Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.     

The fine structure of a microplate-microtubule array,microfilaments and polyhedral body associated microtubules in several species of Anabaena

A microplate-microtubule array was observed in Anabaena sp. (B-378). This structure consists of an arched plate, about 8 nm thick, and various microtubules, 12 nm in diameter and 50 nm long, arranged in rows. The microtubules project at right angles from one side of the plate into the cytoplasm or towards the plasma membrane. Up to twelve microplate-microtubule arrays were observed in a single section of a cell.Microfilaments, about 2.8 nm in diameter and of undetermined length, were observed in four isolates of Anabaena. The microfilaments were always found in bundles, which varied in size, up to 0.63 m across and 0.91 long.Microtubules, 10 nm in diameter and about 150 nm in length, were observed associated with one facet of polyhedral bodies in 8 out of 20 isolates of Anabaena. The microtubules occurred in groups of up to 20 or more, and were always oriented with the long axis parallel to a facet of a polyhedral body. In cross section, the microtubules had an electron transparent lumen 5 nm wide and a wall 2.5 nm thick.These structures are compared to previously deseribed microtubules and microfilaments.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella

This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.     

An update on fibrous flagellar roots in green algae

Summary In flagellate green algae two types of fibrous flagellar roots can be distinguished: system I fibres, cross-striated bundles of 2nm filaments (striation periodicity about 30 nm), which are associated with flagellar root microtubules, and system II fibres, contractile bundles of 4–8 nm filaments which are often cross-striated (striation periodicity variable but greater than 80 nm). The major protein of system II fibres is centrin, a Ca²⁺-modulated phosphoprotein, which is a member of the EF-hand protein family. The major protein of system I fibres (of severalChlamydomonas-type green algae) is a 34 kDa phosphoprotein, named assemblin. Because of the solubility characteristics of system I fibres and the properties of their major protein (paracrystal-formation in vitro, several isoelectric variants, heptad motifs in parts of the amino acid sequence), assemblin is presumably related to the k-m-e-f class of -helical fibrous proteins.Abbreviations NBBC nucleus-basal body connector - SMAC striated microtubule-associated component - k-m-e-f class keratin-myosin-elastin-fibrinogen class - EF-hand protein family Ca²⁺-binding proteins containing one to several Ca²⁺-binding motifs consisting of a peculiar helix-loop-helix configuration - PVDF polyvinyhdene difluoride     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

Oral Apparatus Structure in the Carnivorous Macrostomal Form of Tetrahymena vorax

The structure of the oral apparatus in the carnivorous macrostomal form of Tetrahymena vorax has been investigated using serial thin sections and preparations of isolated oral apparatuses. The cilia of the oral apparatus are organized into an undulating membrane that borders the right and part of the posterior margin of the buccal cavity and three membranelles that project from plateaus on the anterior surface. Each membranelle consists of one short row and two longer rows of hexagonally packed kinetosomes. The organization of the microtubules of the oral ribs is identical to that in the T. vorax microstomal cell type. However, the first oral rib originates near the first kinetosome at the anterior end of the undulating membrane. The fine filamentous reticulum that underlies part of the oral ribs in the macrostomal cell type is not striated, unlike the reticulum in the microstomal form. A band of filaments similar to the fine filamentous reticulum extends around the anterior margin of the large cytostomal opening that occupies most of the posterior part of the oral cavity. The single row of microtubules along the left side of the oral cavity and cytostome also has filaments associated with it. A major difference between the microstomal and macrostomal forms in the structure of the oral apparatus is in the oral connectives. The macrostomal cell type contains only a single cross-connective that joins the three membranelles and the anterior portion of the undulating membrane. The posterior or peripheral connective between the posterior ends of membranelles one and two and the posterior end of the undulating membrane is absent.     

Ciliary filter-feeding structures in adult and larval gymnolaemate bryozoans

Abstract. Ciliary filter-feeding structures of gymnolaemate bryozoans—adults of Flustrellidra hispida and Alcyonidium gelatinosum , larvae of Membranipora sp.—were studied with SEM. In F. hispida and A. gelatinosum , the distal part of each tentacle has a straight row of stiff laterofrontal cilia which carry out "ciliary sieving" to capture suspended food particles that are subsequently transported downward towards the mouth by tentacle flicking; both structure and function resemble those of stenolaemate tentacles. The proximal part of the tentacle and of the ciliary ridge of a cyphonautes larva have strikingly similar structures, except that the laterofrontal cells are monociliate in the adults and biciliate in the larvae. The laterofrontal cells of the tentacles are arranged in a zigzag row and their cilia form two parallel rows, a frontal and a lateral row. The latter probably forms the sieve of stiff filter cilia in front of the water-pumping lateral cilia, whereas the frontal row appears to be held close to the frontal ciliary band of the tentacle. The biciliate laterofrontal cells of the cyphonautes larva have the cilia arranged in similar rows. The detailed morphological similarities between the ciliary bands of adult and larval filtering structures suggest that the feeding mechanisms are similar, contrary to what has been previously thought.     

Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: anisoptera)

Summary The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 . The A-band length is about 3 . The myofibrils consist of thick (250 Å) and thin (85 Å) filaments. Each thick filament is surrounded by 9–12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed.The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.     

THE AXOSTYLE OF SACCINOBACULUS : I. Structure of the Organism and Its Microtubule Bundle

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed.     

Steroid Polyols from the Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O--D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3,6,8,15,16,29-hexaol (sanguinoside A) and (20R,24S)-3-O--D-(2,3,4-tri-O-methylxylopyranosyl)-5-cholestane-3,4,6,8,15,24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahydroxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated -D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds.     

