A distinct class of vesicles derived from the trans‐Golgi mediates secretion of xylogalacturonan in the root border cell

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three‐dimensional structures and macromolecular compositions of these Golgi stacks, we examined high‐pressure frozen/freeze‐substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans‐Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi‐associated vesicles. Margins of trans‐Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan‐I (PGA/RG‐I) are detected in the trans‐most cisternae and TGN compartments. LVs produced from TGN compartments (TGN‐LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN‐LVs containing the XG and PGA/RG‐I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans‐Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans‐cisternae accompanying polysaccharide synthesis with a mathematical model.     

Organization of SNAREs within the Golgi Stack

Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating lipid bilayer merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM proteins. This network of protein interactions spatially and temporally determines the specificity of transport vesicle targeting and fusion within the Golgi.At steady state, the Golgi maintains its structural and functional organization despite a massive lipid and protein flow. A balanced anterograde and retrograde membrane flow are required to constantly recycle the transport machinery and cargo containers (vesicles). In the absence of efficient recycling, directional net cargo transport would cease and the Golgi would collapse. Thus, transport vesicles constantly leave and enter at both sides of the Golgi stack and bud and fuse along the rims of the cisternae. To maintain the compartmental identity, vesicle fusion occurs in a specific and orchestrated manner. These fusion events are mediated by a cascade of reactions centered around the membrane fusion proteins SNAREs (SNAP receptors) (Söllner et al. 1993b).     

A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cis trans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.     

Ultrastructural study on mitosis and cytokinesis in Scytosiphon lomentaria zygotes (Scytosiphonales,Phaeophyceae) by freeze-substitution

Summary. The ultrastructure of mitosis and cytokinesis in Scytosiphon lomentaria (Lyngbye) Link zygotes was studied by freeze fixation and substitution. During mitosis, the nuclear envelope remained mostly intact. Spindle microtubules (MTs) from the centrosome passed through the gaps of the nuclear envelope and entered the nucleoplasm. In anaphase and telophase, two daughter chromosome masses were partially surrounded with endoplasmic reticulum. After telophase, the nuclear envelope was reconstructed and two daughter nuclei formed. Then, several large vacuoles occupied the space between the daughter nuclei. MTs from the centrosomes extended toward the mid-plane between two daughter nuclei, among the vacuoles. At that time, Golgi bodies near the centrosome actively produced many vesicles. Midway between the daughter nuclei, small globular vesicles and tubular cisternae accumulated. These vesicles derived from Golgi bodies were transported from the centrosome to the future division plane. Cytokinesis then proceeded by fusion of these vesicles, but not by a furrowing of the plasma membrane. After completion of the continuity with the plasma membrane, cell wall material was deposited between the plasma membranes. The tubular cisternae were still observed at the periphery of the newly formed septum. Microfilaments could not be observed by this procedure. We conclude that cytokinesis in the brown algae proceeds by fusion of Golgi vesicles and tubular cisternae, not by a furrowing of the plasma membrane. Received September 12, 2001 Accepted November 12, 2001     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Biosynthetic protein transport through the early secretory pathway

 Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation. Accepted: 24 October 1997     

Rab6 functions in polarized transport in Drosophila photoreceptors

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected.

The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

Membrankomplexe dictyosomaler Herkunft als „Matrizen“ für die extraplasmatische Synthese und Orientierung von Mikrofibrillen

Zusammenfassung Es wird ein neu gefundener Typ von Golgi-Vesikeln (F-Vesikeln), die während der Bildung der gemusterten Sekundärwand vonMicrasterias denticulata auftreten, eingehender beschrieben. Es wird gezeigt, daß speziell differenzierte Membranen dieser Vesikel wahrscheinlich als Matrizen für die Bildung der Sekundärwand-Mikrofibrillen fungieren.F-Vesikel entstehen in einem Prozeß der Membranreifung (Matrizen-Bildung) am distalen Pol der Dictyosomen. Von dort werden diese Vesikel, die globuläre Untereinheiten enthalten, welche reihenweise in der Vesikelmembran angeordnet sind, zur Plasmamembran transportiert (Matrizen-Transfer). Dort findet eine Inkorporation und Fusion der F-Vesikelmembran mit der Plasmamembran statt (Matrizen-Inkorporation). Diese inkorporierten und ausgebreiteten Membranareale scheinen bei der Produktion der Reihen von Mikrofibrillen von besonderer Bedeutung zu sein (Matrizen-Realisation).Die elektronenmikroskopischen Ergebnisse beiMicrasterias wurden mit jenen über die Schalenbildung bei Chrysophyceen (Pleurochrysis) verglichen. Dabei wurde eine überraschende Übereinstimmung bei der Wandbildung dieser beiden Organismen festgestellt: In beiden Fällen scheint es, daß Matrizen für die Bildung der Mikrofibrillen an den Membranen von Golgi-Vesikeln gebildet werden. BeiPleurochrysis erfolgt die Bildung der Wandelemente schon innerhalb der Golgi-Zisternen, während beiMicrasterias die Zelluloseproduktion verzögert ist (time gap) und erst nach Inkorporation der Vesikelmembran in die Plasmamembran beginnt.

