Distribution of γ-Tubulin in Higher Plant Cells: Cytosolic γ-Tubulin is Part of High Molecular Weight Complexes

Abstract: γ-Tubulin is a protein found in all eukaryotic cells, where it plays a key role in the nucleation of microtubules. In higher plant cells, γ-tubulin is localized at the nuclear surface, a known microtubule-organizing centre, and is codistributed with all microtubule arrays. Functions of plant γ-tubulin remain to be determined. This study describes some properties of higher plant γ-tubulin. The overall level of γ-tubulin was constant during the cell cycle in synchronized tobacco BY-2 cells. Biochemical analysis of the subcellular distribution of γ-tubulin in maize cells revealed that, in contrast with animal γ-tubulin, plant γ-tubulin is mainly associated with endomembranes. We showed for the first time that the pool of soluble cytosolic γ-tubulin contained two main γ-tubulin complexes. γ-tubulin, Hsp70 and TCP1-related proteins might interact in a small complex of 750 kDa. A second γ-tubulin complex, larger than 1500 kDa was purified. The protein profile of this large complex was very similar to animal γ-tubulin complexes. The putative functions of these two complexes in plant microtubule nucleation are discussed.     

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Summary The distribution of -tubulin throughout cell division is studied in several taxa of higher plants. -Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. -Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, -tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. -Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants -tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to - and -tubulin.Abbreviations Mt microtubule - MTOC microtubule-organizing center - PPB preprophase band     

Microtubule-dependent microtubule nucleation in plant cells

Regulation of microtubule nucleation sites is an essential step in microtubule organization. Cortical microtubule arrays in green plant cells at inter-phase are organized in a distinct manner—the array is formed in the absence of previously recognized organelles for microtubule nucleation, for example the centrosome and spindle pole body. Microtubules in the cortical array were recently found to be nucleated as branches on pre-existing microtubules via recruitment of cytosolic γ-tubulin. In this review we briefly summarize the mechanism of microtubule-dependent microtubule nucleation and discuss a possible role of this mechanism in other cellular processes and their evolution.     

The γ -Tubulin Complex Protein GCP4 Is Required for Organizing Functional Microtubule Arrays in Arabidopsis thaliana

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein γ -tubulin, which forms a complex with GCP2-GCP6 (GCP for γ -Tubulin Complex Protein). To date, it is still unclear how GCP4-GCP6 (the non-core GCPs) may be involved in acentrosomal MT nucleation in plant cells. We found that GCP4 was associated with γ -tubulin in vivo in Arabidopsis thaliana. When GCP4 expression was repressed by an artificial microRNA, transgenic plants exhibited phenotypes of dwarfism and reduced organ size. In mitotic cells, it was observed that the γ -tubulin signal associated with the mitotic spindle, and the phragmoplast was depleted when GCP4 was downregulated. Consequently, MTs failed to converge at unified spindle poles, and the bipolar phragmoplast MT array frequently had discrete bundles with extended minus ends, resulting in failed cytokinesis as reflected by cell wall stubs in leaf epidermal cells. In addition, cortical MTs in swollen guard cells and pavement cells of the leaf epidermis became hyperparallel and bundled, which was likely caused by frequent MT nucleation with shallow angles on the wall of extant MTs. Therefore, our results support the notion that GCP4 is an indispensable component for the function of γ -tubulin in MT nucleation and organization in plant cells.     

Regulation of microtubule nucleation mediated by γ-tubulin complexes

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.     

γ-Tubulin localization changes from discrete polar organizers to anastral spindles and phragmoplasts in mitosis of Marchantia polymorpha L.

