Organisation of Xenopus oocyte and egg cortices

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.     

From egg to pole cells: ultrastructural aspects of early cleavage and germ cell determination in insects.

Insect eggs are giant and very complex cells covered by an extremely resistant shell. Both the egg cell and surrounding eggshell express anteroposterior and ventrodorsal polarity. The molecular and cytoplasmic organization of both axes originates during oogenesis and leads to the production of an ooplasmic system which consists of euplasm and deutoplasm (yolk) and contains a nucleus as well as extranuclear determinants of maternal origin. Both are part of the store of information for early embryogenesis. In addition, the deutoplasm serves as raw material and early nutrient supply for building the embryo. The insect egg cell, which is arrested in the first maturation division when released from the ovary during oviposition, will be activated by different stimuli among different species to complete meiosis and start embryogenesis. The zygote nucleus undergoes a number of synchronous mitotic divisions leading to cleavage energids which initially form a syncytial blastoderm and subsequently the cellular blastoderm. In many insects, prior to blastoderm formation, polar granules (or oosome material) are incorporated in a single cell or a small number of cells which bud off at the posterior pole. These so called pole cells give rise to the primordial germ cells. Therefore, polar granules or the oosome material mark the germ line, and while structural counterparts of determinants of body pattern formation have so far not been found, the polar granules or oosome serve as an autonomous ooplasmic determinant for the pole or germ cells. Anteroposterior body polarity can arise independent of the germ plasm.     

Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope

Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.     

Morphology of mammalian sperm membranes during differentiation,maturation, and capacitation

The mammalian spermatozoon is a highly polarized cell whose surface membrane can be divided into five functionally, structurally, and biochemically distinct domains. These domains are formed during spermatogenesis, continue to be modified during passage through the epididymis, and are further refined in the female reproductive tract. The integrity of these domains appears to be necessary for the sperm to perform its function—fusion with the egg and subsequent fertilization. The domains can be identified morphologically by their surface contours and texture, the content, distribution, and organization of intramembranous particles after freeze-fracture, and by the density of surface and cytoplasmic electron-dense coatings in thin sections. By using a variety of labels that stain carbohydrates (lectins), lipids (filipin and polymyxin B), and monoclonal antibodies to specific membrane constituents, the biochemical composition of these contiguous membrane regions has also been partly elucidated. We review here what is known about the structure, composition, and behavior of each membrane domain in the mature sperm and include some information regarding domain formation during spermatogenesis. The sperm is an excellent model system to study the creation and maintenance of cell polarity, granule exocytosis, and fertilization. Hopefully this review will provide impetus for future studies aimed more directly at addressing the relationship of its morphology to its functions.     

Temperature-dependent imaging of living cells by AFM

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.     

Oogenesis of Diadegma semiclausum (Hymenoptera: Ichneumonidae) and its associated polydnavirus

We present a detailed ultrastructural analysis of ovary and calyx cell differentiation of Diadegma semiclausum. Numerous gametangia in various developmental stages were examined with electron microscopy to characterize the ultrastructure features of oogenesis, the most important of which is the development of nurse cells. In the germarium, the undifferentiated germ cells diversify, and one of the central cells of the cluster differentiates into an oocyte while the remaining become nurse cells. Germ cells continue developing in the vitellarium until mature and then enter into the calyx region. The calyx epithelium contains typical ichneumonid polydnaviruses with the following characteristics: (1) the virus particles assemble in the nuclei of calyx cells where they acquire an initial (inner) membrane, then migrate through the cytoplasm and budd out into the lumen of the ovary, at which time they acquire a second envelope (outer membrane); (2) the particle has a genome comprising several DNA segments. However, this new polydnavirus (Diadegma semiclausum polydnavirus) in the genus ichnovirus was not attached to the surface of the egg chorion.     

Oogenesis and programmed cell death of nurse cells in the endoparasitoid,Pteromalus puparum

In this study, we describe the features of oogenesis in the endoparasitoid, Pteromalus puparum, as well as the distinct type of programmed cell death of the nurse cells through conventional light and fluorescent markers for apoptosis and immunofluorescent analysis. Oogenesis in this endoparasitoid is divided into five stages, of which stages 1–2, 3–4, and 5 are corresponding to previtellogenic growth, yolk uptake, and the formation of egg envelopes, respectively. From these studies, we demonstrate two critical events, which are vitellogenin absorption and rapid transfer of nurse cell content, resulting in remarkable increase in the volume of oocytes during oogenesis in this endoparasitoid. Vitellogenin absorption initiates in the oocyte of early stage 3, and bulk transfer of nurse cell content into the oocyte occurs at stage 4 of oogenesis in P. puparum, which is mainly characterized with the programmed cell death in the nurse cell complex. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

RHO GTPases in the control of cell morphology, cell polarity, and actin localization in fission yeast.

