Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Nuclear lamina at the crossroads of the cytoplasm and nucleus

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.     

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.     

Ultrastructure of the basal lamina and its relationship to extracellular matrix of embryos of the starfish Pisaster ochraceus as revealed by anionic dyes

The ultrastructure of the 51/2–6-day-old embryonic asteroid basal lamina (BL) was studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and after treatment with anionic dyes. Conventional fixation in glutaraldehyde and osmium reveals a BL consisting of a lamina densa separated from the basal cell surface by a lamina lucida. Little or no reticular lamina is present. Material similar in appearance to the basal lamina extends into the blastocoel, forming an extracellular matrix (ECM). Following fixation in the presence of the dye ruthenium red, proteoglycan (PG) granules are visible in the lamina lucida and immediately beneath the lamina densa. The ECM consists of granules of a similar appearance, which are associated with fibers of an intermediate electron density resembling invertebrate collagen. After fixation in the presence of alcian blue under polyanionic conditions, all aspects of the basal lamina and the ECM stain very densely. The use of alcian blue in 0.3 M MgCl₂ (monoanionic condition) or in low concentrations reveals a lamina densa consisting of a fine feltwork and tubule-like structures. A meshwork composed of thick, densely stained and thinner, intermediately stained strands is embedded in the inner aspect (that adjacent to the blastocoel) of the ectodermal lamina densa. Similar elements are present in the endodermal BL, but the dense material is represented by short regions that do not form a meshwork. The dense and intermediate strands of both basal laminae also extend into the blastocoel as ECM. The tubule-like structures extend from the dense material of the inner meshwork into the lamina densa. They also cross both the lamina densa and lucida to associatee with the basal cell membranes. The fact that the basal cell surfaces are often puckered outward at the points of contact suggests that this configuration might be providing a means whereby forces can be transferred from the ECM through the basal lamina to the cells.     

Morphological variability of Undaria pinnatifida (Harvey) Suringar, 1873 (Phaeophyceae, Laminariales) in Peter the Great Bay, Sea of Japan

The morphological variability of the brown alga Undaria pinnatifida was studied in Peter the Great Bay (Sea of Japan). Comparison of the morphology of algae growing at eight sites of the bay that have different water movement revealed three phenotypically different population groups. A discriminant analysis of a set of morphological traits demonstrated that the width of the undivided part of the lamina and the stipe length make the most significant contribution to the discrimination of the detected groups. Analysis of the environmental dependence of morphological traits showed that lamina width and width of undivided part of lamina depend generally on water movement intensity, whereas the stipe length and thallus length are influenced by light intensity and surf degree.     

Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos—Detailed Scanning Electron Microscopical Study

We describe morphological events of the mammalian gastrulation in pre- to middle-primitive-streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.     

An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo

Summary The morphology and intercellular relations of cells in the matrix, lower intermediate, and upper intermediate laminae of the cerebral hemisphere of rabbit embryos was studied with the electron microscope. Models of cells reconstructed from serial sections confirm previous observations made with the Golgi technique. Most cells in the matrix lamina appear to be spongioblasts; there are relatively few neuroblasts and columnar epithelial cells. Neuroblasts predominate in the intermediate lamina. Their short processes are intercalated among axons and spongioblast processes in the lower part. A large process, the preapex, distinguishes nerve cells in the upper part of the intermediate lamina, and its orientation in the direction of movement suggests that it may actively participate in the migration of neuroblasts.Serial section analysis confirms the fact that mitotic cells in the matrix lamina are spherical and have no processes. Assuming that neuroblasts are incapable of further division, it seems probable that intermitotic germinal cells have the form of spongioblasts and columnar epithelial cells and that they give rise to neuroblasts and other spongioblasts.Supported by a postdoctoral fellowship from the United Cerebral Palsy Research and Educational Foundation and a United States National Institutes of Health fellowship No. NB 28,013—Olal.     

