ABSENCE OF CAP MEMBRANES AS A CHARACTERISTIC OF PIT PLUGS OF SOME RED ALGAL ORDERS1

The current general model of the organization of a fully developed rhodophycean pit plug includes a cap membrane associated with each end of the plug. Continuity of the plasmalemma and cap membrane results in the plug core being extracellular. However, routine ultrastructural methods have not convincingly demonstrated cap membranes in the Bangiales or the Corallinales. In this study, a variety of fixation regimes that enhance membrane contrast were used in an attempt to detect cap membranes in representatives of these two orders. Although cap membranes were readily and clearly demonstrable by these methods in a representative of an order previously known to have cap membranes, they could not be detected in members of the Bangiales or Corallinales. Should the absence of cap membranes prove to be an invariant feature of these two orders, presence or absence of cap membranes will constitute a new character for phylogenetic analysis. It is proposed that the ancestral character state of red algal pit plugs was a homogeneous plug without caps and without cap membranes.     

ABSENCE OF CAP MEMBRANES AS A CHARACTERISTIC OF PIT PLUGS OF SOME RED ALGAL ORDERS1

The current general model of the organization of a fully developed rhodophycean pit plug includes a cap membrane associated with each end of the plug. Continuity of the plasmalemma and cap membrane results in the plug core being extracellular. However, routine ultrastructural methods have not convincingly demonstrated cap membranes in the Bangiales or the Corallinales. In this study, a variety of fixation regimes that enhance membrane contrast were used in an attempt to detect cap membranes in representatives of these two orders. Although cap membranes were readily and clearly demonstrable by these methods in a representative of an order previously known to have cap membranes, they could not be detected in members of the Bangiales or Corallinales. Should the absence of cap membranes prove to be an invariant feature of these two orders, presence or absence of cap membranes will constitute a new character for phylogenetic analysis. It is proposed that the ancestral character state of red algal pit plugs was a homogeneous plug without caps and without cap membranes.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

ULTRASTRUCTURE OF CYST FORMATION IN OCHROMONAS TUBERCULATA (CHRYSOPHYCEAE)1

The cysts (statospores) of Ochromonas tuberculata Hibberd are produced within a cytoplasmic silica deposition vesicle (SDV) whose membrane (silicalemma) appears to be formed by the coalescence of golgi vesicles. Silica is first deposited as small nodules and the collar and spines develop by centrifugal growth only after a complete but still thin wall has been laid down. Small vesicles appear to be attached to the SDV only in the region overlying the developing collar; a cap of radially arranged, moderately electron-dense material occurs at the tip of the growing spines. The cyst pore is formed at the anterior end of the flagellate cell, by lack of silica deposition over a small region of the SDV and rupture of the SDV and other membranes crossing this region. When the cyst wall is complete, an extracystic plug is formed in the pore, resulting in the loss of some extracystic cytoplasm and the plasmalemma, and the inner SDV membrane becomes the functional plasmalemma. The plug develops first by coalescence with the cell membrane of golgi-derived vesicles containing dense but apparently nonsiliceous spicules surrounded by amorphous material. During later stages of plug formation only fibrous material is deposited, some of which may be extruded through the pore forcing out some of the spiculate component. Scanning electron micrographs of the mature wall show it is smooth except for the concentrically wrinkled inner face of the flared collar and that the real pore diameter is only ca. half that of the collar. At germination the plug completely disappears in an unknown way and a single cell, similar to a normal vegetative cell emerges through the pore. Chrysophycean cyst formation generally resembles cell wall formation in diatoms, but differs in some details.     

PIT CONNECTION FORMATION IN THE RED ALGA PSEUDOGLOIOPHLOEA

Pit connection formation in the marine red alga Pseudogloiophloea confusa was studied with the electron microscope. The process of formation occurs in 2 stages. First, a septum forms as an annular ingrowth from the lateral walls. Lomasomes are associated with the centripetal accretion of wall material. The completed septum contains a large rimmed aperture, bounded by the continuous plasmalemma, and through which the cytoplasm is continuous from cell to cell. In the second stage, a highly structured plug is formed which completely blocks the aperture. The plug is condensed on flattened vesicles which lie parallel to one another and which traverse the aperture. The mature plug is composed of granules 50–100 A in diameter and surrounded by several dense layers which appear to enclose an 80 A limiting membrane. Once the pit connection is formed, no material is seen to traverse it.     