The fission yeast Schizosaccharomyces pombe undergoes morphogenetic changes during both vegetative and sexual cell cycles that require asymmetric cell growth and actin cytoskeleton reorganisations. Different complex signal transduction pathways participate in S. pombe morphogenesis. The Rho family of GTPases are present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signalling pathways that control actin organisation and morphogenetic processes is well known. In this review, we will briefly summarize the role of the Rho GTPases in the establishment and maintenance of cell polarity and growth of S. pombe. As in other fungi, S. pombe morphogenesis is closely related to cell wall biosynthesis, and Rho GTPases are critical modulators of this process. They provide the coordinated regulation of cell wall biosynthetic enzymes and actin organisation required to maintain cell integrity and polarised growth.     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.     

The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis

Microtubules are an important part of the eukaryotic cytoskeleton, which participates in numerous essential cellular processes. In fungi interphase microtubules mediate cell polarity and participate in polar growth. However, our understanding of their detailed role in fungal growth is just at the beginning. In growing cells of the plant pathogenic fungus Ustilago maydis microtubules are organized by polar microtubule organizing centres that focus the microtubule minus ends at the small bud. Two opposing motor complexes utilize this microtubule polarity. Cytoplasmic dynein and a kinesin of the Unc104/Kif1A family of kinesins mediate rapid bi‐directional transport of early endosomes. A balance of their activity is required for cell cycle‐dependent accumulation of early endosomes at the growth site, the rear cell pole and the region of cell cleavage. Mutant phenotypes suggest that these endosomes participate in polar growth, bud site selection and cell separation. Therefore, our data suggest that endocytotic membrane recycling participates in local exocytosis, and that the microtubule cytoskeleton has a crucial role in this process.     

Enhanced sampling for lipid-protein interactions during membrane dynamics

The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete. Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment. In particular, proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes. In this review, the potential of enhanced molecular dynamics to address cell-biology problems is discussed. The cases of membrane deformation, membrane fusion, and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables. Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented. Finally, novel strategies merging cell biology and physics are considered as future lines of research.     

From oogonia to mature oocytes: inactivation of the maternal centrosome in humans

The fine structure of human oogonia and growing oocytes has been reviewed in fetal and adult ovaries. Preovulatory maturation and the ultrastructure of stimulated oocytes from the germinal vesicle (GV) stage to metaphase II (MII) stage are also documented. Oogonia have large nuclei, scanty cytoplasm with complex mitochondria. During folliculogenesis, follicle cell processes establish desmosomes and deep gap junctions at the surface of growing oocytes, which are retracted during the final stages of maturation. The zona pellucida is secreted in secondary follicles. Growing oocytes have mitochondria, Golgi, rough endoplasmic reticulum (RER), ribosomes, lysosomes, and lipofuscin bodies, often associated with Balbiani bodies and have nuclei with reticulated nucleoli. Oocytes from antral follicles show numerous surface microvilli and cortical granules (CGs) separated from the oolemma by a band of microfilaments. The CGs are evidently secreted by Golgi membranes. The GV oocytes have peripheral Golgi complexes associated with a single layer of CGs close to the oolemma. They have many lysosomes, and nuclei with dense compact nucleoli. GV breakdown occurs by disorganization of the nuclear envelope and the oocyte enters a transient metaphase I followed by MII, when it is arrested and ovulated. Maturation of oocytes in vitro follows the same pattern of meiosis seen in preovulatory oocytes. The general organization of the human oocyte conforms to that of most other mammals but has some unique features. The MII oocyte has the basic cellular organelles such as mitochondria, smooth endoplasmic reticulum, microfilaments, and microtubules, while Golgi, RER, lysosomes, multivesicular, residual and lipofuscin bodies are very rare. It neither has yolk nor lipid inclusions. Its surface has few microvilli, and 1-3 layers of CGs, aligned beneath the oolemma. Special reference has been made to the reduction and inactivation of the maternal centrosome during oogenesis. The MII spindle, often oriented perpendicular to the oocyte surface, is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs, since the maternal centrosome is nonfunctional. However, oogonia and growing oocytes have typical centrioles, similar to those of somatic cells. The sperm centrosome activates the egg and organizes the sperm aster and mitotic spindles of the embryo, after fertilization.     

Polyspermy prevention in marine invertebrates

In marine invertebrates, as in most other organisms, normal development requires that only one sperm nucleus joins with the egg nucleus at fertilization. The principal mechanisms employed are (1) prevention of sperm-egg plasma membrane fusion and (2) modifications of the egg extracellular coat to prevent sperm binding and/or penetration. In a third strategy, fertilization is polyspermic, but only one sperm nucleus fuses with the egg nucleus. Other factors such as gamete density during spawning, chemotaxis, and localized sites for sperm entry may also affect the numbers of sperm reaching the egg.     

