Development of cellular polarity and tight junctions in parotid acinar cells of postnatal rat

The morphogenesis of acino-tubular structures and cytodifferentiation of acinar cells in developing rat parotid glands from the day of birth to the 7th day after birth were studied by conventional ultrathin-section electron microscopy in conjunction with freeze replica and space tracer methods. An ultrathin-section study indicated that the acinar cells developed sequentially in the order of the following three stages: (1) the stage of undifferentiated cell immediately after birth, in which the presumptive acinar cells showed very scanty cell cytoplasm, poorly developed organellae, and no distinctive cellular polarity; (2) the stage about the 3rd day after birth, in which cells were arranged into a single layer, resulting in the establishment of three recognizable domains in the plasma membranes, and developing cellular organellae started to distribute with distinctive polarity; and (3) the stage of the 5th day after birth and thereafter, in which secretory granules were formed, indicating the beginning of exocrine functions. Freeze replica and a tracer study demonstrated that the formation of a sealing strand of tight junctional belt took place in correspondence to the establishment of cellular polarity. These results indicated that the development of cellular polarity, plasma membrane domains, tight junctions, and acino-tubular structure were closely interrelated to each other, and preceded the onset of secretory functions.     

应急大鼠肾上腺髓质嗜铬细胞胞吐特性的超微结构研究

运用透射电镜对制动应急大鼠肾上腺髓质嗜铬细胞内分泌颗粒的胞吐特性进行研究，结果显示：它具有三种不同的胞吐方式：单颗粒胞吐，多颗粒连续胞吐及颗粒内容物通过暂时性融合小孔进行胞吐，用电镜细胞立体形态计量法对正常大鼠肾上腺素细胞内两种不同形态颗粒进行测量，结果显示：肾上腺素细胞中致密圆形颗粒的数目较内容浅淡的颗粒多（P＜0．01），大鼠制动8h时，以致密圆形颗粒的胞吐为主，制动24h时，则两种颗粒均发生胞吐，以上结果提示：这两种不同形态的颗粒可能是大鼠肾上腺髓质嗜铬细胞中同一种分泌颗粒的不同发育阶段。     

Characteristic distribution of gap junctions in rat lacrimal gland in vivo and reconstruction of a gap junction in an in vitro model

We investigated localization of the gap junction in rat lacrimal gland in vivo and in vitro using electron microscopy and immunostaining with anti-connexin32 (Cx32) monoclonal antibody (HAM8). In immunofluorescence study of lacrimal gland tissues, Cx32 protein appeared to exist not only at the intercellular borders of acinar cells, but also in the basal regions, where there apparently was no contact with adjacent acinar cells. Thin sectioning and immunoelectron microscopy revealed that lacrimal acinar cells formed autocellular gap junctions (reflexive gap junctions) in the basal regions and intercellular gap junctions with adjacent acinar cell membranes. In immunofluorescence study of primary culture, Cx32 protein was found on the free surfaces of isolated acinar cells at the early stage of culture. With culturing time, cell aggregates were formed. We observed Cx32 immunoreactivity between acinar cells in these aggregates, but not on their free surface. Electron microscopic study confirmed that these aggregates possessed intercellular gap junctions and morphologically differentiated acinar-like structures. However, reflexive gap junctions were not observed in these aggregates. In conclusion, lacrimal acinar cells form intercellular and reflexive gap junctions in vivo. On the other hand, the existence of an acinar-like structure and intercellular gap junctions indicates that acinar cells differentiated in vitro morphologically. The cells may communicate with each other through these junctions, organizing themselves into an acinar cell network in an in vitro situation.     

Translocation of sLe(x) on the azurophilic granule membrane to the plasma membrane in activated human neutrophils

Using immunogold electron microscopy, we found that human neutrophilic sialyl Lewis x (sLe(x)), an adhesive ligand for selectins, detectable by a monoclonal antibody, KM-93, is present in the sacculi of the Golgi apparatus as well as on the membranes of large electron-lucent azurophilic granules and the plasma membrane, including surface projections and microvilli. Neutrophilic sLe(x), however, was not detected on the membranes of specific granules. In comparison with the distribution of sLe(x), CD18 was localized on the plasma membrane and specific granule membrane but not on the azurophilic granule membrane. We also found by immunogold electron microscopy and flow cytometry that treatment of neutrophils with sialidase resulted in a loss of sLex on the plasma membrane. In contrast, intracellular sLex on the azurophilic granule membrane was not destroyed by sialidase. When sialidase-treated neutrophils were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory mediator peptide, in the presence of cytochalasin B, we observed by immunogold electron microscopy and flow cytometry that sLe(x) again appeared on the plasma membrane. These results indicate that stimulation by fMLP induces the up-regulation of sLe(x) on the cell surface by promoting translocation of sLe(x) from the azurophilic granule membrane to the plasma membrane in human neutrophils.     

