Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis

The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.     

Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures

The intracellular precipitation of nonerythrocyte spectrin has been achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the alpha chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells, and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and two proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the two types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. We conclude that the integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape, or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin, however, did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin- reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.     

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.     

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.     

Immunodetection of spectrin antigens in plant cells

The occurrence of spectrin in plant cells was studied by immunoblotting of extracts, and its localization by immunolabelling of cells, using two polyclonal antibodies raised against spectrin from human and chicken erythrocytes. A variety of plant cells were studied. The two antibodies gave the same results on blots as well as on cells. Western blots of extracts showed weak immunolabelling at 220 kD, where spectrin can be expected, but bands at 85 kD stained more heavily. Because the latter bands were also seen on blots with commercially purified spectrin, we conclude that they were breakdown products of spectrin. Native plant extracts on blots from IEF gels showed a band at pI 4.8, where the purified animal spectrin is also found. Immunolocalizations done on whole cells, PEG-, BMM-, or cryo-sections gave similar data. In most cells the labelling was localized predominantly at the plasma membrane, especially of thin-walled cells. Labelling was also seen in the periphery of a particular class of organelles, probably plastids. This labelling was tissue specific in maize somatic embryos. During carrot somatic embryogenesis cytoplasmic labelling was observed depending on the developmental stage. Many cells with cytoplasmic labelling also had stained nuclei. Labelled nuclei had more condensed chromatin than non-labelled nuclei.     

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane

We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by approximately 3 nm.     

The cycle of follicular and interstitial cells (Leydig cells) in the testis of the marbled newt, Triturus marmoratus (Caudata, Salamandridae)

Ultrastructural examination of the marbled newt (Triturus marmoratus) testis throughout the annual cycle revealed that during the period of testicular quiescence (November-February), primordial germ cells proliferate within cords of filament-rich epithelial cells that will become follicular cells (FCs). Fibroblast-like cells surround the FCs and form the lobule-boundary interstitial cells (ICs). During the period of germ cell development from primordial germ cells to round spermatids (March-June), the FCs surrounding the developing germ cells contain scanty cytoplasm with abundant rough endoplasmic reticulum and scarce filaments. With spermatid elongation (July-August), the FC size grows, its nucleus becomes irregularly outlined, and its cytoplasm displays abundant smooth endoplasmic reticulum, residual bodies, lipid droplets, and large vacuoles. After spermatozoon release by the FCs (August-September), the adjacent ICs increase their size and transform into Leydig cells with abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and lipid droplets. During the period of testicular quiescence (November-February), the Leydig cells undergo involution, eventually developing the morphological attributes of mesenchymal cells. Intermingled among these cells, cords of filament-rich cells are observed. During this period of the cycle, spermatozoon cysts supported by FCs are present. At the beginning of the germ cell proliferation period (March), these spermatozoa are released, and the adjacent ICs undergo a transformation into Leydig cells similar to those observed in August-September. Maturation and involution of ICs occur when testosterone levels are known to be rising and falling, respectively.     

Chromatin and chromosomal fine structure in spermatogenesis of some species of amphibians

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.     

A scanning electron microscopic study of germ cell maturation in the reproductive tract of the male soupfin shark (Galeorhinus galeus)

Germ cell maturation in the reproductive tract of the soupfin shark (Galeorhinus galeus) was studied using scanning electron microscopy (SEM). The SEM showed changes in Sertoli cytoplasm volume during spermatogenic development. In immature spermatocysts in the germinal zone, spermatogonia were embedded in Sertoli cytoplasm. In spermatogonial spermatocysts, Sertoli cells were adluminally located in the spermatocyst, with spermatogonia enveloped in the basal portions of the cytoplasm. During the round spermatid stage, Sertoli cytoplasm was very scanty. Spermatid elongation was accompanied by a progressive increase in the volume of Sertoli cytoplasm, notably around the spermatid heads. In the mature spermatocyst, bundles of spermatozoa are totally enveloped by Sertoli cytoplasm. Spermatozoa occurred randomly in the epididymis. However, in the ampulla ductus deferentis, spermatozoa reaggregated and were embedded in a mucoid substance to form highly ordered spherical bundles. In the sperm bundle, the spermatozoa heads were arranged such that the helical turns of adjacent spermatozoa were precisely aligned, and all the heads in the bundle formed a distinct apex. This study demonstrates the utility of exploring the relationship between germ cells and Sertoli cells in an evolutionarily ancient vertebrate, such as the shark.     

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.     

Ultrastructure of the intact skeleton of the human erythrocyte membrane

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.     

A reassessment of Y chromosomal behaviour in germ cells and Sertoli cells of the mouse as revealed by in situ hybridisation

In situ hybridisation experiments were carried out to reappraise the state of condensation of the Y chromosome in germ cells and Sertoli cells of the mouse. Previous work had suggested that all testicular cells showed a condensed Y chromosome prior to the adult stage. We now demonstrate that, although the Y chromosome is condensed in pre-pubertal Sertoli cells, it is greatly expanded in primordial germ cells (gonocytes). An expanded Y-signal is first seen in Sertoli cell nuclei at or around day 21 of postnatal development, coinciding with the first appearance of spermatids in the germinal epithelium.     

Spermiogenesis in the bullfrog (Rana catesbeiana): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The process by which spermatid cytoplasmic volume is reduced and cytoplasm eliminated during spermiogenesis was investigated in the bullfrog Rana catesbeiana. At early phases of spermiogenesis, newly formed, rounded spermatids were found within spermatocysts. As acrosomal development, nuclear elongation, and chromatin condensation occurred, spermatid nuclei became eccentric within the cell. A cytoplasmic lobe formed from the caudal spermatid head and flagellum and extended toward the seminiferous tubule lumen. The cytoplasmic lobe underwent progressive condensation whereby most of its cytoplasm became extremely electron dense and contrasted sharply with numerous electron-translucent vesicles contained therein. At the completion of spermiogenesis, many spermatids with their highly condensed cytoplasm still attached were released from their Sertoli cell into the lumen of the seminiferous tubule. There was no evidence of the phagocytosis of residual bodies by Sertoli cells. Because spermatozoa are normally retained in the testis in winter and are not released until the following breeding season, sperm were induced to traverse the duct system with a single injection of hCG. Some spermatids remained attached to their cytoplasm during the sojourn through the testicular and kidney ducts; however, by the time the sperm reached the Wolffian duct, separation had occurred. The discarded cytoplasmic lobe (residual body) appeared to be degraded with the epithelium of the Wolffian duct. It was determined that the volume of the spermatid was reduced by 87% during spermiogenesis through a nuclear volume decrease of 76% and cytoplasmic volume decrease of 95.3%.     

Cytology of the human seminiferous epithelium

The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.     

Changes in the distribution of a spectrin-like protein during development of the preimplantation mouse embryo

The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte alpha-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.