Summary. Unlike the astral mitotic spindle that is organized at discrete centriolar centrosomes, the spindle of land plants is typically anastral and its origin has remained obscure. Gamma tubulin (-tubulin), an important component of the centrosome, has been demonstrated at microtubule-nucleating sites in plant cells. Mitotic spindles of certain hepatics are initiated at distinct acentriolar polar organizers (POs) that appear de novo at the onset of mitosis. Data on the relationship of -tubulin to POs and to microtubule arrays throughout the cell cycle were collected from rapidly dividing cells of Marchantia polymorpha (Bryophyta) that were triple-stained for -tubulin, microtubules, and nuclei. POs at opposite ends of the elongated nucleus in early prophase stain brightly for -tubulin and astral microtubules emanating from them initiate the spindle. As the spindle develops, however, the -tubulin becomes dispersed from the highly concentrated spherical form of the POs to more diffusely organized cups at tips of the fusiform nucleus. By the end of prophase, all astral microtubules have disappeared and the -tubulin is located in several minipoles along the now broad polar regions of the spindle. At metaphase, -tubulin extends into the spindle itself. By telophase, the -tubulin has migrated from distal to proximal surfaces of the sister nuclei and extends into the phragmoplast. Upon completion of cytokinesis, -tubulin appears diminished and surrounds the nuclear envelopes. These data show that -tubulin is only briefly concentrated in the PO, migrates in a cell-cycle-specific manner, and is consistently present at all putative sites of microtubule nucleation.     

A ZYG-12–dynein interaction at the nuclear envelope defines cytoskeletal architecture in the C. elegans gonad

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12–mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined γ-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. γ-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules around zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.     

Distinct tubulin genes are differentially expressed during barley grain development

Tubulins, as major components involved in the organization of microtubules, play an important role in plant development. We describe here the expression profiles of all known α-tubulin (TUA), β-tubulin (TUB) and γ-tubulin (TUG) genes of barley ( Hordeum vulgare ), involving eight newly identified TUB sequences, five established TUA genes and one TUG gene. Macroarray and Northern blot-based expression patterns in the pericarp, endosperm and embryo were obtained over the course of the development of the grain between anthesis and maturation. These revealed that the various tubulin genes differed in their levels of expression, and to some extent were tissue specific. Two expression peaks were detected in the developing endosperm. The first and more prominent peak, at 2 days after flowering, included expression of almost all the tubulin genes. These tubulins are thought to be involved in mitoses during the formation of the syncytial endosperm. The second, less pronounced but more extended, peak included only some of the tubulin genes ( HvTUA3 , HvTUB1 and HvTUG ) and might be associated with the cell wall organization in aleurone and starchy endosperm. The HvTUA5 gene is expressed only in embryo of the developing grain and may be associated with shoot establishment. The expression profiles of the tubulin folding cofactors HvTFC A and HvTFC B as well as small G-protein HvArl2 genes were almost perfectly correlated with the global levels of tubulin mRNA, implying that they have a role in the control of the polymerization of α/β-tubulin heterodimers.     

γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.     

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional γ-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon”. Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

On and Around Microtubules: An Overview

Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Immunofluorescence Study of Microtubule Organization in Some Polarized Cell Types of Selected Brown Algae

The microtubule (Mt) organization in apical cells of Sphacelaria rigidula. as well as in branch initials of S. rigidula and Ectocarpus siliculosus, was studied by immunofluorescence. The apical interphase cells of S. rigidula show an impressive cytoskeleton of Mts, converging on the centrosome(s). A number of Mt bundles are perinuclear, but most of them run in axial orientation from the centrosomes to the cell cortex. The anterior Mt system consists of numerous thin Mt bundles, whereas the posterior system contains fewer and thicker bundles. In cells entering prophase, the cytoplasmic Mts gradually disappear. This process is somewhat faster at the posterior than at the anterior pole of the premitotic nucleus. After mitosis, the cytoplasmic Mts of the apical region appear to be re-organized more rapidly than those of the basal part of the cell. The apical daughter nucleus retains a lobed shape and condensed chromatin for a longer time, and increases considerably in size between telophase and cytokinesis, compared to the basal one. Duplication of the centrosomes proceeds more rapidly in the anterior region of apical cells than in the basal part. During branch formation in S. rigidula and E. siliculosus, a new polarity axis is established, and the Mts extend towards the protrusion into which the nucleus migrates before mitosis. After nuclear division, one of the daughter nuclei is positioned at the tip of the branch, where the apical Mt focussing point is localized.     