Palustriella falcata (Brid.) Hedenäs (Amblystegiaceae, Bryopsida) with pluristratose lamina: morphological variability of specimens in springs of the Italian Alps

Pluristratose leaf lamina in pleurocarpous aquatic mosses is a mysterious morphological character state because of its recurrence among unrelated lineages. It has been found sporadically around the world in phylogenetically distant taxa, and is thought to be a mutation and/or adaptation to aquatic habitats. During an extensive survey of bryophytes in spring habitats in the Italian Alps (Province of Trento), we found different numbers of leaf lamina cell layers among specimens of Palustriella falcata. We carried out a thorough study, measuring a set of morphological characters that identify variability among specimens within the same spring and among multiple springs. The main goals were to assess the amount of morphological variability, to quantify the concordance among morphological traits, and to test to what extent environmental variables account for morphological variability. Our results showed that, in many cases, morphological characters differed even among shoots within a spring. We found positive and significant partial correlation between pluristratose lamina and width of costa, but negative correlation between pluristratose lamina and length of cells. Constrained multivariate analysis showed that 40.3% of this morphological variation was explained by a set of environmental variables, but most importantly, we observed extensive pluristratose laminae in constantly submerged habitats. We interpreted the different numbers of cell layers in the leaf lamina as a phenotypic continuum from P. falcata, with a single layer of cells, to Palustriella pluristratosa Stech & Frahm, with a multilayered lamina. In addition we offer a point of view concerning the evolutionary significance of this trait, its possible origin, and its evolution in aquatic mosses.     

In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.     

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Remodeling of neural, glial, and tracheal cell populations in the developing Manduca wing

In the rat larynx, plasma exudation and edema formation were studied by light and electron microscopy after i.v. injections of the mast cell activator compound 48/80, substance P, and capsaicin. The morphological effects of substance P and capsaicin on connective tissue mast cells in vivo were also examined. Of the drugs tested, only compound 48/80 degranulated the connective tissue mast cells. All drugs induced a subepithelial plasma exudation in the subglottic region, with edema in the lamina propria and widened intraepithelial intercellular spaces, though the tight junction regions seemed intact. In the epiglottis, 10 min after compound 48/80 injection, there was edema in the lamina propria on the lingual side, with an intact and tight epithelial lining. No morphological sign of edema was found in the epiglottis after injection of substance P or capsaicin. The pronounced effect found in the epiglottic region after compound 48/80 injection was due to the release of mediators such as histamine and 5-hydroxytryptamine from the connective tissue mast cells. This study supports the belief that substance P in vivo mediates an increased vascular permeability by a direct effect on the blood vessels – a mechanism distinct from mast cell degranulation.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.     

Microsporidian Spore Wall: Ultrastructural Findings on Encephalitozoon hellem Exospore

A study of the spore wall of Encephalitozoon hellem was performed on thin sections, freeze-fracture, and deep-etched samples to obtain information on spore wall organization and composition. Our observations demonstrate that the spore wall is formed by an inner 30–35 nm electron-lucent endospore and an outer 25–30 nm electron-dense exospore. The exospore is a complex of three layers: an outer spiny layer, an electron-lucent intermediate lamina and an inner fibrous layer. Freeze-fracture and deep-etching techniques reveal that the intermediate lamina and the inner fibrous layer result from the different spatial disposition of the same 4-nm thick fibrils. In thin sections the endospore reveals a scattered electron-dense material that appears in the form of trabecular structures when analyzed in deep-etched samples. The presence of chitin in the exospore is discussed.     

The calcium binding proteins calbindin,parvalbumin, and calretinin have specific patterns of expression in the gray matter of cat spinal cord

Calcium binding proteins (CBPs) regulate intracellular levels of calcium (Ca²⁺) ions. CBPs are particularly interesting from a morphological standpoint, because they are differentially expressed in certain sub-populations of cells in the nervous system of various species of vertebrate animals. However, knowledge on the cellular regulation governing such cell-specific CBP expression is still incomplete. In this work on the L7 segment of the cat spinal cord, we analyzed the localization and morphology of neurons expressing the CBPs calbindin-28 KD (CB), parvalbumin (PV), and calretinin (CR), and co-expressing CB and PV, CB and CR, and PV and CR. Single CBP-positive (⁺) neurons showed specific distributions: (1) CB was present in small neurons localized in laminae I, II, III and X, in small to medium size neurons in laminae III–VI, and in medium to large neurons in laminae VI–VIII; (2) PV was present in small size neurons in laminae III and IV and in medial portions of laminae V and VI, medium neurons and in lamina X at the border with lamina VII, in medium to large neurons in laminae VII and VIII; (3) CR labeling was detected in small size neurons in laminae I, II, III and VIII, in medium to large size neurons in laminae I and III–VII, and in small to medium size neurons in lamina X. Double labeled neurons were a small minority of the CBP⁺ cells. Co-expression of CB and PV was seen in 1 to 2% of the CBP⁺ cells, and they were detected in the ventral and intermediate portions of lamina VII and in lamina X. Co-localization of CB and CR was present in 0.3% of the cells and these cells were localized in lamina II. Double labeling for PV and CR occurred in 6% of the cells, and the cells were localized in ventral part of lamina VII and in lamina VIII. Overall, these results revealed distinct and reproducible patterns of localization of the neurons expressing single CBPs and co-expressing two of them. Distinct differences of CBP expression between cat and other species are discussed. Possible relations between the cat L7 neurons expressing different CBPs with the neurons previously analyzed in cat and other animals are suggested.     