A COMPLEMENTARY FREEZE-FRACTURE STUDY OF CHRYSOCHROMULINA CHITON (HAPTOPHYCEAE)

Complementary freeze-fracture replicas and high resolution tantalum-tungsten shadowing have been used in a study of the membanes of the marine alga Chrysochromulina chiton. Membrane particle populations range from 38/100 nm² in the plastid to 2/100 nm² in the pyrenoid cap membrane. Membrane asymmetry was evident in all membranes, but was most obvious in those with higher particle numbers. In all complementary replica pairs, particles were always more numerous on protoplasmic fracture faces. Small, particle-free areas with bordering particles were also seen as recurring membrane features. Complementarity of matching fracture faces was seen for very small background granularity patterns and for large membrane components, but not for particles. Complementarity can also be seen in non-membranous fracture faces both within and external to the cell, suggesting the presence of polymeric materials in these areas that produce “particles” due to plastic deformation.     

The Fine Structure of Conidia of Botryodiplodia ricinicola with Observations on Effects of Chilling

Conidia of Botryodiplodia ricinicola (Saccardo) Petrak havebeen studied, principally by freeze-etch electron microscopy.Freshly harvested conidia have a thin scaly surface layer, freeof rodlets, which covers an otherwise homogeneous-looking wallwhich is continuous with the single centrally-perforate septum.The contours of the plasmalemma are usually smooth. Nuclei andsmall vacuoles are numerous. Hydrophobic fracture faces of theplasmalemma, tonoplasts and nuclear membranes variously revealintra-membrane particles or corresponding depressions or both.Lipid inclusions are small and numerous. Compact orderly stacksof membranes are present, sometimes one in each locule of theconidium. Conidia of a strain insensitive to chilling were seento differ only in respect of the distribution of intra-membraneparticles on fracture faces of tonoplasts. Chilled and chilled-and-soakedconidia of the wild type showed fine-structural differencesfrom untreated conidia, most obviously in respect of the greatersize of some of the lipid inclusions, but also in respect offeatures of the plasmalemma which after chilling contained plasmalemmasomesand, after subsequent immersion for 15 min, showed annular depressions.Also, intra-membrane particles in some membrane systems showedaltered distribution between the two hydrophobic fracture faces.It is concluded that cell lipids and cytoplasmic membrane systemsmay be involved in the previously demonstrated chilling sensitivityof conidia of this species. Botryodiplodia ricinicola, conidia, ultrastructure, chilling effects     

RHODOPHYCEAN PIT PLUGS: AN ULTRASTRUCTURAL SURVEY WITH TAXONOMIC IMPLICATIONS

The fine structure of pit plugs was examined in 63 species of red algae representing 34 families. The number of plug cap layers was found to be a taxonomically reliable character when tested against a recent revision of the Rhodymeniales which established the order Palmariales. In a broader survey involving all the orders of red algae possessing pit plugs, three basic plug cap configurations were detected. These differ in having one, two, or no plug cap layers. Some taxa have plug caps in which the outer cap layer is greatly elaborated, however, this layer was shown to be positionally and cytochemically identical, thus probably homologous, to the outer cap layer of the typical two-layered plug cap. The pit plug character confirms the validity of the order Palmariales and indicates that the Nemaliales and Cryptonemiales are heterogeneous assemblages. Revision of these groups along lines indicated by the plug character is supported by significant features of morphology and reproduction. These correlations lead us to propose that Rhodophysema and Coriophyllum be transferred to the Palmariales, that the Gelidiales be resurrected, that the Bonnemaisoniales (including the Naccariaceae) is deserving of recognition, that the Batrachospermaceae be raised to a higher rank which will also receive the Lemaneaceae and the Thoreaceae, and that the Corallinaceae and Hildenbrandiaceae both be raised to ordinal status.     

