Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Histochemical Studies on Sialic Acids in the Epididymis During Post-natal Development of the Rat

A variety of sialic acids contained in the rat epididymis during post-natal development were examined by means of lectin and carbohydrate histochemistry. Epididymides from male Sprague– Dawley rats on post-natal days 14, 21, 30, 39, 49, 56 and 70 were fixed in Bouin's fluid and embedded routinely in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Sambucus sieboldiana lectin or Maackia amurensis lectin or to the selective periodate oxidation– phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The results revealed that sialic acids appeared in the epididymal epithelium at day 14, followed by particular distribution patterns corresponding to cell differentiation during days 21–39. High-level O-acetylation of sialic acids was observed in the principal cells of the initial segment and proximal caput after day 39. These results suggest that sialic acids with different linkages and O-acetylation become adult in distribution at the differentiation period under the influence of androgen, before spermatozoa reach the epididymal lumen. Such carbohydrates may be correlated, at least in part, with sperm-binding sialoproteins, which increase dramatically during the window between days 21 and 39.     

附睾局部组织肾素血管紧张素系统(英文)

附睾内液体微环境对精子的成熟和贮藏是相当重要的。附睾液体的形成取决于附睾上皮的吸收与分泌功能,而先前的实验已经证明：这些吸收与分泌的活动是受到除神经激素以外旁分泌与自分泌的调控。虽然肾素血管紧张素系统（ＲＡＳ）在很多组织上旁分泌与自分泌的作用已多有报导,但其在附睾的存在及作用仍鲜为人知。本综述总结了本实验室在这方面的研究结果,通过使用不同的实验方法,例如,免疫组织化学,放射免疫分析,分子生物学及短路电流电生理学方法,我们得到的结果显示了ＲＡＳ主要成员在附睾的分布（Ｆｉｇ．１）和表达,并阐明了血管紧张素ＩＩ对附睾阴离子分泌的调控作用（Ｆｉｇｓ．２＆３）。本综述还就血管紧张素ＩＩ对附睾旁分泌与自分泌的作用及其机制（Ｆｉｇ．４）,以及对精子功能的作用进行了讨论。     

Release of Photosynthetic Protein Catabolites by Blebbing from Thylakoids

Thylakoid proteins and their catabolites have been detected in lipid-protein particles isolated from the stroma of intact chloroplasts obtained from primary leaves of 2-week-old bean seedlings (Phaseolus vulgaris L. cv Kinghorn). The lipid-protein particles bear morphological resemblance to plastoglobuli seen in the chloroplasts of senescing leaves, but they are much smaller. They range from 10 to 320 nm in radius, are uniformly stained in thin sections visualized by transmission electron microscopy, and are discernible in the stroma of chloroplasts in corresponding thin-sectioned leaf tissue. The lipid-protein particles contain thylakoid lipids and are enriched in free fatty acids. Specifically, the free-to-esterified fatty acid ratio is about 1:1 in the particles compared to only 1:18 for corresponding thylakoid membranes. Western blot analyses indicate that these particles also contain thylakoid proteins and, in some cases, catabolites of these proteins including the CF1 [beta] and [gamma] subunits of ATPase, cytochrome f, and the 31- and 33-kD proteins of PSII. Lipid-protein particles with similar properties were generated in vitro from isolated, light-stressed thylakoids. Collectively, these data suggest that blebbing of lipid-protein particles may be a means of removing potentially destabilizing macromolecular catabolites from thylakoid membrane bilayers.     

Development During Vegetative Growth in the Apical Region of the Shoot of Lupinus albus

This investigation was designed to show the nature of the developmentin terms of cell numbers and of rates of division that proceedsin the terminal region of the shoot during the vegetative phasein Lupinus albus. Data are assembled that show that with eachsuccessive plastochron in the dome the number of cells increasesand the rate of division decreases; in the first internode againthe number of cells increases and the rate of division decreases,and in the first primordium both the number of cells and therate of division decrease. The data also show that with thetransition from one plastochron to the next the rate of divisionin any particular intemnode or primordium decreases. It is asignificant feature that all the changes in the system can becharacterized through constant numerical factors. The significanceof the results is discussed.     

Polysialylation of NCAM Characterizes the Proliferation Period of Contractile Elements during Postnatal Development of the Epididymis

Polysialic acid (polySia) attached to the neural cell adhesion molecule (NCAM) regulates inter alia the proliferation and differentiation via the interactions with neurotrophins. Since in postnatal epididymis neurotrophins and their receptors like the Low-Affinity Nerve Growth Factor Receptor p75 and TrK B receptor are expressed, we wanted to analyze if the polysialylation of NCAM is also involved during the development of the epididymis. To this end, we monitored the developmental changes in the expression of the polysialyltransferases and NCAM polysialylation using murine epididymis at different time points during postnatal development. Our results revealed that during postnatal development of the epididymis both polysialyltransferases, ST8SiaII and ST8SiaIV, were expressed and that the expression levels dropped with increasing age. In agreement with the expression levels of the polysialyltransferases the highest content of polysialylated NCAM was present during the first 10 days after birth. Interestingly, proliferating smooth muscle cell populations prevalently expressed polysialylated NCAM. Furthermore, we observed that inverse to the decrease in polysialylation of smooth muscle cells a strong up-regulation of collagen takes place suggesting a functional relationship since collagen was recently described to induce the turnover of polysialylated NCAM via an induction of endocytosis in cellulo. The same time course of polySia and collagen synthesis was also observed in other regions of the male reproductive system e.g. vas deferens and tunica albuginea (testis). Together, we identified a spatio-temporal expression pattern of polySia-NCAM characterized by high proliferation rate of smooth muscle cells and low collagen content.     

Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.     

Cell Blebbing upon Addition of Cryoprotectants: A Self-Protection Mechanism

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.     

Cryopreservation of Boar Semen

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.     

Cell Blebbing and Membrane Area Homeostasis in Spreading and Retracting Cells

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.     

Apical Dominance in the 'Rogue' Tomato

The rogue tomato exhibits less apical dominance than the normalplant though the degree of correlative inhibition varies considerablybetween winter- and summer-sown plants. An examination of thelevel of endogenous hormones in both rogue and normal plantsat both times of year indicates that the degree of branchingis strongly associated with the levels of auxin in the tissue.It is suggested that this hormone has an effect on apical dominanceby virtue of its role in hormone-directed transport and by itseffect on the formation of abscisic acid in the region of thelateral buds. The results are discussed in relation to currenthypotheses of the mechanism of apical dominance.     

猪精子冷冻损伤研究进展

综述了猪冻存精子顶体、质膜和线粒体的形态学变化及其对精子受精能力的影响,同时分析了冻融精子中所发生的蛋白质、DNA等生物大分子的变化及其可能对冻存精子质量及受精能力的影响,指出冷冻保存过程对精子蛋白质、DNA等生物大分子质和量的影响是精子冷冻损伤的实质,应用先进的蛋白组学进行猪精子冷冻损伤机理研究,有助于深刻揭示冷冻损伤的分子机理,为推动猪精液冷冻保存技术研究取得突破性进展提供理论依据。     

Apical Growth in Algae

Cytological and physiological aspects of apical growth in algae are reviewed. Data on morphology and ultrastructural organization of siphonous thalli are presented. Special attention is paid to the organization of microtubules and microfilaments and the role of the cytoskeleton in morphogenesis. A comparative study of apical growth in algae, fungi, and higher plants is presented.