Charles E. Bracker

Origin and Continuity of Golgi Apparatus

The Golgi apparatus in its most familiar form is that part of the cell’s endomembrane system1 consisting of regions of stacked cisternae (dictyosomes) which lack ribosomes. It is a complex structure with unique functions in compartmentalizing products of synthesis, serving as a site of cytomembrane differentiation and producing exocytotic vesicles whose membranes are capable of fusing with plasma membrane. Unlike semiautonomous organelles such as chloroplasts and mitochondria, the function of the Golgi apparatus in secretion depends on functional continuity with other components of the endomembrane system.

Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrils in Vitro

Small spheroidal structures (mostly 40–70 nm in diameter) similar in shape, size, and chitin-synthesizing ability to the chitosomes described earlier for yeast cells of Mucor rouxii were isolated from four other fungi: Allomyces macrogynus (Emerson) Emerson and Wilson, Saccharomyces cerevisiae Meyen ex Hansen, Neurospora crassa Shear and Dodge, and Agaricus bisporus (Lange) Imbach. Chitosomes were also found in the mycelial form of M. rouxii . Evidence of zymogenicity of chitin synthetase was found in crude preparations from all five fungal genera in that protease treatment caused an increase in chitin synthetase activity. There were, however, specific differences among the fungi in the response of the zymogen to acid or neutral proteases and in the retention of zymogenicity during chitosome isolation. Upon addition of substrate (uridine diphosphate N -acetyl- D -glucosamine) and activating protease (if needed), the isolated chitosome samples produced chitin microfibrils. Both “fibroid” coils and extended microfibrils were seen. The isolation of functional chitosomes from five widely diverse genera, representing five major fungal groups (Zygomycetes, Chytridiomycetes, Hemiascomycetes, Ascomycetes, and Basidiomycetes) and three distinct developmental forms (hyphase, yeast cells, and basidiocarp), suggests that the chitosomes are a ubiquitous component of the cytoplasmic mechanism that conveys chitin synthetase to the sites of microfibril assembly at the surface of fungal cells.

Ultrastructural specialization at the host-pathogen interface in rust-infected flax

SummaryUltrastructure of the association between the rust fungus, Melampsora lini, and a compatible variety of flax, Linum usitatissimum, was studied to clarify the structural relationships and interactions at the site of host penetration and at the host-parasite interface. Results of freeze-etching as well as a special section-staining procedure consisting of periodate-chromate-phosphotungstate (PACP) are shown with a host-parasite combination for the first time. The host plasma membrane is invaginated by the fungus and forms a continuous boundary around the fungal haustoria which penetrate the host cells. No morphological continuities are observed linking the protoplasts of host and fungus. With both freeze-etching and the PACP stain, the invaginated portion of the host plasma membrane at the host-parasite interface shows distinctive features that are not characteristic of the non-invaginated portion of the membrane. This localized specialization of host plasma membrane in response to the fungus appears as a significant and consistent feature of the host-parasite interaction. The host plasma membrane is separated from the haustorial wall by an amorphous layer of sheath material which covers the body but not the neck of the haustorium. This sheath provides the environment in which the haustorium exists and functions during the course of the host-parasite association. Occasionally, a collar of wall-like material derived from the host cell forms around the haustorial neck. The collar is continuous with the host wall and is distinct and discontinuous from the haustorial sheath. In fewer than 5% of the infected cells this wall material encases entire haustoria. The fungal wall is structurally specialized around the site of host penetration, and it becomes intimately associated with the host wall where the fungus penetrates into the lumen of the host cell. During penetration, the host and fungal walls appear to be fused so that the interface between them is not clearly delineated. The haustorial wall is continuous, via the haustorial neck, with the wall of the haustorial mother cell which lies outside the host cell. Different staining properties reveal this wall continuum to consist of several well-defined regions having different structure or composition. A ring of fungal wall material midway along the haustorial neck stains densely with lead citrate, but is preferentially etched away by periodic acid. The neck ring denotes a transition in the staining reaction of the fungal wall, from that present in the region of host penetration to that of the wall surrounding the haustorium. The findings demonstrate specialization of the fungal wall in the area of host penetration as well as specialization of the host plasma membrane at the host-parasite interface to a degree not previously realized from ultrastructural information.

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

SummaryThe 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.

