ER Cisternae Research Articles

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.

Involvement of myosin in intracellular motility and cytomorphogenesis in Micrasterias.

Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM. Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements.

Clusters of VIP-36-positive vesicles between endoplasmic reticulum and Golgi apparatus in GH3 cells.

The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.

Nature's ingenuity: bypassing the classical secretory route via apocrine secretion.

Although it has been suggested that epithelial cells of the male reproductive system are involved in apocrine secretion, this method of secretion is not fully understood. In the present study, apocrine secretion was investigated in epithelial principal cells lining the epididymis and vas deferens (VD) of adult mice. The tissues were fixed by cardiac vascular perfusion with glutaraldehyde for routine electron microscope (EM) analysis and Bouin's fixative for light microscope (LM) immunocytochemistry to access functional roles. In the epididymis and VD, the apex of principal cells revealed protrusions of cytoplasm referred to as apical blebs (ABs). The latter contained solely numerous free ribosomes, 20 nm vesicles and few ER cisternae, suggesting segregation of their contents. While some ABs displayed wide areas of contact with the apical principal cell cytoplasm, others showed thin stalk-like attachment points as well as fissures at the junction of the two areas. Together with images of ABs and their contents deep in the lumen, it is suggested that ABs detach from principal cells whereupon they breakdown to release their contents therein. As ABs of the epididymis were immunoreactive for glutathione-S-transferases (GSTs) and ubiquitin, it is proposed that these proteins are synthesized on free ribosomes in ABs and that apocrine secretion represents the manner whereby they enter the lumen to effectively protect sperm from free radical injury and ubiquitinate proteins for degradation, respectively. ABs of the VD were immunoreactive for 3beta-HSD, suggesting that they are also capable of synthesis of steroids with their release via apocrine secretion. Taken together the data provide evidence for apocrine secretion in the adult mouse epididymis and VD that could play important roles in relation to sperm maturation, protection and viability.

Ergastoplasmic paracrystalline inclusion bodies in the adipose gonadal envelope and fat body of the glow worm, Lampyris noctiluca (Insecta, Coleoptera)

The gonads of glow worm larvae are enveloped by adipose tissue which represents a specialized fat body. The adipose gonadal envelope, and also to a lesser extent the fat body cells, contain tubular paracrystalline inclusion bodies (PIBs). Cells of other tissues are devoid of such inclusions. The PIBs form in the cisternae of rough ER. In young larvae PIB formation is sparse, but at advanced larval stages PIBs often occur as bundles in stacks of ergastoplasm. Typically, a PIB within a cisterna consists of four to seven parallel tubules. The outer diameter of a tubule is ca 28.8 nm and the width of the tubule lumen ca 12.2 nm. The “wall” of a tubule contains globular protein subunits of ca 8.3 nm diameter; the subunits are arranged helically. Since the adipose gonadal envelope progresses through a cytological differentiation process during differentiation and maturation of the gonads, the increased number of PIBs may indicate enhanced metabolic activity of the tissue related to nutrition of the growing gonads.

An Integral Membrane Protein from the Nuclear Pore Complex Is Also Present in the Annulate Lamellae: Implications for Annulate Lamella Formation

Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. Söderqvist et al., 1997, Eur. J. Biochem. 250, 808–813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP3) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP3 had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP3 located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP3 into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0.1 to 2 μm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.

Functional morphology of the secretory pathway organelles in yeast.

The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division. It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport. These resident membrane proteins return by retrograde vesicular transport. Apart from this, the pathway involves endocytosis. The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical. Biochemists have postulated that, in S. cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector-mediated transport into the functional cascade of the Golgi system cisternae and between them. This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome-like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome. The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration. The quality of organelle imaging in S. cerevisiae ultra-thin sections depends on the fixation technique used and on specimen contrasting by metals. The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase. Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway. The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S. cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed.

Cytochemical Localization of Phenolic Compounds in Columella Cells of the Root Cap in Seeds of Brassica napus—Changes in the Localization of Phenolic Compounds during Germination

Changes in the localization of phenolic compounds were investigated in columella cells of embryonic roots in 1-year-old Brassica napus seeds during 48 h of imbibition and germination. In dry, dormant seeds, phenolic compounds were located in the apoplastic compartment between the cell wall and plasmalemma in the outermost layer of the columella. These apoplastic phenolic deposits disappeared during the activation processes associated with imbibition and germination, but new deposits appeared successively in the nucleus, ER cisternae, protein bodies and on the outer surface of the root cap. A large number of phenolic deposits were observed in the outermost part of the columella, becoming less frequent towards the initial centre. Their appearance coincided with restoration of the ER and immediately preceded cytological activation. After the primary root had emerged from the seed coat, deposits of phenolic compounds disappeared from the cytoplasm, but simultaneously appeared in the vacuoles.

Structural elements common to mitosis and apoptosis

Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40–60 (~50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100–200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of ~50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of ~50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Effect of temperature on apical membrane remodeling in ADH-stimulated toad urinary bladders.

Pretreatment and removal of vasopressin (ADH) in toad urinary bladder renal model tissues induces endocytosis at 25 degrees C. The objective of the current study is to determine if apical membrane remodeling, as well as transepithelial water flow, can be affected by lowering the temperature to 15 degrees C. Control toad urinary bladders in the presence of an osmotic gradient at either 25 degrees C or 15 degrees C when visualized by scanning electron microscopy (SEM) show a typical apical membrane surface with no apparent surface differences. ADH-treated tissues following 15-min stimulation at 25 degrees C or 15 degrees C revealed a propagation of apical microvilli on their surface membranes. After 15 min following removal of ADH, bladder tissues at 25 degrees C or 15 degrees C showed surface invaginations involving over 44% and 80% of granular cells, respectively. The rate of water flow in tissues at 15 degrees C remained elevated compared to tissues held at 25 degrees C. This was consistent with the observation that ADH-stimulated tissues following washout at 15 degrees C still had marked apical membrane surface involvement. However, at 30 min and 60 min postwashout, ADH-stimulated tissues at 15 degrees C recovered considerably, with a reduction in the number of shallow apical membrane invaginations involving fewer than 33% and 20% of granular cells respectively. This may indicate that the membrane undergoes continuous remodeling even at cold temperature conditions but with a different half-time. Control bladder tissues subjected to transmission electron microscopy (TEM) reveal a dense cytoplasmic profile with a scattered distribution of secretory granules, rough ER cisternae, mitochondria, and little or no vacuolation. In contrast, ADH-stimulated bladder tissues displayed a vacuolated cytoplasm, expanded rough ER cisternae, and ruffled basolateral membranes. These observations suggest that the apical membrane undergoes considerable reorganization following cessation of hormone action and that lowering the temperature reduces the rate of membrane remodeling and thus may provide a means to monitor the processes of endocytosis and the mechanisms responsible for water channel retrieval.

The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum.

Autocrine motility factor receptor (AMF-R) is a marker for a distinct smooth membranous tubule. Ilimaquinone (IQ) is a sea sponge metabolite which induces the complete vesiculation of the Golgi apparatus and we show here that the addition of IQ to MDCK cells also results in the disruption of the AMF-R tubule. By immunofluorescence microscopy, the resultant punctate AMF-R label resembles the products of IQ-mediated vesiculation of the trans-Golgi network, however, the two labels can be distinguished by confocal microscopy. AMF-R tubule fragmentation occurs after nocodazole or taxol treatment of the cells demonstrating that the action of IQ on AMF-R tubules is not related to the ability of IQ to depolymerize microtubules. IQ activity is therefore not Golgi-specific. Electron microscopy of IQ-treated cells reveals that AMF-R is distributed to fenestrated networks of narrow interconnected tubules which are distinguishable from the uniform Golgi-derived vesicles and morphologically equivalent to smooth ER. Distinct fenestrations are visible in incompletely fragmented tubules which may represent intermediates in the fragmentation process. Smooth AMF-R labeled tubules exhibit continuity with rough ER cisternae and IQ selectively targets smooth and not rough ER. AMF-R tubules can be distinguished from the intermediate compartment labeled for ERGIC-53 by confocal microscopy and thus constitute a distinct IQ-sensitive subdomain of the smooth ER.

Microinjection of profilins from different sources into the green algaMicrasterias causes transient inhibition of cell growth

Recombinant profilins from different sources (Betula verrucosa, Schizosaccharomyces pombe, Acanthamoeba castellani, or man) cause marked effects on cell growth and morphogenesis when microinjected into growing cells of the green algaMicrasterias denticulata. Whereas control injections with β-lactoglobulin only result in a slight delay of cell growth, when profilin is injected cell differentiation ceases and only resumes about 1 to 2 h after the injection, depending on the dose. The resulting cell does not show any malformations, but is reduced in size and retarded in differentiation compared to controls. As a consequence of the profilin microinjection the pattern of cytoplasmic streaming and cytoplasmic structure are also altered. Gelsolin, injected for comparison, leads to minor retardation of cell development but produces less marked effects than profilin. Microinjection of fluorescently labeled profilin shows even distribution throughout the cytoplasm and more intense fluorescence in the nucleus. Electron microscopical investigations of cells fixed immediately after profilin injection show a normal distribution of dictyosomes, ER cisternae, microtubules, and secretory vesicles compared to noninjected controls at the same developmental stage. Our results indicate that disturbance of the natural actin turnover by the injection of actin-binding proteins strongly affects development ofMicrasterias, corroborating a key role of actin in the morphogenetic process.

Osmoregulatory mutants that affect the function of the contractile vacuole inChlamydomonas reinhardtii

Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.

Effects of UV irradiation on cell development and ultrastructure of the green alga Micrasterias

The unicellular freshwater green alga Micrasterias denticulata growing in small acid bog ponds at different altitudes has been subjected to different UV conditions experimentally produced in a sun simulator in the presence of white light. The results show that the cells are resistant to UV cut-off wavelengths down to 284 nm even when exposed during the most sensitive stage of cell development for 5 h. Under these conditions cell growth, morphogenesis, cytoplasmic streaming and organelle distribution remain unaffected. When lowering the cut-off wavelength to 280 or 275 nm or prolonging the time of exposure cytoplasmic streaming is retarded, vacuoles are formed, chloroplast distribution is altered and gradually cell development is inhibited. At a 90 min irradiation with 280 nm cut off or a 60 min exposure to irradiation with a 275 nm cut off cytoplasmic streaming ceases. At an ultrastructural level especially dictyosomes and ER cisternae are targeted at 280 nm cut-off and at 275 nm cut-off. The dictyosomes become bent, the number of their cisternae decreases and vesicle production is reduced or completely inhibited. Large sheets of ER cisternae are formed in cortical cell regions. In contrast to these drastic alterations in structure and function of the endomembrane system the microtubule cytoskeleton remains unaffected. Even after a “275 nm” irradiation microtubule bundles are abundant in the cytoplasm. However, microtubule repolymerization after experimental destruction with a microtubule inhibitor is prevented under UV conditions which is indicated by an irreversible dislocation of the nucleus during recovery from drug treatment.

The organization of endoplasmic reticulum export complexes.

Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI.

Transformations of membrane-bound organelles in sec 14 mutants of the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

In early descriptions of ultrastructural alterations of secretory (sec) mutants of the yeast Saccharomyces cerevisiae, two mutants, sec7 and sec14, were shown to produce cell structures, the so-called Berkeley bodies thought at first to correspond to Golgi structures. In sec7 mutants grown at restrictive temperature, secretion granules soon dis-appeared, whereas networks of Golgi tubules increased in size and transformed into stacks of seven to eight flattened elements. At these time intervals, structures resembling Berkeley bodies appeared to be extensions of the endoplasmic reticulum (Rambourg et al., 1993). It is the purpose of the present study to examine by electron microscopy S. cerevisiae sec14 mutants and to compare the modifications along their secretory pathway with those occurring in a homologous mutant of Yarrowia lipolytica. S. cerevisiae sec 14 mutant cells coming from exponentially growing cultures were examined either at 24 degrees C or after shifting at 37 degrees C for 0, 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. Y. lipolytica mutant cells were first cultured in YNB in 5000 medium and then transferred for 0, 6, 8, 12, 20, and 24 hr, in a phosphate-buffered YPD medium, which allows wild cells to differentiate from yeast to mycelian form. In both cases, cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, post-fixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, stereopairs were prepared from section stained with lead citrate and tilted at +/- 15 degrees from the 0 degree position of the goniometric stage of the electron microscope. In S. cerevisiae mutant cells shifted for 2 min at the restrictive temperature, faintly stained networks of thin anastomosed tubules were located at close proximity and often continuous with faintly stained ER cisternae. More intensely stained tubular networks with nodular dilations having the size of secretion granules were dispersed throughout the cytoplasm. Later on, the faintly stained ER elements and related tubular networks decreased in number, whereas the intensely stained nodular tubular networks increased in frequency. The incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Widemeshed, intensely stained fenestrated spheres were often encountered and increased in number, in parallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as they seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cerevisiae sec14 mutant, the main ultrastructural modification observed in Y. lipolytica transferred to the YPD medium was the formation of deep plasma membrane invaginations. It appears that two functionally homologous PI/PC transfer proteins (Sec14psc and Sec14pyl) control distinct physiological processes in the two sec14 mutants examined. Such differences are perhaps related to the regulatory role of these proteins in different target organelles, i.e., the Golgi apparatus in S. cerevisiae or the plasma membrane in Y. lipolytica.

Transformations of membrane‐bound organelles in sec14 mutants of the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica

Background In early descriptions of ultrastructural alterations of secretory (sec) mutants of the yeast Saccharomyces cerevisiae, two mutants, sec7 and sec14, were shown to produce cell structures, the so-called Berkeley bodies thought at first to correspond to Golgi structures. In sec7 mutants grown at restrictive temperature, secretion granules soon disappeared, whereas networks of Golgi tubules increased in size and transformed into stacks of seven to eight flattened elements. At these time intervals, structures resembling Berkeley bodies appeared to be extensions of the endoplasmic reticulum (Rambourg et al., 1993). It is the purpose of the present study to examine by electron microscopy S. cerevisiae sec14 mutants and to compare the modifications along their secretory pathway with those occurring in a homologous mutant of Yarrowia lipolytica. Methods S. cerevisiae sec 14 mutant cells coming from exponentially growing cultures were examined either at 24°C or after shifting at 37°C for 0, 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. Y. lipolytica mutant cells were first cultured in YNB in 5000 medium and then transferred for 0, 6, 8, 12, 20, and 24 hr, in a phosphate-buffered YPD medium, which allows wild cells to differentiate from yeast to mycelian form. In both cases, cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, post-fixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, stereopairs were prepared from sections stained with lead citrate and tilted at ± 15° from the 0° position of the goniometric stage of the electron microscope. Results In S. cerevisiae mutant cells shifted for 2 min at the restrictive temperature, faintly stained networks of thin anastomosed tubules were located at close proximity and often continuous with faintly stained ER cisternae. More intensely stained tubular networks with nodular dilations having the size of secretion granules were dispersed throughout the cytoplasm. Later on, the faintly stained ER elements and related tubular networks decreased in number, whereas the intensely stained nodular tubular networks increased in frequency. The incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Wide-meshed, intensely stained fenestrated spheres were often encountered and increased in number, in parallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as they seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cerevisiae sec14 mutant, the main ultrastructural modification observed in Y. lipolytica transferred to the YPD medium was the formation of deep plasma membrane invaginations. Conclusions It appears that two functionally homologous PI/PC transfer proteins (Sec14psc and Sec14pyl) control distinct physiological processes in the two sec14 mutants examined. Such differences are perhaps related to the regulatory role of these proteins in different target organelles, i.e., the Golgi apparatus in S. cerevisiae or the plasma membrane in Y. lipolytica. © 1996 Wiley-Liss, Inc.

Developmental features of protophloem sieve elements in roots of wheat (Triticum aestivum L.)

Protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L.) are arranged in single vertical files. The number of PSEs within the files increases by symmetrical divisions, which take place after the completion of asymmetrical (formative) divisions and before the initiation of differentiation. The divisions are preceded by well defined pre-prophase bands (PPB) of microtubules, which surround the nucleus in an equatorial position. In the cytoplasmic region between the nuclear surface and the PPB, perinuclear and endoplasmic microtubules were observed. The perinuclear microtubules are considered as part of the developing spindle, while the endoplasmic ones interlink the perinuclear microtubules with the PPB. Dividing cells do not show any signs of incipient differentiation. The first and most reliable indication of a commencing differentiation is provided by the sieve-element plastids that begin to accumulate dense crystalloid inclusions in the very young PSEs. In mature PSEs plastids contain two kinds of crystalloid inclusions, dense and thin, in a translucent stroma. Depending on the plastid-inclusions criterion it was shown that: (a) the PSEs of a given root do not initiate differentiation at exactly the same stage, (b) the developmental sequence extends to a span of 7–9 actively differentiating PSEs arranged in a single vertical file, and (c) each PSE needs about 16–21 h to pass through the whole developmental sequence. In the last two differentiating PSEs of a file, mitochondria were found to be enveloped by single cisternae of ER. The association is temporary as it is lost in the first PSEs with an autolysed lumen. During differentiation, Golgi bodies were abundant and active in producing vesicles involved in cell wall development. Golgi vesicles were also found among the microtubules of the PPB, but no local thickening was observed. Golgi bodies disorganize in the last stages of autolysis and disappear in mature sieve elements.

Influence of methyl derivatives of coumarin on mitotic activity and ultrastructure of meristematic cells of Allium cepa root tips

7-Methoxy-, 7-methyl- and 6-methylcoumarins retarded mitosis in Allium cepa root promeristem and caused chromosomal aberrations. Chromosomes became shorter and thicker, and often were lost during anaphase, forming micronuclei during interphase. Chromatid bridges were observed in anaphase and telophase, leading to the formation of a restitutional nucleus, often of irregular shape. Abnormal fragmopasts, often discontinuous, prevented normal cytokinesis. There was increased vacuolization and the mitochondria contained an electron-dense matrix. Parts of the cytoplasm, which were surrounded by ER cisternae, contained mitochondria and pieces of ER membrane. Autophagous vacuoles showed a lower density of cytoplasm, but still contained numerous pieces of ER cisternae.