γ-Tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti--tubulin, to detect Mts, and an antibody to -tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. -Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.Abbreviations CLSM confocal laser scanning microscope - FITC fluorescein isothiocyanate - Mt microtubule - MTOC microtubule-organizing center This work was supported by National Science Foundation grant DCB-9019285 to B.A.P., National Institutes of Health (NS30009) and American Cancer Society (CD6255) grants to H.C.J., and a University of Georgia Graduate School Assistantship to B.L. We thank Dr. Mark Farmer and the University of Georgia Center for Advanced Ultrastructural Research for the use of the confocal microscope.     

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

Background  

Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

Tubulin and actin protein patterns in Scots pine (Pinus sylvestris) roots and developing ectomycorrhiza with Suillus bovinus

The role of tubulin and actin in the development of Scots pine ( Pinus sylvestris ) roots and in the formation of the ectomycorrhiza with the basidiomycete Suillus bovinus was studied by immunoblotting of 2D-gels with anti-tubulin and anti-actin antibodies. In the short roots the α-tubulin pattern was different from that in the other root types due to the more acidic pI of the two α-tubulins. During the formation of the ectomycorrhiza, two new α-tubulins were detected in the acidic α-tubulin cluster. No such variation occurred in the plant β-tubulin patterns. The fungal tubulins dominated in the ectomycorrhiza, but no changes in tubulin polypeptide patterns from those in the S. bovinus mycelium were observed. Contrary to the tubulins, plant actin dominated in the mycorrhiza. The specific α-tubulin patterns of uninfected and infected short roots indicate that α-tubulin is involved in the morphogenesis of Pinus sylvestris short roots. The high level of plant actin at early stage of the mycorrhiza formation suggests a significant role of this protein in the interaction between plant cells and fungal hyphae.     

A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant

Fluorescence microscopy was used to study meiosis in microsporocytes from wild-type Arabidopsis thaliana and a T-DNA-tagged meiotic mutant. Techniques for visualizing chromosomes and β-tubulin in other plant species were evaluated and modified in order to develop a method for analyzing meiosis in A. thaliana anthers. Like most dicots, A. thaliana microsporocytes undergo simultaneous cytokinesis in which both meiotic divisions are completed prior to cytokinesis. However, two unique events were observed in wild-type A. thaliana that have not been reported in other angiosperms: (1) polarization of the microsporocyte cytoskeleton during prophase I prior to nuclear envelope breakdown, and (2) extensive depolymerization of microtubules just prior to metaphase II. The first observation could have implications regarding a previously uncharacterized mechanism for determining the axis of the metaphase I spindle during microsporogenesis. The second observation is peculiar since microtubules are known to be involved in chromosome alignment in other species; possible explanations will be discussed. A T-DNA-tagged meiotic mutant of A. thaliana ( syn1 ), which had previously been shown to produce abnormal microspores with variable DNA content, was also cytologically characterized. The first observable defect occurs in microsporocytes at telophase I, where some chromosomes are scattered throughout the cytoplasm, usually attached to stray microtubules. Subsequent developmental stages are affected, leading to complete male sterility. Based on similarities to synaptic mutants that have been described in other species, it is suggested that this mutant is defective in synaptonemal complex formation and/or cohesion between sister chromatids.     

Functional Characterization of Wiskott-Aldrich Syndrome Protein and Scar Homolog (WASH), a Bi-modular Nucleation-promoting Factor Able to Interact with Biogenesis of Lysosome-related Organelle Subunit 2 (BLOS2) and γ-Tubulin

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal γ-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.