Ultrastructural studies of the commensal suctorian,Choanophrya infundibulifera hartog

Summary The feeding tentacles of Choanophrya contain a central canal lined by microtubules. Only one tentacle develops during metamorphosis of the embryo into the adult, but others develop at intervals throughout adult life. Each tentacle forms adjacent to a solitary, subcortical kinetosome which lies parallel to the body surface, lacks accessory elements and never develops a cilium. Small condensations of electron-dense material and short bundles of microtubules form adjacent to the cartwheel region of the kinetosome. Initially these bundles are orientated randomly but later they become radially arranged and curved into prolamellae around a disc-shaped condensation centre, to form a paddlewheel-like tentacle primordium 0.8–1.1 m in diameter. The condensation centre consists of alternating concentric electron-dense and electron-transparent zones, and lies with its axis perpendicular to both the kinetosome and the cortex. The microtubules in each prolamella increase in number and pairs of short tip microtubules develop between adjacent prolamellae. Subsequently the developing lamellae become enclosed by a cylinder of ring microtubules. Once all the microtubule components of the tentacle primordium are established it increases in length by addition of material to the basal ends of the microtubules to form a short microtubule canal. As the canal elongates the epiplasm above it disappears and the pellicle membranes become uplifted around the protruding tentacle. An epiplasmic collar differentiates around the growing tentacle whilst spheroid vesicles and solenocysts begin to accumulate in the surrounding cytoplasm.This investigation was supported by the J.S. Dunkerley Fellowship in Protozoology, awarded by the University of Manchester.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex     

Functional organization of battery cell complexes in tentacles of Hydra attenuata

Ultrastructural and light microscopic observations on the organization of thick and thin regions of hydra's tentacles, made on serial sections and on whole fixed, plastic-embedded tentacles, reveal the existence of two levels of anatomical order in the tentacle ectoderm: (1) The battery-cell complex (BCC), composed of a single epitheliomuscular cell (EMC) and its content of enclosed nematocytes and neurons; and (2) the battery cell complex ring (BCC ring), an arrangement of 4 or more BCCs into larger units organized as rings around the circumference of the tentacle. All EMCs of the distal tentacle appear to contain batteries of nematocytes, and are, therefore, called “battery cells.” Apart from battery cell complexes and migrating nematocytes, there are no other cell types in the tentacle ectoderm. Battery cells are composed of three distinct regions: the cell body, peripheral attenuated extensions and myonemes. Thick tentacle bands are composed of cell bodies, whereas thin bands are made up of attenuated extensions. Myonemes contribute to both thick and thin regions. It was confirmed that each battery cell has several myonemes, which appear to interdigitate with myonemes of other more proximal and distal battery cells, but not with battery cells of the same BCC ring. Nematocytes have several basal processes. Some processes insert between myonemes and contact the mesoglea; other processes insert into cuplike extensions of myonemes, and are connected to myonemal cups by desmosomal junctions. These observations are discussed in relation to mechanical and electrical aspects of tentacular contraction and bending.     

Ultrastructural Features of Allantosoma intestinalis,a Suctorian Ciliate Isolated from the Large Intestine of the Horse1

ABSTRACT. Allantosoma intestinalis, a suctorian ciliate isolated from the intestine of the horse, was studied utilizing light and electron optical methods. These small sausage-shaped organisms have a varying number of tentacles (between one and 12) located at each extremity of the body. The microtubular axoneme of each tentacle in cross-section consists of two files of microtubules arranged in a daisy-like configuration. Haptocysts occur in the tentacle shaft, abutted to the plasma membrane of the knob of the tentacle, and in the cell body. The haptocysts are bottle-shaped, with prominent annular striations around their midportion. The cell is covered by three membranes, an outer plasma membrane, an outer alveolar, and an inner alveolar membrane. A thin epiplasmic layer is found beneath the inner alveolar membrane, and a single row of microtubules underlies the epiplasm. The subpellicular microtubules are arranged parallel to each other forming a corset around the cell along the long axis: such a system is not characteristic of suctorians. A field of diminutive kinetosomes (each 180 nm long, max. of 15 per field), lacking cilia, was found below the cortex. The function of these prokinetosomes is unknown. A ciliated swarmer has not been observed, only the nonciliated adult. The characteristics of Allantosoma are compared with those of other suctorian genera.     

The fine structure of Crithidia fasciculata with special reference to the organelles involved in the ingestion and digestion of protein

Summary As in other trypanosomatids, the cell membrane of Crithidia fasciculata overlies a single layer of microtubules. Each microtubule possesses a large number of periodically arranged drumstick-like appendages and adjacent microtubules are joined by fibrillar connectives. Anteriorly, the microtubules gradually taper to terminate just before or just after entering the reservoir. An attempt is made to correlate microtubule tapering with maintenance of form of the truncated anterior end of the cell. Smooth and coated vesicles are proliferated from the Golgi saccules and the prominent contractile vacuole lies nearby. The single mitochondrion is extensive and expanded at one point to form a capsule for the kinetoplast. The cristae are predominantly plate-like but other configurations do occur. The cytostome, a shallow invagination of the reservoir membrane, is found between two constrictions in the reservoir wall. Supporting the cytostome are several microtubules which penetrate deeply into the cytoplasm. Ingestion of ferritin occurs by pinocytosis from the cytostome and by coated vesicle formation from the reservoir membrane. Digestion probably occurs in multivesicular bodies which contain acid phosphatase activity.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside