Outer Nuclear Envelope Research Articles

Age-related changes in ultrastructural features of cathepsin B- and D-containing neurons in rat cerebral cortex

The present study examines age-related changes in the subcellular localization of cathepsin B (cath B) and cathepsin D (cath D), as well as morphological features of the cathepsin-immunoreactive (ir) neurons in rat cerebral cortex. Sprague–Dawley rats were studied at 3 and 26 months. By immunoelectron microscopy cath B- or cath D-immunoreactivities were found in many, but not all, pyramidal neurons. In young rat cerebral cortical neurons, cath B was observed not only in lysosomal systems such as multivesicular bodies, dense bodies, and lipofuscin granules, but also in extralysosomal sites. By contrast, cath D was confined mainly to lysosomal systems in young rats. In aged rats, cath B showed a similar pattern in its subcellular localization compared to the young control, but some of the dense bodies containing cath B was closely apposed to the outer nuclear envelope. These cells exhibited a relatively normal appearance. Regardless of subcellular localization, approximately 10% of cath B-ir neurons displayed ultrastructural disturbances presumed to indicate an early stage of degeneration. The nucleus was indented, nuclear boundary was indistinct, nuclear pore structures appeared separately with high frequency, and the endoplasmic reticulum appeared to be affected. In addition to its presence in lysosomal structures, cath D-immunoreactivity in aged cerebral cortex was noted prominently in the cytosol as diffuse granules. About 37% of cath D-ir cells showed this age-related change. Among the neurons with the diffusely scattered form of cath D, approximately 70% of cells exhibited the degenerating features. These cells were characterized by large amounts of diffuse cath D, reduced cellular size, loss of the nuclear boundary, scattered nuclear pore structures, an often fragmentation of the nucleus, disturbances of endoplasmic reticular system, and in advanced stages, condensed nucleus and poor preservation of almost cytoplasmic organelles. Though some of these features were also found in cath B-ir neurons, findings of overt degeneration, such as fragmented and condensed nuclei and impaired almost cytoplasmic organelles, were generally not observed in cath B-ir neurons. In addition, lipofuscin aggregates containing cath D were observed frequently in the extracellular space close to sites of ruptured plasma membrane, whereas in the sections stained with anti-cath B antibodies, large-sized lipofuscin aggregates showed only very weak or no cath B-immunoreactivity at all. Taken together, the present results suggest that cath D and cath B may be regulated differently and play their specific roles in the aging of the brain, especially, the change in location of cath D from the lysosomal system to the cytosol in the aged brain may play an important role in age-related cell death.

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca2+ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.

Rickettsia rickettsii infection of the EA.hy 926 endothelial cell line: morphological response to infection and evidence for oxidative injury.

EA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium. Rickettsia rickettsii can efficiently infect and cause a cytopathic effect in EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA.hy 926 cells at 10 d post-infection (p.i.) following application of a secondary agarose overlay containing 2 micrograms emetine ml-1 and 40 micrograms NaF ml-1 on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication of R. rickettsii resulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i. These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously for R. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA.hy 926 cells were also used to investigate the influence of the antioxidant alpha-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 microM alpha-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 microM alpha-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 microM alpha-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing alpha-lipoic acid concentrations. Furthermore, treatment with 500 microM alpha-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. In conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced by R. rickettsii infection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.

Effects of the antioxidant alpha-lipoic acid on human umbilical vein endothelial cells infected with Rickettsia rickettsii.

Rickettsia rickettsii infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy (D. J. Silverman, Infect. Immun. 44:545-553, 1984). These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems. In this study, it was shown that R. rickettsii infection of human umbilical vein endothelial cells resulted in a significant depletion of intracellular reduced glutathione (thiol) content at 72 and 96 h and decreased glutathione peroxidase activity at 72 h postinfection. Infected cells displayed a dramatic increase in the concentration of intracellular peroxides by 72 h. Supplementation of the cell culture medium with 100, 200, or 500 microM alpha-lipoic acid, a metabolic antioxidant, after inoculation with R. rickettsii restored the intracellular levels of thiols and glutathione peroxidase and reduced the intracellular peroxide levels in infected cells. These effects were dose dependent. Treated infected monolayers maintained better viability at 96 h after inoculation with R. rickettsii than did untreated infected cells. Moreover, supplementation of the cell culture medium with 100 microM alpha-lipoic acid for 72 h after infection prevented the occurrence of morphological changes in the infected cells. The presence of 100 or 200 microM alpha-lipoic acid did not influence rickettsial growth in endothelial cells, nor did it affect the ability of R. rickettsii to form lytic plaques in Vero cells. Treatment with 500 microM alpha-lipoic acid decreased by 50% both the number and size of lytic plaques in Vero cells, and it also decreased the recovery of viable rickettsiae from endothelial cells. However, under all treatment conditions, a significant number of rickettsiae could be detected microscopically. Furthermore, the rickettsiae apparently retained their capacity for intracellular movement, since they possessed long polymerized actin tails after 72 and 96 h of treatment regardless of the concentration of alpha-lipoic acid used. Since alpha-lipoic acid does not seem to exhibit direct antirickettsial activity except with long-term exposure at very high concentrations, the mechanism of its protective activity for endothelial cells infected with rickettsiae may involve complex changes in cellular metabolism that only indirectly affect rickettsiae.

Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

2418 Effect of abdominal vagotomy on interleukin-1β induced noradrenaline release in the paraventricular nucleus in conscious rats

Seasonal breeding mammals exhibit a reversible annual cycle of fertility, and thus represent valuable models for investigating the organization of luteinizing hormone-releasing hormone (LHRH) neurons which mediate the neuroendocrine control of reproduction. Electron microscopic immunocytochemistry was used to examine the ultrastructure of LHRH neurons and their projections in a seasonal breeder, the golden hamster, housed under photoperiodic conditions which are reproductively stimulatory. LHRH perikarya in the diagonal band, medial septum, and preoptic area were bipolar or unipolar cells which possessed nuclei with prominent indentations and multiple nucleoli. LHRH reaction product within these cells was associated with neurosecretory granules and the rough endoplasmic reticulum (RER), particularly those portions of RER adjacent to the outer nuclear envelope. LHRH cell bodies and dendrites in the hamster received an extremely limited amount of synaptic input; of a total of twenty-five cells analyzed, we found only five instances of morphologically identified synapses onto immunoreactive dendrites. Occasionally close somatic appositions were seen between LHRH cells and non-immunoreactive neurons, and in one instance between two LHRH somas. In the organum vasculosum of the lamina terminalis (OVLT), LHRH fibers appeared as isolated strings of varicosities. In contrast, within the median eminence immunoreactive axonal profiles were frequently bundled together in fascicles although synaptic specializations were not observed between individual axons. The existence of contacts between LHRH axons in the median eminence, coupled with the extreme paucity of synaptic inputs onto LHRH neurons in this species, suggests that the median eminence may be a site where neural or hormonal signals could influence the coordinated release of LHRH from cells of dispersed origin.

Ultrastructural changes in aging leaves of a light‐grown achlorophyllous mutant of barley