Eyespot membranes in newly released zoospores of the green algaChlorosarcinopsis gelatinosa (Chlorosarcinales) and their fate during zoospore settlement

Summary Eyespot membranes in zoospores of the green algaChlorosarcinopsis gelatinosa were studied with the freeze-fracture technique. The PF of the plasmalemma overlying the eyespot lipid globules contains significantly greater numbers of intramembraneous particles (IMP; 8,200 IMP/m²) compared to other areas of the plasmalemma (2,100 IMP/m²). In the eyespot area the EF of the plasmalemma reveals no IMP, but regularly arranged depressions corresponding to the PF particles. Sizes of PF particles are not significantly different between the eyespot area and other areas of the plasmalemma. Zoospore settlement starts approximately two hours after release and involves in sequence, rounding up of the cells, retraction of the flagella and secretion of a cell wall. Eyespot membrane specializations on the PF of the plasmalemma disappear during flagellar retraction and before cell wall secretion.The functional significance of eyespot membrane specializations is discussed in accordance with the view that these membranes are engaged in photoreception and primary sensory transduction relating to green algal phototaxis.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

Quantitative analysis of intramembranous particles in the membranous system of rat liver cells at different stages of development and aging

The density of intramembranous protein particles was studied by freeze-fracture. Particle density on the fracture faces of the plasmalemma and the rough endoplasmic reticulum (RER), as well as the outer and inner membranes of the nucleus and the mitochondria in rat hepatocytes were quantified. Comparison among different age groups sampled days postcoitum (dpc), days postpartum (dpp), and months postpartum (mpp) shows age-related changes in particle density in each membrane system. With the exception of the RER, particle densities increased after the 16th dpc, reached a maximum at birth, and then decreased with increasing age. Simultaneously, the number nuclear pores shows a positive correlation with the particle density of the nuclear membranes. The particle density on the membranes of the RER shows a maximum on the 16th dpc, and on the 6th dpp. Thereafter, the density of the RER decreases slightly. In all membrane systems, the density of the particles on the external fracture faces is more variable than the density of the particles on the protoplasmic fracture faces.     

A PROPOSAL FOR A NEW RED ALGAL ORDER,THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

The ultrastructure of the salt gland of Spartina foliosa

Summary The salt gland in Spartina foliosa is composed of two cells, a large basal cell and a smaller, dome-shaped cap cell which is located on a neck-like protrusion of the basal cell. There is no cuticular layer separating the salt gland from the mesophyll tissue. The basal cell has dense cytoplasm which contains numerous mitochondria, rod-like wall protuberances, and infoldings of the plasmalemma which extend into the basal cell and partition the basal cell cytoplasm. The protuberances originate on the wall between the basal and the cap cells and are isolated from the basal-cell cytoplasm by the infoldings of the plasmalemma. While the cap cell has no partitioning membrane system or wall protuberances, it resembles the basal cell by having dense cytoplasm and numerous mitochondria.The basal cell seems to be designed for efficient movement of ions toward the cap cell. The long, dead-end extracellular channels in the basal cell of Spartina appear comparable to surface specializations seen in the secreting epithelium of animal cells which carry out solute-linked water transport. The number of mitochondria and their close association with the plasmalemma extensions suggest that they have an important role in the transfer of ions through the basal cell.The accumulated ions would move into the extracellular spaces along an osomotic gradient where the accompanying passive flow of water would move the ions into the cap-cell wall and from there the solution would pass out through the pores in the cuticle.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