Continuity between cytoplasmic endomembranes and outer mitochondrial membranes in fungi

SummarySeveral types of intimate association are shown between endoplasmic reticulum or endoplasmic reticulum-like membranes and the outer membranes of mitochondria in fungal hyphae. These include close physical association, contact, thread-like continuity, and direct luminal continuity. Membranes are smooth surfaced in the immediate region of association or continuity, and some have ribosomes at other sites along their surfaces. The continuities represent sites of membrane interaction which may facilitate exchange between adjacent membrane components. Additional continuities are shown between mitochondria and other endomembrane components. Observations are discussed in relation to the body of information linking mitochondria and endoplasmic reticulum in a variety of eukaryotic cells.

Mapping the Growth of Fungal Hyphae: Orthogonal Cell Wall Expansion during Tip Growth and the Role of Turgor

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.

Protoplasmic changes during zoospore encystment and cyst germination in Pythium aphanidermatum

The addition of nutrients to a suspension of zoospores of Pythium aphanidermatum induces immediate encystment followed by cyst germination. A combination of light and electron microscopic techniques shows that the changes associated with these events include an early loss of motility, loss of the characteristic zoospore shape, and rearrangement of protoplasmic organization. Secretion of a cyst coat is accompanied by the loss of large vesicles from the peripheral cytoplasm. Then coated invaginations appear on the plasma membrane, and a system of peripheral cisternae is gradually lost. Several minutes after the beginning of encystment, a cyst wall forms beneath the cyst coat. Meanwhile, a sequence of changes in the endomembrane-system provides for the modification or resorption of unused peripheral vesicles and the generation of a new polar growing region which becomes the incipient germ tube. Significant in the initiation of polarized growth is the peripheral accumulation of a cluster of secretory vesicles. The cell wall adjacent to this cluster of vesicles changes in stainability and then expands to form a germ tube which elongates and becomes a new hypha. The cluster of secretory vesicles is maintained in the growing apex by a continual supply of vesicles from the Golgi apparatus. Storage vacuoles with characteristic “fingerprint” inclusions then give rise to the first population of hyphal vacuoles, and the initiation of a growing mycelium is completed. The sequential appearance and disappearance of various cell components are correlated with developmental events during encystment and germination. These observations increase the understanding of the roles of endomembrane components in morphogenesis.

The Ultrastructure and Development of Sporangia In Gilbertella Persicaria

SUMMARYThe ultrastructural features of sporogenesis in the mucoraceous fungus Gilbertella persicaria are illustrated and described, with emphasis on cytokinesis and spore wall formation. During cleavage, the principal structural changes involve pattern transformations of protoplasmic membranes. Endoplasmic reticulum (ER) is one of the most changeable membrane components, transforming from a complex fenestrated and branching form to a simpler sheet-like form during the course of cleavage.During precleavage, small vesicles are formed, apparently from special cisternae. The disappearance of these initial vesicles coincides with the appearance of cleavage vesicles. Cleavage vesicles are distinguished by the presence of granules on the inner surface of the vesicle membrane. Prior to cleavage, the nuclei, ER, and cleavage vesicles form an inter-associated system. Cleavage is initiated endogenously by the coalescence of cleavage vesicles to form a ramifying tubular cleavage apparatus. The cleavage apparatus dema...

Dissociation of chitosomes by digitonin into 16 S subunits with chitin synthetase activity

Digitonin exerts profound effects on chitosomes (microvesicular structures with chitin synthetase activity isolated from the fungus Mucor rouxii). At low concentrations, it stimulates chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity; at higher concentrations, it inhibits it. Digitonin also causes disintegration of the chitosome and the release of a homogeneous population of chitosome subunits with chitin synthetase activity. These chitosome subunits have a sedimentation coefficient of 16 S, compared to 105 S for whole chitosomes, as determined by centrifugation in sucrose density gradients, and measure 7--12 nm in diameter. After dissociation, chitin synthetase remains in a zymogenic state, and requires treatment with a protease for activation. No change in sedimentation coefficient of chitosome subunits was observed after proteolytic activation. The product synthesized by the chitosome subunits was characterized by X-ray diffractometry ad alpha-chitin and was by the criterion indistinfuishable from chitin made by preparations of undissociated chitosomes. However, in the electron microscope, the chitin microfibrils made from chitosome subunits were, in general, much shorter than those produced by undissociated chitosomes and often exhibited a needle-like appearance.

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm3. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.