The pattern and sequence of cellular degradation during the course of leaf senescence remains obscure and the nature of the trigger that induces cell senescence is unknown. In order to probe the pre‐mortem phase of senescence temporal changes in cell ultrastucture were studied in aging leaves of light‐grown achlorophyllous Hordeum vulgare L. cv. Dyan mutant seedlings. Electron microscope examination of the ultrastructure of mesophyll cell plastids revealed the absence of ribosomes and a highly disorganized prolamellar body. Both the number and size of plastoglobuli increased with aging and this change coincided with depletion of starch grains and dilation of lamellar membranes. Aging of mesophyll cells occurred coincident with a decline in ribosome content of the cytoplasm and loss of matrix granularity. Loss of ribosomes associated with the outer nuclear envelope membrane and a reduction in chromatin were also apparent. Only after 10 days was there evidence of loss of internal membrane integrity and swelling of mitochondrial cristae. Compartmentation was thus maintained during the aging process with membrane dissolution occurring late in senescence. These results suggest that an inability to produce chlorophyll and carotenoids and form thylakoid stacks due to the absence of plastid ribosomes, contributes to the rapid onset of senescence in light‐grown achlorophyllous seedlings. Furthermore, disruption of chloroplast ribosome synthesis/assembly may constitute part of the plastid signal involved in triggering cell senescence.

Comparison of ultrastructural characteristics of gonadotropin-releasing hormone neurons in prepubertal and adult male rats

Gonadotropin-releasing hormone neurons from prepubertal (29-day-old) and adult (three-month-old) male rats were demonstrated immunocytochemically using the LR1 antibody, and prepared for electron microscopic examination. Gonadotropin-releasing hormone neurons were equally immunoreactive in the two age groups, but there were heavy deposits of reaction product in the outer nuclear envelope of these neurons in prepubertal animals. Point count stereology on electron micrographic montages of gonadotropin-releasing hormone neurons at x25,000 was used to compare the relative proportion of cytoplasm containing various subcellular organelles. More of the cytoplasm was occupied by Golgi apparatus and secretory vesicles in the prepubertal animals. The representation of mitochondria was equal in the two age groups, while there were more lysosomes in the gonadotropin-releasing hormone neurons from adult animals. The density of synaptic input to the neurons was estimated using quantitative morphometrics on electron micrographs of three levels of section through the neuron, magnified x25,000. The percentage of the perikaryal membrane with synaptic contacts was greater in the gonadotropin-releasing hormone neurons from adults. Most strikingly, there were gonadotropin-releasing hormone terminals on gonadotropin-releasing hormone soma of these neurons in prepubertal animals, but not in the adults. The highly immunoreactive outer nuclear envelope and relative larger representation of Golgi and secretory vesicles in gonadotropin-releasing hormone neurons in prepubertal animals suggest that these cells are actively synthesizing peptides, including gonadotropin-releasing hormone. The large representation of Golgi apparatus may also reflect the active biosynthesis of membrane in association with the elaboration of neuronal processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of gonadotropin-releasing hormone during the rat estrous cycle: a cellular analysis.

In an attempt to understand the regulatory events that control the synthesis of gonadotropin-releasing hormone (GnRH) during the estrous cycle of the rat, we undertook a cellular analysis of time of translation of GnRH mRNA into protein. A specific antiserum, Rb 1076, which recognizes both the extended proGnRH as well as the processed form of the decapeptide was used for immunocytochemical staining. A cell was considered to be actively translating the pro-GnRH mRNA if elements of the rough endoplasmic reticulum (RER), including the outer nuclear envelope, were filled with reaction product. A synthetically quiescent cell contained only immunopositive neurosecretory granules. Cycling rats were killed at various times and all GnRH cells scored as being RER positive (+) or negative (-). On the morning of estrus almost all GnRH neurons in 5 out of 6 of the animals studied were synthesizing their unique peptide. The immunostaining in many of the cells at this time was very pale, suggesting a prior depletion. This was the only time point examined where near uniformity among individuals in a group was observed. At all other times considerable heterogeneity was observed among animals within a group. For example, at 17.30 h on the afternoon of proestrus 50% or more of the GnRH neurons were RER+ in half of the animals; the other half had values of 16-45% RER+. Synthetically active and inactive cells were found in close proximity in all animals. No regional differences were observed; all GnRH cell subpopulations from the level of the diagonal band of Broca through the hypothalamus reflected the population as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical Differentiation and Intercellular Interactions of Migratory Gonadotropin-Releasing Hormone (GnRH) Cells in the Mouse