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Dictyosome-Derived membrane complexes as templates for the extraplasmatic synthesis and orientation of microfibrils

Summary A newly found type of Golgi vesicles (F-vesicles), occuring during the formation of the highly patterned secondary wall ofMicrasterias denticulata is described in more detail. It is shown that specifically differentiated membranes of these vesicles are probably functional as templates for the formation of the secondary wall inMicrasterias. F-vesicles are formed in a process of membrane maturation (template formation) on the distal pole of the dictyosomes. From there these vesicles, carring globular subunits orientated in rows on the vesicle membrane, are transported to the plasma membrane (template transfer). Here an incorporation and fusion of the F-vesicle membrane with the plasma membrane occurs (template incorporation). These incorporated and exposed membrane areas seem to be involved in the production of the patterned arrays of microfibrils (template realization).The electron microscopic results inMicrasterias are compared with those described for scale formation in the chrysophycean algaPleurochrysis. A surprising relationship between the types of wall formation in these two organisms could be demonstrated: in both cases templates for the production of microfibrils seem to be formed on membranes of Golgi cisternae. InPleurochrysis the formation of wall elements takes place already within the Golgi cisterna, while inMicrasterias cellulose production is delayed (time gap) and begins just after the incorporation of the vesicle membrane into the plasma membrane.

     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

Golgi vesicles of uncommon morphology and wall formation in the red alga,Polysiphonia

Summary Golgi bodies of immature carposporangia ofPolysiphonia sp. are composed of a polarized stack of six to ten curved cisternae. The cisternae are surrounded by 50–200 nm diameter slightly granular vesicles.Hypertrophied, fibrillar Golgi cisternae occur in mature carposporangia. Secretory vesicles originate from ends of cisternae and by complete vesiculation of terminal cisternae; 0.6–1.2 m diameter, fibrous vesicles, many with electron dense nucleoids are abundant throughout the cytoplasm of mature sporangia. Vesicles expand, fuse with each other and cluster around starch granules. Some vesicles secrete their content into the spore wall. Morphological analyses of starch granules as well as topographical relations between vesicles, starch granules and the adjacent cytoplasm suggest that these Golgi vesicles function like lysosomes. The significance of these observations is discussed in relation to the composition of plant cell walls and cellular expansion.     

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

Summary Secretory 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.     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

EFFECT OF LATRUNCULIN B AND BREFELDIN A ON CYTOKINESIS IN THE BROWN ALGA SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

In zygotes of the brown alga Scytosiphon lomentaria (Lyngb.) Link, cytokinesis proceeds by growth of membranous sacs, which are formed by fusion of Golgi vesicles and flat cisternae accumulated at the future cytokinetic plane. It has been reported that depolymerization of actin filaments by latrunculin B does not inhibit mitosis. However, this molecule prevents the formation of the actin plate, which appears at the region of intermingled microtubules from each centrosome just before and during cytokinesis. In this study, zygotes treated with latrunculin B were observed using EM. Remarkably, this reagent inhibited the formation of flat cisternae. Golgi vesicles gathered around the midzone between the two daughter nuclei and fused with the plasma membrane there. As a result, the plasma membrane invaginated, in a complicated manner, into the cytoplasm. However, these invaginations of the plasma membrane never produced a continuous partition membrane. The ultrastructure of zygotes treated with brefeldin A, which prevents Golgi‐mediated secretion, was also examined. Flat cisternae appeared at the future cytokinetic plane, and a new cell partition membrane was formed. However, the partition membrane became thick, because it was filled with amorphous material rather than the normal rigid fibrous material. These results suggested that actin is involved in the formation of flat cisternae, where it is necessary for completion of the new cell partition membrane, and that Golgi vesicles may play an important role in the deposition of cell wall material.     

Ultrastructural Studies of a Vesicle System Associated with Endoplasmic Reticulum in Exo-Erythrocytic Forms of Plasmodium berghei1

ABSTRACT. Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

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     

Macromolecular differentiation of Golgi stacks in root tips ofArabidopsis andNicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

Summary The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to young and old, polysaccharideslime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips ofArabidopsis andNicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types.Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (30 nm) and are the least stained. Medial cisternae are narrower (20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4–6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network.Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.Dedicated to the memory of Professor Oswald Kiermayer