缺铁水稻根细胞膜泡相关基因群的分析与电镜观察

在缺铁固体培养基中,可清楚地观察到水稻根表面的分泌物和根的形态变化以及侧根增多.为了从分子水平深入了解水稻受缺铁胁迫后的反应机理,对缺铁水稻根进行了10531个EST芯片的微点阵分析,发现膜泡相关的基因群相对转录表达率最高.利用超薄切片技术,在透射电镜下观察了缺铁胁迫下水稻根尖细胞的结构变化.结果显示：缺铁根尖细胞中,膜泡好似起源于质膜和囊泡的组装,然后在质膜上凸起,并且是双方向性的.膜泡表面有衣被.虽然正常供铁情况下也有膜泡出现,但与缺铁比较有很大区别.缺铁根尖细胞的膜泡明显大,膜泡和凹陷泡上有较清晰的衣被.同时还观察到在单层膜的膜泡内有管状和圆球形的内含物质.内含物的来源有可能,至少是部分通过内吞作用来源于细胞外基质.     

Cytochemical detection of endogenous peroxidase in the intralobular ducts of hamster major salivary glands

Light and electron microscopic cytochemical investigation of endogenous peroxidase activity in the intralobular ducts of hamster major salivary glands was carried out using the diaminobenzidine-hydrogen peroxidase method. The peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, the Golgi apparatus and secretory granules in both the intercalated duct cells and the striated duct light cells of all glands. These results suggest the ability of the intralobular duct cells to secrete peroxidase the same as that of acinar cells in hamster salivary glands.     

Ciliogenesis in the human oviduct epithelium during the normal menstrual cycle.

Ciliogenesis has been investigated in the human oviduct epithelium during the normal menstrual cycle. Both centriolar and acentriolar pathways were involved in the replication of basal bodies. The centriolar pathway, in which procentrioles generate with the aid of preexisting diplosomes, played a minor role in the human oviduct. In the acentriolar pathway, fibrous granules were the first structure which appeared in the course of ciliogenesis and they initially occurred in association with the Golgi apparatus or free ribosomes. Subsequently deuterosomes arose in the aggregates of fibrous granules or apart from fibrous granules, and then microtubules-containing procentrioles originated around deuterosomes. Newly formed centrioles migrated to the apical cytoplasm with accompanying deuterosomes, and ciliary shafts extended first at the periphery of the luminal surface of ciliogenic cells. Deuterosomes as well as fibrous granules were considered to be related to the rootlet formation. Replicaion of basal bodies and protrusion of ciliary shafts mostly occurred during the proliferative phase of the menstrual cycle; however, a small number of fibrous granules indicating the ciliogenesis were still observed in some ciliated cells during the secretory phase. Ciliogenic cells in early stages of ciliogenesis contained secretory granules-like vesicles in the apical cytoplasm, suggesting that the ciliated cells are differentiated from secretory cells in the late secretory phase on demand.     

The relationship between intramembranous particles and aquaporin molecules in the plasma membranes of normal rat skeletal muscles: a fracture-label study

The plasma membrane of skeletal muscles contains water channels such as aquaporin 4 (AQP4), aquaporin 3 (AQP3) and aquaporin 7 (AQP7). In dehydrated mice, we have recently reported the altered distribution of the aggregations of intramembranous particles (IMPs), such as orthogonal array (a crystal-like structure) and IMP cluster (a rosette-like structure) on the freeze-fractured skeletal muscle plasma membranes. In this fracture-label study, we first tested whether the orthogonal arrays (OAs) were composed of AQP4 in skeletal muscles and further analyzed the relationship between IMPs including IMP clusters and AQP3 molecules. As a result, many of the gold particles indicating AQP4 was associated with OAs (79%) by our fracture-label technique. On the other hand, approximately 50% of gold particles indicating AQP3 were associated with IMP clusters. Thus we confirmed that the OAs are composed of AQP4 in skeletal muscles, and further demonstrated that some of the IMP clusters are composed of AQP3 and may participate in maintaining osmotic homeostasis in skeletal myofibers. The fracture-label method is useful in investigating the molecular identification of membrane proteins such as AQP3 and AQP4.     

