiomycete fungi

In most filamentous basidiomycetes, clamp cells are found at the septa of dikaryotic mycelia. Clamp cell formation starts at hyphal tip cells with the development of a lateral bulge at a position slightly apical to the future septum. Relative to the growth direction of the hypha, the protrusion expands backwards into a hook-like structure. Next, the two genetically different haploid nuclei within the hyphal tip cell divide. A septum appears between clamp cell and hyphal tip cell, thereby trapping one nucleus within the clamp cell. Another septum is laid within the hypha, separating a nucleus of the other type in the newly generated subapical hyphal cell from the two different nuclei kept together in the new apical hyphal cell. Through fusion of clamp and subapical cell, the two solitary nuclei become united within the subapical hyphal compartment. In 1933, Buller described subapical formation of a peg to which the clamp cell fuses as an additional, subsequently neglected step in this series of events. In this study, we represent evidence for subapical peg formation and its role in clamp cell fusion. Our observations potentially indicate a B mating type regulated extracellular communication between clamp and subapical hyphal cell.

Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis.

A morphologically structured model is proposed to describe trends in biomass growth, substrate consumption, and antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis. Filamentous biomass is structured into three morphological compartments (apical, subapical, and hyphal), and the production of retamycin, a secondary metabolite, is assumed to take place in the subapical cell compartment. Model accounts for the effect of glucose as well as complex nitrogen source on both the biomass growth and retamycin production. Laboratory data from bench-scale batch and fed-batch fermentations were used to estimate some model parameters by nonlinear regression. The predictive capability of the model was then tested for additional fed-batch and continuous experiments not used in the previous fitting procedure. The model predictions show fair agreement to the experimental data. The proposed model can be useful for further studies on process optimization and control.

Immunofluorescence microscopy of the microtubule cytoskeleton during conjugate division in the dikaryon Pleurotus ostreatus N001

Fluorescence microscopy was used to describe the distribution of nuclei and the organization of the microtubule network in hyphae of Pleurotus ostreatus. Dikaryotic hyphae of P. ostreatus N001 grow by tip extension with two closely spaced nuclei moving slowly forward with the growing hyphal tip. During vegetative growth of the hyphae, cytoplasmic microtubules are found as long filaments oriented longitudinally within fungal hyphae. When the apical cell reaches a length of approximately 150 μm, the two nuclei divide synchronously. Mitosis occurs in association with clamp connection formation, with one of the nuclei dividing in the hook of the developing clamp connection and the other in the main hypha. After mitosis, two daughter nuclei move forward to approximately the center of the apical cell, while the other two move backward to a central position in the subapical cell. Two septa are formed, one in the clamp and the other across the main axis of the hypha to delimit the apical cell. The use of fluorescence microscopy made it possible to examine the changes in the cytoplasmic microtubules, the configuration of the mitotic apparatus, the site of septation and the post-mitotic nuclear migrations during conjugate division in P. ostreatus dikaryotic hyphae.

Immunofluorescence Microscopy of the Microtubule Cytoskeleton during Conjugate Division in the Dikaryon Pleurotus ostreatus N001

Fluorescence microscopy was used to describe the distribution of nuclei and the organization of the microtubule network in hyphae of Pleurotus ostreatus. Dikaryotic hyphae of P. ostreatus N001 grow by tip extension with two closely spaced nuclei moving slowly forward with the growing hyphal tip. During vegetative growth of the hyphae, cytoplasmic microtubules are found as long filaments oriented longitudinally within fungal hyphae. When the apical cell reaches a length of approximately 150 μm, the two nuclei divide synchronously. Mitosis occurs in association with clamp connection formation, with one of the nuclei dividing in the hook of the developing clamp connection and the other in the main hypha. After mitosis, two daughter nuclei move forward to approximately the center of the apical cell, while the other two move backward to a central position in the subapical cell. Two septa are formed, one in the clamp and the other across the main axis of the hypha to delimit the apical cell. The use of fluorescence microscopy made it possible to examine the changes in the cytoplasmic microtubules, the configuration of the mitotic apparatus, the site of septation and the post-mitotic nuclear migrations during conjugate division in P. ostreatus dikaryotic hyphae.

The anatomy of Neotenophycus ichthyosteus gen. et sp. nov. (Rhodomelaceae, Ceramiales), a bizarre red algal parasite from the central Pacific

A minute parasite of Neosiphonia poko (Hollenberg) Abbott from a shallow lagoon on the central-Pacific Johnston Atoll is described as Neotenophycus ichthyosteus Kraft et Abbott, gen. et sp. nov. The infective parasite cell first connects to a central-axial cell of the host, then emerges from between host pericentral cells at a node before dividing into a three- or four-celled primary axis. Epibasal cells of the parasite divide to form three pericentral cells whose derivatives produce a globular head on the basal cell and on which reproductive structures differentiate almost immediately. Trichoblasts on any life-history stage are completely lacking. Spermatangia are borne on mother cells across the whole thallus surface. Procarps consist of four pericentral cells that encircle a subapical fertile-axial cell in an ampullar configuration, one of the pericentral cells serving as the supporting cell and bearing a four-celled carpogonial branch and a single sterile cell. Diploidization results in a longitudinal/concave division of the auxiliary cell and formation of an arching linear series of inner gonimoblast cells, each dividing toward the thallus surface into gonimoblast filaments of very narrow, horizontally aligned cells terminated by initially monopodial, later by sympodial, carposporangia, the whole of the mature female gametophyte consisting of an amalgam of several cystocarps within a lax jacket of sterile gametophytic tissue. Tetrasporophytes are composed of lobes of pericentral-cell-derived filaments, each axial cell of which is ringed by three pericentral cells producing tetrahedral tetrasporangia enclosed by two pre-sporangial cover cells. Affinities of the new genus are discussed and comparison is made particularly to the enigmatic parasite Episporium centroceratis Möbius. It is concluded that relationships with any previously described tribe are so remote or obscure that the new tribe Neotenophyceae should be proposed for it.