GnRH cells are first detected in the olfactory placode of the mouse on Gestational Day 11.5 (E11.5). Between E12.5 and E15.5 they migrate across the nasal septum and by E16.5 attain their adult distribution within the forebrain. In the present study, we used immunocytochemistry at the light and electron microscopic level to study the biochemical and morphological differentiation of gonadotropin-releasing hormone (GnRH) neurons and the cellular associations that they make during this migratory process. On E12.5, when the majority of GnRH cells are still in the nasal septum, only 15% of the population can process the pro-GnRH precursor to the amidated decapeptide, Two days later (E14.5), when most of the cells have advanced into the forebrain, 79% contain mature GnRH. In keeping with these light microscopic observations, ultrastructural analysis reveals that on E12.5 GnRH immunologic reaction product is confined to the outer nuclear envelope and rough endoplasmic reticulum. By E14.5 these migratory cells in the nasal septum have more reaction product in the rough endoplasmic reticulum and some of the Golgi cisternae are also immunopositive. Neurosecretory granules, some of which are immunoreactive, also appear at this stage. We had anticipated that the expression of GAP-43 would coincide with axonal elongation and pathfinding in GnRH neurons. Instead, GAP-43 was expressed at the early migratory stage of GnRH cells and its expression declined rapidly after these neurons had entered the forebrain and commenced axonal outgrowth. Hence, on E12.5, 62.3% of GnRH cells in the nasal septum are immunopositive for GAP-43, while only 12.6% of the forebrain population at the same embryonic stages express the protein. Similarly, on E14.5 GAP-43 is expressed in 50.7% of the nasal septum GnRH cells but only 11.6% of cells in the E14.5 forebrain express this protein. While in the nasal septum, GnRH neurons migrate only within the confines of the olfactory and vomeronasal axonal fascicles. During this part of the migratory route, the cells maintain close membrane apposition with each other and with the axons and ensheathing glia of the nerve fascicles. Once in the forebrain, GnRH neurons no longer maintain these associations, nor do they follow any defined anatomical structure. These findings indicate that although GnRH cells express their unique neuropeptide early in their ontogeny, their differentiation continues and is coordinated with their migration. The migration of these neurons across the nasal septum relies on axonal fascicles of the olfactory and vomeronasal nerves. In the forebrain, however, GnRH cells must utilize alternative guidance mechanisms to complete their migration.

Mycorrhiza‐like interaction between the achlorophyllous gametophyte of Lycopodium clavatum L. and its fungal endophyte studied by light and electron microscopy

SUMMARYThe mycorrhiza‐like association of mature gametophytes of Lycopodium clavatum and an endophytic fungus was studied by means of light and electron microscopy. Fine aseptate hyphae of the fungus formed very regular dense hyphal coils and numerous small vesicles of different stages in the cortical cells. Extended infections occurred within preformed spaces between the cells of the storage tissue. These spaces probably arose by degeneration of the host cells. Several hyphae grew within them and were partly swollen or irregularly lobed and branched. They were embedded in a heterogeneous material, probably of host origin. Arbuscles were not established during the interaction. All fungal structures degenerated in the older host tissues that remained intact. The fungus contained bacterium‐like organelles. Elliptic interphase spindle‐pole bodies of the fungus were attached to the outer nuclear envelope. The host‐fungus interaction in the gametophyte of L. clavatum cannot be related to any type of mycorrhizal association described to date. It is therefore called the ‘Lycopodioid mycothallus interaction’.

Cryofixation and freeze substitution of teliospores of Gymnosporangium clavipes: an ultrastructural investigation

Teliospores of the rust fungus Gymnosporangium clavipes were cryofixed using high-pressure freezing or cold-metal slamming techniques and prepared for transmission electron microscopy by freeze substitution. Both cryofixation techniques provided excellent ultrastructural preservation of the teliospore cytoplasm. Gross cytoplasmic freeze damage was not observed in any spore frozen in a dextran solution under high pressure and only a low number had minor freeze damage. More spores exhibited cytoplasmic freeze damage using the cold-metal slamming technique. A comparison of these cryotechniques indicated that dextran induced no obvious ultrastructural artifacts during high-pressure freezing. Artifacts induced by high-pressure freezing were identified as breaks in the spore wall and disruptions of the nuclear envelope. In most teliospores, regions of the cytoplasm separated from the inner surface of the teliospore wall creating electron-transparent gaps. Teliospores consisted of two cells separated by a transverse septum. Each cell contained a single, spherical nucleus with a well-preserved nucleoplasm. A spindle pole body and oblong, fibrillar bodies were associated with the outer nuclear envelope. Ribosomes, glycogen particles, mitochondria, microbodies, lipid bodies, strands of smooth endoplasmic reticulum, complex membranous networks, double membraned cisternae and numerous vesicles were present throughout the cytoplasm. Golgi equivalents were not observed. Microtubules were observed in the cytoplasm and often seen near the outer surfaces of nuclei. Microfilaments were not detected in the cytoplasm but were observed in the nuclear matrix. The utility and fidelity of high-pressure freezing in preserving fungal cells for ultrastructural analysis are discussed.

HRSEM of the nuclear envelope (NE): Nuclear pore substructure; baskets and fibrous components

The nuclear envelope (NE) of eukaryotic cells has been studied for many years by a variety of em techniques yielding a three dimensional model of the nuclear pore complex (NPC) consisting of two rings (∼120nm diameter), one at the outer NE and one at the inner NE. Between the rings are eight spoke structures and a central plug. The cytoplasmic ring may be decorated with up to eight particles. The NPCs are embedded in a proteinaceous network: the nuclear lamina. Recently, low voltage HRSEM was used to show the existence of a basket-like structure attached to the nucleoplasmic ring. SEM is an ideal technique for the study of membrane surfaces. High resolution can be achieved in SEMs by the use of a field emission source which produces a high brightness probe of less than lnm diameter and a specimen stage within the objective lens, reducing chromatic abberations and production of SEIII electrons. Resolution of biological specimens can be further enhanced by coating with thin, continuous films of refractory metals such as chromium or tantalum which allows the use of higher accelerating voltages and magnifications. The NEs of Xenopus oocyte germinal vesicles have been prepared as previously described for HRSEM without detergent except they have been coated nominally with 3nm of tantalum by magnetron sputtering instead of ion beam sputtered platinum. NEs have then been examined at 30kV. The ring, plug/spoke complex and particles can all be seen at the cytoplasmic surface as well as details of the outer membrane structure and particles associated with it (Fig. 1). On the nucleoplasmic surface (Fig. 2) the inner ring is observed. It has a subunit appearance with eight filaments extending from between the subunits to a third ring structure: these make up the basket-like structure. When ‘baskets’ are close together they are joined by fibres at the ‘basket ring’ (Fig. 2). When several baskets are in close proximity these fibres form a network like a canopy over the baskets (Fig. 3). Other fibres are present on the inner membrane surface which may be membrane associated fibres or canopy fibres that have collapsed. It is uncertain which, if any, of these fibres are lamins as a further level of fibres is observed at the level of the nucleoplasmic ring when the membrane is removed with detergent (Fig. 4). These fibres are consistent with previously described lamina.

The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors

We have isolated two cytosolic fractions from Xenopus oocytes that contain all of the activity necessary to support both steps of nuclear import in digitonin-permeabilized mammalian cells: binding at the nuclear envelope and translocation through the nuclear pore. The first cytosolic fraction (fraction A) interacts with an import-competent, but not a mutant, nuclear localization sequence-bearing conjugate and stimulates its accumulation at the nuclear envelope in an ATP-independent fashion. The second cytosolic fraction (fraction B) gives no discernible effect when added alone; but when added either together with fraction A, or after fraction A, stimulates the passage of the conjugate from the outer nuclear envelope to the interior of the nucleus in an ATP-dependent fashion.

Localization of interferon-induced lupus inclusions demonstrated by computer image reconstruction of monensin-treated daudi cells

A structural analysis of cells that contained the interferon-α-induced lupus inclusions (LI) was performed using a high-voltage electron microscope to determine the exact cellular location of LI and their association with normal cell organelles. LI were induced in the human B lymphoblastoid cell line, Daudi, by culturing with the pure recombinant human leukocyte interferon, IFLrA. Just prior to harvesting, a portion of the cells was treated with monensin to selectively swell the Golgi apparatus, and thereby simplify their identification using the electron microscope. Organellar associations between LI and the outer nuclear envelope and Golgi apparatus were identified in stereopairs of 1-μm sections prepared from both cells that were not treated with monensin and those that were treated with monensin. Serial 0.25-μm sections of the monensin-treated cells were prepared, and seven arbitrarily chosen cells were examined. Each of these cells contained a single LI, and it formed throughout an endoplasmic-reticulum region that made contact with both the outer nuclear envelope and the Golgi vesicles. Reconstruction of a cell by computer from the digitized negatives of serial sections clearly illustrated these relationships. This study reports the first determination of the association between LI and the Golgi apparatus. It also identifies the presence of only one LI in every cell, and the routine association of the LI with both the outer nuclear envelope and the Golgi apparatus. The unique cell location of LI formation suggests their functioning in membrane biogenesis, the trafficking of proteins to the plasma membrane or to cytoplasmic vesicles, or the processing of proteins for secretion.

On the use of human autoantibodies in the determination of cytoskeletal-cell surface interactions

Intermediate filaments (IF) form a cytoskeletal system which appears to connect the nuclear surface with the cell surface. At the level of the nuclear surface, IF appear to be anchored to the outer nuclear envelope membrane and/or to nuclear pore complexes. In turn, these cytoplasmic IF are also thought to be linked in some unknown fashion to the nuclear lamina which is composed primarily of a polymer of the Type V IF proteins, the nuclear lamins. At the level of the cell surface, IF are connected to plasma membrane associated structures at sites of cell-cell and cellsubstrate adhesion. In epithelial cells, adhesion sites between cells include desmosomes and the major adhesion sites between basal cell surfaces and the basement membrane are termed hemidesmosomes.

Relation of nucleolar structure and position to the cytoplasmic microtubule system in Dictyostelium

AbstractThe cytoplasmic microtubule system seems to influence the position and structure of nucleoli in Dictyostelium discoideum amoebae in several growing and migrating states. For example, nucleoli were usually excluded from the nuclear periphery near the microtubule‐organizing center (MTOC) in all cases; and in migrating adherent cells, more than half the nucleoli were located opposite the MTOC. This localization was disrupted by nocodazole treatment, after which the nucleoli were largely dispersed except near the MTOC. More extensive effects of microtubules on nucleolar structure were seen in aggregating cells. In contrast to the normal oval structure in growing cells, nucleoli took on a different morphology: they protruded from the leading edge of nuclei and elongated to form nozzle‐like structures. Analysis by rapid‐freeze substitution and indirect immunofluorescence showed each nozzle surrounded by more than 10 microtubules; and in the presence of nocodazole, the microtubules shortened as expected and the nozzles disappeared. Between microtubules and the outer nuclear envelope, various‐sized cross‐bridges were seen. The implication that microtubules were associated with the nucleoli in aggregating cells was verified in vitro: nuclei isolated from growing cells contained the MTOC but few if any detectable microtubules; but nuclei from aggregating cells were surrounded by them. These data are consistent with the notion the microtubule system may help regulate the position and conformation of nucleoli during early development of Dictyostelium.