水通道蛋白8在人胎盘和胎膜中的表达

目的:探讨水通道蛋白8(aquaporin 8,AQP8)在正常人胎膜及胎盘中的表达情况。方法:采集6例正常足月剖宫产的胎膜和胎盘组织,采用逆转录聚合酶链反应(RT-PCR)技术检测AQP8mRNA在胎膜、胎盘中的表达,用免疫组织化学的方法检测AQP8蛋白在胎膜、胎盘中的定位表达。结果:AQP8mRNA在羊膜、绒毛膜和胎盘均有表达,AQP8蛋白主要表达于羊膜上皮细胞、绒毛膜滋养细胞和胎盘合体滋养细胞的胞浆和胞膜中。结论:AQP8在羊水平衡的调节和母儿液体交换过程中可能起重要作用。     

与利它素分泌相关的桑蚕雌蛾性附腺超微结构研究

本文用扫描电镜和透射电镜对与野蚕黑卵蜂识别利它素分泌相关的桑蚕雌蛾性附腺分泌部进行了详细的研究。表明附腺分泌部中柱形腺上皮分泌细胞的超微结构与其功能具有非常好的一致性。大量发育良好的粗面内质网、高尔基复合体及具包膜的囊泡表明这些腺上皮分泌细胞是同其蛋白质合成密切相关的,同时大量存在的线粒体和微绒毛表明由腺上皮分泌细胞中粗面内质网合成的蛋白质在此处分泌入腺腔并分泌很活跃。根据透射电镜对一系列切片的观     

Distal surface of the rat ruffle-ended ameloblasts at the stage of enamel maturation observed by scanning electron microscopy

Distal surface of the rat ruffle-ended ameloblasts was observed by high resolution scanning electron microscopy. Specimens fixed by perfusion with 0.5% formaldehyde and 0.5% glutaraldehyde were decalcified with ethylenediamine tetraacetic acid and freeze-fractured using dimethyl sulfoxide. They were treated with 0.1% osmium tetroxide for 96 hr to remove excess cytoplasmic matrices, dehydrated, and critical-point dried. The present method was useful for observing both surface and intracellular structures simultaneously. The dense lamina lining the distal surface of the ruffle-ended ameloblasts having been dissolved in this preparation, the surface was clearly demonstrated in three dimensions under SEM. The surface was characterized by a complex labyrinth formed by protrusion and invagination of the plasma membrane. At high magnification, two kinds of minute granules are visible: small and larger granules measured as 10-20 nm and 70 nm in diameter, respectively. The former were more numerous than the latter. Furthermore, microfibrils connecting the protrusions of the plasma membrane were observed on the distal surface. The small granules probably connect the dense lamina with the distal surface of the ameloblasts. In addition, a denuded area free from the granules was sometimes recognized on the distal surface. These surface structures of the distal end of the ameloblasts appeared to be concerned with the enamel maturation.     

Role of matrix metalloproteinase-7 in angiogenesis associated with age-related macular degeneration

To investigate the possible role of matrix metalloproteinase-7 in choroidal neovascularization associated with age-related macular degeneration, immunoelectron microscopy using ultrathin frozen sections and conventional transmission electron microscopy were performed in subfoveal fibrovascular membranes from patients with age-related macular degeneration. immunoelectron microscopy revealed that matrix metalloproteinase-7 was expressed within basal laminar deposits and amorphous materials around the retinal pigment epithelial cells. The results support a role for matrix metalloproteinase-7 in the development of choroidal neovascularization in age-related macular degeneration.     

Culture prior to transplantation preserves the ultrastructural integrity of monkey pancreatic islets

Transplantation of pancreatic islets represents a promising way of curing type I diabetes (insulin-dependent diabetes mellitus). Culture enables the survival of endocrine tissue awaiting islet transplantation and reduces islet immunogenicity prior to xenografting. In this study, attempts were made to preserve the monkey islets in culture for 7 days and to study the ultrastructure by electron microscopy. The islets were isolated from monkey pancreas by the collagenase digestion method and were separated from acinar cells by dextran density gradient centrifugation. These islets were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was CMRL-1066. After 7 days of culture the islets were processed for light and electron microscopic studies, which revealed that the cultured islets were intact and maintained their structural integrity. Semi-thin sections of the cultured islets showed morphology with occasional structural alterations at the periphery. Dithizone staining of the cultured islets showed crimson red colour, proving that the islets were pure and without any exocrine contamination. Electron microscopy showed that the cultured islets had well-preserved alpha-, beta- and delta-cells. Different cell types of the monkey pancreatic islets were identified by the presence of their characteristic secretory granules. The ultrastructural characteristics present in hormone-synthesizing cells, i.e. rough-endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules, were observed as in native islets.     

