I. Brent Heath

Variant Mitoses in Lower Eukaryotes: Indicators of the Evolution of Mitosis?

Publisher Summary Mitosis is defined as all those types of nuclear division that produce two, or rarely more, daughter nuclei, each containing a chromosome complement approximately similar to that of the original nucleus. The greatest range of variations by which mitosis is accomplished, occurs in the protistan and fungal kingdoms, some members of which are probably most similar to the ancestors of higher plants and animals. The variations in the higher organisms are secondarily derived from the division patterns of typical plants and animals. This chapter discusses the characteristics and evolution of mitosis. The efficiency of mitosis consists of two basic components; the frequency with which each daughter nucleus receives the necessary complete genome complement (genetic efficiency) and the amount of energy and materials expended in the synthesis and operation of the mitotic apparatus. The chapter also discusses the use of mitosis as a phylogenetic marker. It is applicable to all eukaryotic cells and thus is valuable across boundaries where other structures are absent on one side and present in various forms on the other side.

Nucleus-Associated Organelles in Fungi

Publisher Summary This chapter discusses the nucleus-associated organelles (NAOs) of fungi. In many fungi, at various stages in the life cycle, the nuclei are associated with Golgi bodies, flagella root systems, mitochondria, microbodies, and nuclear caps. Nucleus-associated organelle is a variously shaped, primarily osmiophilic structure, which is located outside of, or within, the nuclear envelope and typically lies at the spindle poles during nuclear division. Fungal NAOs are involved with three main activities; nuclear division, nuclear movements including karyogamy, and spore delimitation. Centrioles are involved in flagellum production and the consequent cell movements. The NAO is involved in both mitosis and meiosis; the replicated NAOs separate and the spindle develop between them such that there is always at least one NAO at each spindle pole. The NAO commonly undergoes some morphological change during the transition from interphase to mitosis and meiosis. One of the most common changes is enlargement, which is most clearly seen in basidiomycetes, such as Boletus and Trametes .

Effects of exogenous calcium ions on tip growth, intracellular Ca2+ concentration, and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca2+ and the growth rate increased with increased external Ca2+ up to 5 × 10−2 m Ca2+. When Ca2+ was greater than 5 × 10−2 m, growth rates decreased. Internal membrane-associated Ca2+ was localized with chlortetracycline. Internal Ca2+ became depleted in hyphae grown in the absence of Ca2+ and was increased in hyphae grown in high concentrations of Ca2+, showing that internal Ca2+ can be modulated by external Ca2+. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca2+ in the presence of a large concentration gradient. In the absence of external Ca2+, growth can occur for a limited time through use of internal Ca2+. The actin cytoskeleton was altered in hyphae grown in both high and low Ca2+. Hyphae grown in 10−3 m Ca2+ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10−1 m and 0 Ca2+. The tips of hyphae growing in low Ca2+ also had a tendency to swell, giving these hyphae irregular shapes. Ca2+ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca2+ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca2+, thus affecting the actin cytoskeleton and possibly other growth processes.

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

SummarySecretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.

Evidence that actin reinforces the extensible hyphal apex of the oomycete Saprolegnia ferax

SummaryFilamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.

The relation between turgor and tip growth inSaprolegnia ferax: Turgor is necessary, but not sufficient to explain apical extension rates

Abstract Linear growth rate ofSaprolegnia was reduced in direct proportion to increased osmotic pressure (II) of the medium, when sorbitol or PEG-400 was used as osmotica. However, increasing medium II reduced hyphal turgor only to a minimum positive level, which was maintained while extension rates continued to decline. TPA, a K+-channel agonist effective onSaprolegnia protoplasts, also caused dose-dependent linear growth rate reductions but did not substantially affect turgor. When turgor was compared with linear growth rate in the osmoticum experiments, there was a positive correlation only for hyphae growing faster than 12 μm/min; below this, there was a twofold range in extension rate despite essentially constant turgor. As well, TPA-treatments produced a twofold reduction in hyphal extension rate without substantially affecting turgor. Turgor should be consistent within a coenocyte, and is steady under constant growth conditions. However, under such conditions, we found average variations of fivefold in extension rate between hyphae, and twofold for hyphae over time. These results suggest that turgor is not the prime determinant of tip extension rate, and they are consistent with cytoskeletal regulation of that rate. Linear growth rates ofSaprolegnia colonies were similar on basal medium containing 1% (w/v) glucose, sorbitol, or PEG and only slightly faster than without added carbohydrate. Increasing medium II with glucose also reduced hyphal extension rate.

Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae

Vegetative and reproductive stages of Caecomyces equi gen. nov., sp. nov. isolated from the horse caecum were examined by light and electron microscopy. This organism, which is similar to isolates known as Sphaeromonas communis, produces uniflagellate, uninucleate zoospores whose perikinetosomal structures, i.e. circumflagellar ring, spur, struts and scoop, are similar in many respects to those described in species of Neocallimastix. Microtubular roots extend basally from the spur and associate with hydrogenosomes and the nucleus. Another group of microtubules radiates laterally in a fan-shaped array close to the plasmalemma. Zoospores encyst, shedding their flagella with basal bodies, and germinate to diglobular thalli. Either coralloid or bulbous rhizoids form in plant material, but only the latter in axenic culture. Incipient zoospores are produced from a multinucleate eucarpic thallus and devlop within cleavage vacuoles containing flagella. An isolate from the cow rumen was found to be similar to C. equi in morphology and zoospore ultrastructure. On the basis of zoospore ultrastructure, we assign the new genus to the Neocallimasticaceae of the order Spizellomycetales. Organisms previously described as Sphaeromonas communis and Piromonas communis are renamed Caecomyces communis and Piromyces communis and assigned to the same family.

Predicting the distribution, conservation, and functions of SNAREs and related proteins in fungi

Hyphal tip growth, the hallmark of the fungi, requires highly polarized and localized exocytosis, but how this requirement is met is unknown. Members of conserved protein families called SNAREs and Rabs mediate vesicle trafficking and fusion at virtually every step of the intracellular pathway in all examined eukaryotes. We have searched the available nearly complete fungal genomes, established the presence or absence of members of the SNARE and Rab families in these genomes, and predicted their evolutionary relationships to one another. Comparisons with the extensively studied Saccharomyces cerevisiae indicate that, in general, most of the members of these families (including those involved in mediating exocytosis) are conserved. The presence of exceptional SNAREs and Rabs in some fungi that are not conserved in S. cerevisiae may be indicative of specialized steps that occur in these fungi. The implications of these findings for current tip growth models are discussed.

Cytoskeletal and Ca2+ regulation of hyphal tip growth and initiation

Hyphal tip growth is a complex process involving finely regulated interactions between the synthesis and expansion of cell wall and plasma membrane, diverse intracellular movements, and turgor regulation. F-actin is a major regulator and integrator of these processes. It directly contributes to (a) tip morphogenesis, most likely by participation in an apical membrane skeleton that reinforces the apical plasma membrane, (b) the transport and exocytosis of vesicles that contribute plasma membrane and cell wall material to the hyphal tips, (c) the localization of plasma membrane proteins in the tips, and (d) cytoplasmic and organelle migration and positioning. The pattern of reorganization of F-actin prior to formation of new tips during branch initiation also indicates a critical role in early stages of assembly of the tip apparatus. One of the universal characteristics of all critically examined tip-growing cells, including fungal hyphae, is the obligatory presence of a tip-high gradient of cytoplasmic Ca2+ that probably regulates both actin and nonactin components of the apparatus, and the formation of which may also initiate new tips. This review discusses the diversity of evidence behind these concepts.

An electron microscopic and spectroscopic study of murine epiphyseal cartilage: Analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution

Newborn mice epiphyseal growth plates were preserved by slam freezing/freeze substitution and examined by conventional electron microscopy, stereopsis, high voltage electron microscopy, and electron spectroscopic imaging (ESI). To illustrate the improved ultrastructure of this cryogenic procedure, conventional, aqueously fixed growth plates were included showing collapsed hypertrophic chondrocytes surrounded by a depleted and condensed extracellular matrix. In contrast, the cryogenically prepared epiphyses contain chondrocytes and extracellular matrix vesicles both in direct contact with proteoglycan filaments retained in an expanded state. ESI is an electron microscopic technique which enables the direct localization of atomic elements superimposed over fine structural details. This technique was used to examine the colocalization of calcium and phosphorus within matrix vesicles and within their associated extracellular environments. Matrix vesicles appeared in three distinct diameter ranges. The integrity of the matrix vesicles was examined at various stages of mineralization and also within the mineralized zone of provisional calcification.