Compositional mapping of bitumen using local electrostatic force interactions in atomic force microscopy

In recent years, many researchers have investigated bitumen surface morphology, especially the so‐called bee‐like structures, in an attempt to relate the chemical composition and molecular conformation to bitumen micromechanics and ultimately performance properties. Even though recent studies related surface morphology and its evolution to stiffness and stress localization, the complex chemical nature of bitumen and its time‐ and temperature‐dependent properties still engender significant questions about the nature and origin of the observed morphological features and how they evolve due to exposure to various environmental and loading conditions. One such question is whether the observed surface features are formed from wax or from the coprecipitation of wax and asphaltene. Our prior work was mainly theoretical; it used density functional theory and showed that the coprecipitation theory may not stand, mainly because wax–asphaltene interactions are not thermodynamically favourable compared to wax–wax interactions. This paper presents a comprehensive approach based on experiments to study surface morphology of bitumen and conduct compositional mapping to shed light on the origin of the bee‐like surface morphological features. We used Atomic Force Microscopy (AFM), with the main focus being on single‐pass detection and mapping of local electric properties, as a novel approach to enhance existing compositional mapping techniques. This method was found to be highly effective in differentiating various domains with respect to their polarity. The results of our study favour the hypothesis that the bee‐like features are mainly composed of wax, including a variety of alkanes.     

On the nature of hyaline zones in the cytoplasm of Amoeba proteus.

Investigations with the Nomarski DIC (differential interferece contrast) microscope and the electron microscope on the nature of hyaline zones in the cytoplasm of Amoeba proteus revealed that these regions represent pure ground cytoplasm. Differences between specimens 1) treated with 2% ethanol, 2) released from high hydrostatic pressure or 3) preincubated at 35 degrees C for 30 minutes could not be observed. Only dying cells undergoing lysis contained a watery solution within the zones of hyaline appearance. The existence of a so-called cell surface complex composed of the plasma membrane and an electron dense filamentous layer of groundplasm was demonstrated by the electron microscopical analysis of narcotized and pre-heated amoebae. This complex corresponds morphologically to the cell surface complexes in tissue cells. Hence it seems possible that the cell surface complex of amoebae is also responsible for changes of the cell shape and movements of the cell membrane. Observations with the DIC microscope also revealed the existence of two types of hyaline caps in A. proteus: in pseudopodia extending during normal locomotion the hyaline cap consists of pure ground cytoplasm, whereas in specimens showing fountain-like streaming the cap is built up by a large vacuole containing a watery fluid.     

Mechanobiology of the cell surface: Probing its remodeling dynamics using membrane tether pulling assays with optical tweezers

Mammalian cell surfaces consist of the plasma membrane supported by an underneath cortical cytoskeleton. Together, these structures can control not only the shape of cells but also a series of cellular functions ranging from migration and division to exocytosis, endocytosis and differentiation. Furthermore, the cell surface is capable of exerting and reacting to mechanical forces. Its viscoelastic properties, especially membrane tension and bending modulus, are fundamental parameters involved in these responses. This viewpoint summarizes our current knowledge on how to measure the viscoelastic properties of cell surfaces employing optical tweezers-based tether assays, paving the way for a better understanding of how cells react to external mechanical forces, with a glance on their remodeling dynamics and possible consequences on downstream cellular processes.     

GCr15钢轴承内套圈滚道不规则碳化物的成因

对锻造、球化退火、淬回火等工序后GCr15钢轴承内套圈组织中的碳化物形貌进行观察,结合热膨胀试验和热力学分析,研究二次碳化物的溶入、析出与长大行为,分析了带尖角大颗粒不规则碳化物形成的原因.结果表明:GCr15钢轴承内套圈在较低温度辗挤成形时,其滚道表层合金元素偏析区中已析出的二次碳化物发生破碎,导致滚道表层组织中出现...     

Confocal microscopy and 3-D reconstruction of the cytoskeleton of Xenopus oocytes

Xenopus oocytes contain a complex cytoskeleton composed of three filament systems: (1) microtubules, composed of tubulin and at least three different microtubule-associated proteins (XMAPs); (2) microfilaments composed of actin and associated proteins; and (3) intermediate filaments, composed of keratins. For the past several years, we have used confocal immunofluorescence microscopy to characterize the organization of the oocyte cytoskeleton throughout the course of oogenesis. Together with computer-assisted reconstruction of the oocyte in three dimensions, confocal microscopy gives an unprecedented view of the assembly and reorganization of the cytoskeleton during oocyte growth and differentiation. Results of these studies, combined with the effects of cytoskeletal inhibitors, suggest that organization of the cytoskeleton in Xenopus oocytes is dependent upon a hierarchy of interactions between microtubules, microfilaments, and keratin filaments. This article presents a gallery of confocal images and 3-D reconstructions depicting the assembly and organization of the oocyte cytoskeleton during stages 0-VI of oogenesis, a discussion of the mechanisms that might regulate cytoskeletal organization during oogenesis, and speculates on the potential roles of the oocyte cytoskeleton during oogenesis and axis formation.     

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.