The role of chromatoid bodies and cytoskeleton in differentiation of rat spermatozoids

An ultrastructural and immunocytochemical study of rat male germ cells at different stages of development has been carried out. Investigation of morphological changes of spermatogenic cells showed the presence of close associations between chromatoid bodies (CBs) and other cell organelles, particularly with the nucleus and Golgi apparatus. In addition, a connection of manchette noncentosomal microtubules (MTs) with spermatid perinuclear ring plasma membrane (PM) in the zone of adhesion intercellular contact, zonula adhaerens (ZA), was revealed. These results, as well as the available literary data, make it possible to analyze expected pathways of noncentrosomal MT nucleation in the late spermatids. It is possible to suggest that noncentorosomal MT are nucleated on the sites of perinuclear ring ZA. The immunocytochemical analysis revealed two novel proteins for these cells: BASP1 and MARCKS. It was shown that these proteins were present in CBs in early spermatids. During spermatozoid differentiation, these proteins are located along the outer dense fibers (ODFs) of the sperm tail. BASP1 and MARCKS are believed to be involved in the processes of calcium accumulation in CBs and ODFs. Calcium ions seem to play a significant role in RNA processing and protein synthesis in spermatids. Calcium is also necessary for sperm mobility defined mainly by ODFs.     

Argonaute2 Protein in Rat Spermatogenic Cells Is Localized to Nuage Structures and LAMP2-Positive Vesicles Surrounding Chromatoid Bodies

Localization of Argonaute2 (AGO2) protein—an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in nuage of rat spermatogenic cells—was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments.     

Localization of mouse vasa homolog protein in chromatoid body and related nuage structures of mammalian spermatogenic cells during spermatogenesis

The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70–90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.     

CLASPing microtubules at the trans-Golgi network

Microtubule (MT) arrays can be formed either from centrosomes or from noncentrosomal locations. In this issue of Developmental Cell, Efimov and colleagues report a role of CLASPs, the MT plus end-binding proteins, in MT formation from Golgi, implicating Golgi-originated MT arrays in efficient cell migration (Efimov et al., 2007).     

The RNA binding protein SAM68 transiently localizes in the chromatoid body of male germ cells and influences expression of select microRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68(-/-) germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis.     

CAMSAP3-dependent microtubule dynamics regulates Golgi assembly in epithelial cells

The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.     

Spetex-1: a new component in the middle piece of flagellum in rodent spermatozoa

Spetex-1 has recently been isolated by differential display and screening of cDNA library. It encodes a protein of 556 amino acid residues possessing coiled-coil motifs. In the rat seminiferous tubules (ST), Spetex-1 was expressed in the cytoplasm of elongating spermatids. To examine the subcellular distribution of Spetex-1 in mature spermatozoa, we performed biochemical and immunocytochemical approaches. We found that Spetex-1 that was synthesized in the cytoplasm of elongating spermatids was subsequently integrated as a middle piece component into spermatozoa during spermiogenesis. After integration, the majority of Spetex-1 in spermatozoa could be extracted by 6M urea under reduced condition but not released by the treatment of 1% Triton X-100. Immunoelectron microscopy demonstrated that Spetex-1 seemed to locate at the inner side of outer dense fibers (ODFs) in the middle piece or the narrow space between ODFs and axoneme. Spetex-1 might be involved in the stability of the structural complexity comprising axoneme and ODFs in the middle piece of sperm flagellum.     

NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells

Nanos and pumilio bind each other to regulate translation of specific mRNAs in germ cells of model organisms, such as D. melanogaster or C. elegans. Recently described human homologues NANOS1 and PUMILIO2 form a complex similar to their ancestors. This study was aimed to identify the proteins interacting with NANOS1-PUMILIO2 complex in the human spermatogenic cells. Here, using the yeast two-hybrid system we found that NANOS1 and PUMILIO2 proteins interact with RNA DEAD-box helicase GEMIN3, a microRNA biogenesis factor. Moreover, GEMIN3 coimmunoprecipitates with NANOS1 and PUMILIO2 in transfected mammalian cells. By double immunofluorescence staining, we observed that complexes built of NANOS1, PUMILIO2 and GEMIN3 are located within cytoplasmic region of germ cells. These proteins condense to form a compact aggregate in the round spermatids of the human and mouse germ cells. This aggregate was reminiscent of the chromatoid body (CB), a perinuclear structure present in the mammalian male germ line. This structure is considered evolutionary remnant of germ plasm, a hallmark structure of germ cells in lower metazoan. Using a CB marker VASA protein, we demonstrated that CBs are present in the human round spermatids, as they are in the mouse. Moreover, NANOS1, PUMILIO2 and GEMIN3 colocalize with VASA protein. We demonstrated for the first time that a mammalian Nanos-Pumilio complex functions within CB, a center of RNA storing and processing, involving microRNAs. NANOS1-PUMILIO2 complex, together with GEMIN3 and small noncoding RNAs, possibly regulate mRNA translation within CB of the human germ cells.     

Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein marcks

The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotpe.     

Efficient formation of bipolar microtubule bundles requires microtubule-bound gamma-tubulin complexes

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.     

Ultrastructural and cytochemical changes of the head components of human spermatids and spermatozoa

The ultrastructural study of chromatin condensation simultaneously with the evolution of the perinuclear organelles was conducted in the spermatids and epididymal and ejaculated spermatozoa of man with the aid of the “en bloc” alcoholic PTA staining and the EDTA regressive method. The round nuclei of young spermatids (steps 1, 2) were characterized by the persistence of nucleoli that were PTA positive, and the presence of a subacrosomal layer of well-stained peripheral chromatin. In the beginning of the phase of nuclear elongation (step 3), the central chromatin also became dense, like the peripheral chromatin, while the nuclear ring and the associated manchette and the two anlages of the postacrosomal dense lamina and the posterior ring appeared. During steps 4 and 5, the sliding of the nuclear ring and the manchette, the growth of the postacrosomal dense lamina, and the progression of the posterior ring towards the base of the nucleus were seen along with structural and cytochemical modifications of the chromatin. In the flattened nuclei of step 4 spermatids, coinciding with the loss of the nucleolar components, the chromatin achieved maximum compactness in the entire nucleus and was PTA positive. In the spermatids of step 5, the disappearance of peripheral dense chromatin and the specific staining of the chromatin granules marked the beginning of the second stage of transformation of the basic nucleo-proteins. The condensed nuclei of the mature spermatids were partially stained by PTA in step 6 and totally unstained in step 7. The PTA staining revealed the persistence of PTA-positive chromatin areas in the nuclei of certain spermatids otherwise mature. The morphological aspect of the chromatin then remained the same in the nuclei of epididymal and ejaculated spermatozoa. These observations suggest that in man, as in other mammals studied, new proteins accumulate in the elongating nuclei of spermatids and are replaced at the phase of maturation by sperm-specific nucleoproteins. The defects in condensation of the chromatin that occur during spermiogenesis could be related to the modalities of accumulation of intermediate nucleoproteins.     

Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells

The formation of a belt-like junctional complex separating the apical from the lateral domain is an essential step in the differentiation of epithelial cells. Thus protein complexes regulating this event are of first importance for the development of cell polarity and physiological functions of epithelial tissues. In Drosophila, the discovery of a gene, crb, controlling the coalescence of the spots of zonula adherens (ZA) into a adhesive ring around the cells was a major step. We know now that Crumbs, the product of crb is an apical transmembrane protein conserved in mammals and that it interacts by its cytoplasmic domain with two cortical modular proteins, Stardust (Sdt) and Discs lost (Dlt) that are also essential for the correct assembly of the ZA. These two proteins are also conserved in mammals and it is most likely that the Crumbs complex plays a similar role in very different species. Recently, we have shown that Crumbs interacts with the cortical cytoskeleton made of DMoesin and beta heavy-Spectrin and this connection could explain in part the role of Crumbs in building the ZA. Future work will help to understand several aspects of the Crumbs complex that are still unknown, like the role of the large extracellular domain or the precise function of Sdt and Dlt in the building of the ZA. Finding an answer to these questions will help to find new therapies for Retinitis pigmentosa and other retina degeneration in which CRB1, the human homologue of crb, has been involved.     

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.     

Differential Expression of MARCKS and Other Calmodulin-Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [³H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional ³H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca²⁺, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [³H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.     

Formation of division spindles in higher plant meiosis

Depolymerisation of the MT cytoskeleton during late prophase makes it impossible to follow the cytoskeleton cycle in centrosomeless plant meiocytes. This paper describes rearrangements of the MT cytoskeleton during plant meiotic spindle formation in normally dividing pollen mother cells in various higher plant species and forms in which the cytoskeleton does not depolymerise at prophase. In such variants of the wild-type, cytoskeleton rearrangements can be observed at late prophase/early prometaphase. Radial MT bundles coalesce in the meridian plane, reorientate tangentially, curve and give rise to a developed ring-shaped perinuclear cytoskeleton system at the meridian. During nuclear envelope breakdown this ring disintegrates and splits into a set of free MT bundles. Three sub-stages of prometaphase are indicated: early prometaphase (disintegration of perinuclear ring and invasion of MTs into the former nuclear area), middle prometaphase or chaotic stage (formation of bipolar spindle fibres), and late prometaphase (formation of bipolar spindle). Analysis of a range of abnormal phenotypes (disintegrated, multiple, polyarchal, chaotic spindles) reveals two previously unknown processes during late prometaphase: axial orientation and consolidation of the spindle fibres.     

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.