High levels of morphological variation despite close genetic relatedness between Zoanthus aff. vietnamensis and Zoanthus kuroshio (Anthozoa: Hexacorallia)

Recent investigations into the encrusting anemone genus Zoanthus using molecular and morphological techniques have begun to bring order to this taxonomically neglected group. Previous studies have confirmed the existence of three distinct species present in southern Japan: Z. sansibaricus, Z. kuroshio, and Z. gigantus. Results from such studies show species of Zoanthus to be highly morphologically plastic, often incorporating morphotypes with varying oral disk color and oral disk diameter. Literature lists the species Z. aff. vietnamensis as occurring in southern Japan and throughout the western Pacific Ocean, but due to the morphological plasticity of Zoanthus species, a re-examination of Z. aff. vietnamensis using molecular techniques was needed. Here, using mitochondrial 16S rDNA and the nuclear internal transcribed spacer of ribosomal DNA (ITS-rDNA) sequences, as well as morphological data, we have examined several nominal Z. aff. vietnamensis samples collected from Kagoshima Bay and Yakushima Island, Japan. Based on polyp length and diameter, oral disk diameter, mesentery and tentacle numbers, and colony form, Z. aff. vietnamensis is easily distinguishable from Z. sansibaricus, Z. kuroshio, and Z. gigantus. However, despite these clear morphological differences, our mitochondrial and nuclear sequence-based phylogenies indicate that Z. aff. vietnamensis and Z. kuroshio are very closely related (perhaps conspecific), highlighting the morphological plasticity of this genus and the difficulty of species identification based on morphological data alone.     

Extracellular Matrix Components Regulating Glandular Differentiation and the Formation of Basal Lamina of a Human Pancreatic Cancer Cell Line in Vitro

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extracellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.     

Antisera to basal lamina and glial endfeet disturb the normal extension of axons on retina and pigment epithelium basal laminae

In order to determine the role of the extracellular matrix in regulating the directed growth of embryonic neurites, antisera to retina (a-RBL I and II), to pigment epithelium (a-PBL) and to glomerular (a-GBL) basal lamina were probed for an effect on the ordered extension of neurites. In the assays, retina explants from chick and quail were cultured on basal lamina from embryonic chick retina and pigment epithelium either in the presence of anti-basal lamina antisera or in the presence of the corresponding preimmune sera. In the presence of all anti-basal lamina antisera, normal extension of axons was greatly inhibited both on retina and on pigment epithelium basal lamina. The antisera affected the growth pattern and the morphology of the individual axons in two ways: in the presence of a-RBL I the short axons were less directed, developed more and longer side branches, and the lamellipodia of the growth cones were reduced in size compared to axons from control cultures. In the presence of a-RBL II and a-GBL, axons grew slowly out from the explants as very thick bundles, strikingly different from axons in control cultures. The antiserum to pigment epithelium basal lamina induced both strong fasciculation and disorganization of the linear fiber extension, being intermediate between the two types of effects observed after antiserum addition. The results suggest that adhesive matrix molecules in basal laminae have important functions in elongation, fasciculation and in the morphology of growing axons.     

Light- and electronmicroscopic investigations of the development of the stria vascularis in the ductus cochlearis of the guinea pig fetus

Our studies on exactly dated guinea pig fetuses show: Already in early stages of development (37th day), in the anlage of the vascular stria vesicular invaginations of the apical plasmalemma of epithelial cells are noted and regarded as a sign of an exchange of substances between epithelial cells and the endolymph. As to the direction of substance transport, the morphological finding allows no decision. From the 42nd to the 44th day of development, the subepithelial basal lamina becomes progressively indistinct. From the 45th day onward, the basal lamina is not yet demonstrable. The largest part of the chromophobe cells of the vascular stria derives from the mesenchyme. The question, whether some of the chromophobe cells are of epithelial origin, as some authors propose, will hopefully be clarified in further studies.