FREEZE-FRACTURED CHLOROPLAST MEMBRANES OF GONYAULAX POLYEDRA (PYRROPHYTA)1

The chloroplast membranes of Gonyaulax polyedra Stein were studied in replicas of rapidly frozen and fractured cells. The thylakoid EF_s face lacked the large 15–16 nm particles characteristic of plants with the light-harvesting chlorophyll a/b protein, presumably because the principal light-harvesting protein of Gonyaulax is the small water-soluble peridinin-chlorophyll-protein and the chlorophyll a/b protein is absent. As in other plants, the EF_s thylakoid fracture face carried more particles (4 ×) than EF_uface. The PF faces of the thylakoid showed twice as many particles as did the EF_s faces. No circadian differences in the number or size of thylakoid membrane particles could be detected. Three membranes comprise the chloroplast envelope in Gonyaulax. They could be clearly differentiated in freeze-fractured cells. The middle envelope membrane carried many fewer particles on both the EF and PF faces than did the other two envelope membranes. The PF faces of both the outer and inner envelope membranes showed more particles than the EF faces, as do many other membranes which have been examined.     

The fine structure and cytology of the association between the parasitic red algaHolmsella australis and its red algal hostGracilaria furcellata

Summary Holmsella australis Noble andKraft ms. is a colourless red algal parasite, forming whitish pustules on its photosynthetic red algal host,Gracilaria furcellata Harvey. In the infected region, host cortical tissue continues to grow and enclose the expanding pustule. Filaments of both host and parasite grow apically, the cells being connected by primary pit connections (PCs). Secondary PCs form between cells of the same species, and in addition,H. australis initiates the formation of secondary PCs with cells ofG. furcellata. All three types of secondary PC are morphologically distinct. In hostparasite PCs the surface adjoining the host cell is similar in structure to a host-host PC, while that adjoining the parasite cell has the structure of a parasite-parasite PC. The plasma membrane is continuous between the cells of the unrelated host and parasite. In addition, a cap membrane is typically produced only on the host surface, though occasionally the parasite side is enclosed by a cap membrane as well. Cap membranes are absent from parasite-parasite PCs (making them intracellular), while host-host PCs are typically extracellular, both cells producing cap membranes. The presence or absence of a cap membrane in certain positions appears to vary, and suggests that cells may be able to regulate its presence. Since transport of nutrients would be expected to occur from host to parasite cells, and between parasite cells, the morphological evidence presented here suggests the PCs may be the pathway.     

The subacrosomal granule and its evolution during spermiogenesis in a lizard

Summary Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.This research forms part of project N. 31.26.S1-0244 supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas     

Fine structure of the noctuid moth ear. I. The transducer area and connections to the tympanic membrane in Feltia subgothica haworth

The sensillum of the ear of Feltia subgothica contains two ciliated receptor cells, the A cells. The cilium of each is enclosed within a well developed scolops consisting of a cap, a set of scolopalial rods and a collar, an unusual structure contained within the dendrite. The tip of the cilium is inserted in a channel in the scolopalial cap. The cap is linked to the tympanic membrane by a series of three structures: a cap cell, a microtubular shaft and a microfibrillar plug. The two latter structures are heavily reinforced by cytoskeletal elements and the microfibrillar plug is actually continuous with the tympanic membrane. These three structures transmit the vibration of the tympanic membrane to the scolops. The simplicity and accessibility of the ear suggests that it might be a good system in which to investigate cellular events associated with transduction of sound in these receptors.     

The ultrastructure of the plasmodesmata of the salt glands ofTamarix as revealed by transmission and freeze-fracture electron microscopy

Summary Numerous plasmodesmata occur in the walls between the secretory cells ofTamarix salt glands. The plasmalemma bounds the plasmodesmata and is continuous from cell to cell. In freeze-fracture, the e-face of the plasmalemma within the plasmodesmata is virtually devoid of intramembranous particles while, in contrast, the p-face is decidedly enriched with particles. The axial components appear to be a tightly curved membrane bilayer, as judged from measurements and their appearance in freeze-fracture, and the e-face of this membrane is also devoid of particles. Observations from both thin sections and freeze-fracture replicas indicate the presence of a circular cluster of six particles around the axial component near the cytoplasmic termini of the plasmodesmata. These particles extend from the p-face of the axial component to the p-face of the plasmalemma. These observations are summarized in a model.