家兔黄体的扫描电镜观察

本文用二甲基亚砜冷冻割断法对兔非妊娠性黄体进行了扫描电镜观察。发现黄体细胞多排列成索,索间有窦状毛细血管。在被割断的黄体细胞内可见圆形核,其核孔及核内染色质纤维与核仁的纤维部与颗粒部均可清晰地显示出来。胞质内含有大量滑面内质网(SER)。线粒体嵴呈管泡状。高尔基复合体形态多样立体感特强,在一个细胞内可见多个高尔基体网的不同面,并可见有不同粗细的小管与远处的小泡相连。此外还发现由SER和RER的扁平池所形成的轮纹结构。在上述膜性结构之间尤可见有由微丝,微管交织成网的细胞支架,其末端可附于膜上。值得注意的是在细胞间隙中除可见有以细茎与胞膜相连的球状突起外,尚有许多大小不一的泡状结构,最大的可达4μm,其膜可局部塌陷,如膜破裂,可见其中含有由多个小泡相连而成的串珠样结构,并可见串珠由泡内移出的图象。血窦内皮含核部突向腔内,在内皮细胞之间的间隙处和窦周间隙内,亦可见有小圆形颗粒。根据观察所见结合文献资料对黄体细胞的超微结构及其分泌方式进行了讨论。     

Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.     

Immunocytochemical studies on co-localization of alpha-granule membrane alphaIIbbeta3 integrin and intragranular fibrinogen of human platelets and their cell-surface expression during the thrombin-induced release reaction

We employed a double-staining method to immunocytochemically study the role of alpha-granule membrane alphaIIbbeta3 integrin in the expression of intragranular fibrinogen (Fbg) and albumin on the surface of thrombin-activated human platelets. In unstimulated platelets, alphaIIbbeta3 was distributed along the alpha-granule membrane as well as on the cell-surface membrane, including the open canalicular system (OCS), while Fbg and albumin were exclusively and evenly detected in the granules. At 30 s after activation by 0.1 U ml(-1) thrombin under non-stirred conditions, a small amount of Fbg in the granules was redistributed in association with alpha-granule membrane alphaIIbbeta3, suggesting that co-localization between alphaIIbbeta3and Fbg occurs at this period during platelet activation. At 1-5 min, the shape of the platelets changed from discoid to spheroid with pseudopodia and intact alpha-granules were no longer observed in these cells. Fibrinogen associated with alphaIIbbeta3 became increasingly expressed on the membranes of both the swollen OCS and the cell surface. Nevertheless, abundant Fbg still remained in the lumen of the swollen OCS, the membrane of which had already been depleted of alphaIIbbeta3. By western blot analysis using anti-human Fbg antibody, we detected no Fbg in the surrounding medium of the thrombin-stimulated platelets for 5 min. Unlike Fbg, abundant albumin in the alpha-granules was released into the surrounding medium without association with the membrane of either the alpha-granules or the cell surface. These results suggest that a small portion of the intragranular Fbg may bind to an Fbg binding site expressed on the activated aIIbbeta3 on the alpha-granule membrane, resulting in the formation of an alphaIIbbeta3-Fbg complex, which is then moved through the OCS membrane and expressed on the cell-surface membrane. Thus, alpha-granule membrane aIIbbeta3 may act as a carrier of intragranular Fbg.     

An osmium-ferricyanide staining method for the intercellular space in the small intestinal epithelium.

The post-fixation with osmium tetroxide and potassium ferricyanide (OsFeCN) produced dense deposits on the outer surface of the lateral plasma membrane in the mouse small intestinal epithelium. The deposits also filled up the intercellular space. No deposits were found on the surface of apical and basal plasma membranes. This staining pattern was highly reproducible when pH of the OsFeCN solution was adjusted to 7.0 by cacodylate buffer without calcium ion. Thus, our modified OsFeCN method is useful to selectively stain the intercellular space in epithelial tissues.     