Endogenous opioids and neural cancer: An immunoelectron microscopic study

[Met 5]-enkephalin, an endogenous opioid peptide derived from proenkephalin A, participates in tumorigenic events by serving as a natural trophic factor that inhibits cell replication. In order to understand how endogenous opioids function in modulating neoplasia, the present study examined the fine structural association of enkephalin with the cellular components of a tumor cell. Immunoelectron microscopic studies were undertaken using antibodies recognizing [Met 5]-enkephalin-like substances, and murine S20Y neuroblastoma cells that are known to be responsive to endogenous opioid modulation. Enkephalin was found throughout the cell body and process. Immunoreactivity was associated with the plasma membrane, outer nuclear envelope, and a variety of organelles. With the exception of aggregates of immunoreactivity subjacent to the inner nuclear envelope, the nucleus was not reactive. These results establish that growth-related enkephalins are localized discretely within neuroblastoma cells. Since neuroblastoma cells produce and secrete enkephalins, and enkephalins interact with receptors to mediate actions on cell replication, this study examined enkephalins involved in two different patterns of traffic, further work will be needed to examine each aspect.

Cytochemical demonstration of increased phospholipid content in cell membranes in chlorphentermine-induced phospholipidosis.

We recently introduced a novel cytochemical approach to high-resolution cytochemistry of phospholipids in biological tissues. The technique consists of adsorption of bee venom phospholipase A2 to colloidal gold particles (PLA2-gold complex) and subsequent application of this complex for localization of the enzyme substrate, i.e., glycerophospholipids. In the present study, this technique was applied at the post-embedding level, in both light (LM) and transmission electron microscopy (TEM), to investigate drug-induced phospholipidosis, an experimental disorder in which the lysosomal catabolism of phospholipids is inhibited. Rats received one week of daily treatment (40 mg IP/kg) with chlorphentermine (CP), a cationic amphiphilic drug known to induce phospholipidosis in several tissues. Glutaraldehyde- and osmium-fixed lung and kidney tissues from both treated and control animals, were embedded in Epon and sections processed for labeling by PLA2-gold. In CP-treated specimens the presence of large osmiophilic inclusions in several cell types of lung parenchyma and kidney cortex confirmed the onset of phospholipidosis. These inclusions were densely labeled by PLA2-gold at both LM and TEM levels. Two general types of abnormal inclusions were distinguished on the basis of their ultrastructure and labeling pattern by PLA2-gold, suggesting different content or configuration of phospholipids. Moreover, quantitative evaluation of labeling density over various membrane compartments in lung alveolar cells evidenced significantly increased phospholipid content after CP treatment. In type II pneumocytes, such increases were measured in membranes of the RER, Golgi complex, outer and inner nuclear envelope, and the basolateral and apical domains of the plasma membrane. In capillary endothelial cells, the basal and luminal domains of the plasma membrane also showed an increase in labeling density. These results further demonstrate the potential usefulness of the PLA2-gold technique for in situ ultrastructural localization of phospholipids in normal and pathological tissues.

Potential for free radical-induced lipid peroxidation as a cause of endothelial cell injury in Rocky Mountain spotted fever.

Cells infected by Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, display unusual intracellular morphological changes characterized by dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope. These changes are consistent with those that might be expected to occur following peroxidation of membrane lipids initiated by oxygen radical species, such as the hydroxyl radical or a variety of organic radicals. Using a fluorescent probe, we have found significantly increased levels of peroxides in human endothelial cells infected by R. rickettsii. Studies with desferrioxamine, an iron chelator effective in preventing formation of the hydroxyl radical from hydrogen peroxide and the superoxide free radical, reduced peroxide levels in infected cells to those found in uninfected cells. This observation suggests that the increased peroxides in infected cells may be lipid peroxides, degradation products of free radical attack on polyenoic fatty acids. The potential for lipid peroxidation as an important mechanism in endothelial cell injury caused by R. rickettsii is discussed.