隐失波荧光显微镜及其在植物细胞生物学中的应用

隐失波荧光显微术（evanescent wave fluorescence microscopy,EWFM）是一种对细胞膜附近的生物学过程进行选择性观测的光学显微成像技术,该技术是基于全内反射原理,即当光在生物样品表面产生全内反射时,虽然在样品表面未被光直接照亮,但它仍会产生一种隐失波,这种隐失波却有足够能量将样品表面100～200 nm范围内的荧光基团激发。由于这一特性使得隐失波荧光显微镜能对靠近细胞膜的生物学过程,如胞吞胞吐、单个分子与细胞膜的结合和解离,以及膜上受体的动态变化过程等进行更好的观测。本文简略地介绍了隐失波荧光显微术的原理,同时对其在细胞生物学,特别是在植物细胞生物学中的应用作了初步总结。随着隐失波荧光显微镜的商业化及其观测技术的不断改进,它将在生命科学领域的研究中发挥越来越大的作用。     

Use of taxol and collagenase for better three-dimensional visualization of microtubules in the enterocyte and Brunner's gland cell, with special reference to their relation to the Golgi apparatus

Cytoskeletal microtubules were visualized in the mouse duodenal mucosa by an improved immunofluorescence method using a microtubule-stabilizing reagent, Taxol, and collagenase as an enzymatic epitope retriever. The improvement in immunostaining was shown morphologically and statistically by comparing fluorescence intensities of specimens prepared with or without Taxol and collagenase treatment. In free cells in the epithelium and in the lamina propria, microtubules radiated from the gamma-tubulin-immunostained organizing center. Enterocytes and Brunner's gland cells double-stained with an anti-alpha-tubulin antibody and a lectin (Helix pomatia agglutinin, soybean agglutinin or Ulex europaeus agglutinin-I) showed that microtubules ran along the cell axis and were abundant between the Golgi apparatus and the apical surface. The microtubules appeared to provide a structural support to hold the Golgi apparatus in position and to act as railways for secretory granules, which are transported towards the apical surface. In addition, gamma-tubulin-like immunoreactivity was associated with the Golgi apparatus in the enterocytes. These results show that the method using Taxol and collagenase is effective for visualizing microtubules in epithelial cells, and that microtubules may play important roles in both positioning of the Golgi apparatus and transport of secretory granules. Our results also support the idea that the Golgi apparatus may act as an organizing center for microtubules.     

Clathrin sheets on the protoplasmic surface of ventral membranes of osteoclasts in culture

Physical cell-shearing resulted in various degrees of disruption of the basolateral (upper) membranes, cytoskeletons or cell organelles and exposed the protoplasmic surface of ventral (adhesion) membranes of osteoclasts that were attached to the underlying substratum, such as coverslips, mica or synthetic apatite plates. Freeze-dried replicas of the ventral membranes left behind on the substratum after cell-shearing provided three-dimensional information on the ultrastructure of the protoplasmic membrane surface of cultured osteoclasts. An extensive area of the protoplasmic surface and various amounts of cytoskeletal structures attached to the adherent ventral surface of the plasma membrane were visible. In particular, the most characteristic finding of the present study is that numerous clathrin sheets displaying various sizes, shapes and curvature were revealed on the ventral membrane. The polygon substructures of the clathrin lattices appeared to be composed of hexagons with a few pentagons interspersed. They were seen at the peripheral membranes where they were situated at the sites of close contact with the underlying substratum. In addition, clathrin lattices were never observed on the basolateral (upper) membranes. In favourable stereo views, most cytoskeletons were not in direct contact with the clathrin sheets. However, a few observations indicated possible remnants of cytoskeletons attached to clathrin lattices. Podosomes did not have a direct structural relationship to clathrin lattices. Although it is generally accepted that cytoskeletal podosomes in motile cells, such as osteoclasts, play a major role in cell adhesion, the present study indicates that membrane-associated clathrin might also function during attachment to the substrate. In this regard, clathrin is thought to be required for receptor-mediated endocytosis, but whether it might also function in cell attachment is still a matter for debate. This type of clathrin-related adhesion appears to be a previously unrecognized site of cell/substrate adhesion in osteoclasts. To assess this possible function, we focused on clathrin and related cytoskeletal elements on the ventral membranes of cultured osteoclasts.