A morphometric analysis of microtubules in relation to the inhibition of lysosome movement caused by colchicine

Colchicine was administered intraperitoneally to rats in doses which are known to inhibit the basal migration of lysosomes in uterine epithelial cells. The fractional volume of microtubules in the cells was then measured by morphometry. Colchicine at 0.10 mg/kg reduced the microtubule content of the cells from 0.22% down to 0.15%, and 1.0 mg/kg reduced microtubule content to 0.03%. Microtubules were essentially absent from the cells after colchicine doses of 3.0 and 10.0 mg/kg. The microtubule content of uterine epithelial cells thus decreased in the colchicine dose range from about 0.10 to 1.0 mg/kg, the same dose range in which an inhibition of lysosome migration has been observed. These results support the suggestion that microtubules are necessary for the basal migration of lysosomes in uterine epithelial cells. In addition, colchicine at 1.0 mg/kg caused a redistribution of the Golgi complex and a class of electron-transparent, 130 to 450 nm vesicles. These organelles were restricted to the apical halves of the cells in untreated rats, but they were dispersed throughout the cells after drug treatment. The change in the position of the organelles may be caused by a loss of cytoskeletal function of the microtubules.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

FINE STRUCTURAL STUDIES ON THE INTERPHASE AND DIVIDING APICAL CELLS OF SPHACELARIA TRIBULOIDES (PHAEOPHYTA)1

The apical cells of Sphacelaria tribuloides Menegh. are larger than other thallus cells, contain more organelles and appear polarized. Their tip portion, where they grow, contains a well developed Golgi apparatus, abundant endoplasmic reticulum (ER) membranes, mitochondria, chloroplasts and a large number of small vacuoles. It seems likely that a continuous flow of membranous material from the ER membranes to the dictyosomes and from the latter to the plasmalemma of the extending tip portion takes place. In contrast, the basal pole possesses fewer organelles and is occupied mainly by large-sized, sometimes central vacuoles. The apical cells undergo two distinct types of highly asymmetrical differential divisions giving rise to cells of the thallus and hair initials. During the early stages of mitosis the nuclear envelope remains intact, except for fenestrated poles. Microtubules pass through the fenestrae into the nucleoplasm. During meta-phase, a typical chromosome plate is organized. The sites of attachment of spindle microtubules to the chromosomes are structurally different from the rest of the chromosomes. At late anaphase, the nuclear envelope breaks down completely. During telophase, a new membrane encloses the chromosomes which are decondensed and the nucleoli are reorganized. Cytokinesis proceeds long after mitosis at a stage in which the nuclei have increased in size and have moved farther apart. A membranous furrow develops centripetally, without the participation of microtubules. However, microtubules traverse the thin cytoplasmic strands which, in both interphase and cytokinetic cells, meander among the vacuoles of the basal pole of the cell and the internuclear space. Dictyosomes appear to be involved in the subsequent wall deposition.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

Ultrastructural effects of colchicine on perikarya and initial segment of rat retinal ganglion cells.

Our ultrastructural study was focused on the perikaryal region and initial segment of the axon of rat retinal ganglion cells in controls and after intraocular injections of colchicine. In control rats that region contained, among other organelles, elements of the Golgi complex and, close to them, short isolated microtubules oriented preferentially toward the axon where they funnel and aggregate in bundles. One day after sufficient doses of colchicine to inhibit axoplasmic transport (2-20 micrograms) these cytoplasmic microtubules were absent, whereas some axonal microtubules were still present but reduced in number. In addition, colchicine induced an altered distribution of organelles, leaving empty spaces in the periphery and most organelles concentrated in the perinuclear region, especially around Golgi elements where numerous vesicles and tubules accumulate at the trans face of Golgi elements. These results suggest that the vesicles that leave the Golgi and have been directed towards axoplasmic transport may need the cytoplasmic microtubules located between Golgi elements and the axonal initial segments to reach the axon.     

The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect

A structure for a generalized insect epidermal cell during the formation of the epicuticle is proposed, based on studies of several different epidermal cell types. The protein epicuticle is defined as the dense homogeneous layer below the cuticulin. The formation of the protein epicuticle involves secretory vesicles arising in Golgi complexes, and marks an interlude in the involvement in cuticle formation of plasma membrane plaques. The plaques are concerned in cuticulin formation before and in fibrous cuticle formation after the deposition of the protein epicuticle. The epidermis is characterized by the possession of a cytoskeleton of microtubules and a matrix of microfibers. In the elongated cells forming bristles and spines, the microfibers are often oriented in bundles with an axial banding which repeats every 120 Å. The microtubules are also arranged in columns with a trigonal packing and center to center spacing of about 800 Å. These cytoskeletal structures separate the other organelles into channels which may restrict the pathways open for the movement of secretory and pinocytotic vesicles. The protein epicuticle arises from the secretory vesicles which discharge at the apical surface. The contents disperse and reaggregate below the cuticulin. The Golgi complexes in the basal and central regions have many secretory vesicles and a small saccular component, differing from those nearer the apex which are smaller and have fenestrated saccules. The small coated vesicles (800 Å in diameter) associated with both sorts of complex, probably move to the apical and basal faces of the cell where they may give rise to the large coated vesicles (2000 Å in diameter) inserted in the plasma membrane. Pinocytosis occurs from both apical and basal faces but most lytic activity is in the apical region. Plant peroxidase injected into the haemocoel is taken up basally and transported to the apical MVBs. The large coated vesicles on the apical face may be concerned in the control of the extracellular subcuticular environment. They appear to fill up and detach, fusing to become the apical MVBs.     

Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.     

Colchicine-induced tubular,vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. I. Morphology and phosphatase cytochemistry

Treatment of rats with colchicine administered intraperitoneally at a dosage of 0.5 mg per 100 g of body weight for 6 hr induces extensive accumulations of tubular-vesicular and cisternal organelles in the absorptive cells of the small intestine. The formation of these organelle aggregates coincides with a reduction of microtubules and massive changes in the cellular organization including alterations of the Golgi apparatus and the plasma membrane. In most cases the accumulated tubules and vesicles contain a homogeneous electron-dense matrix, the cisternae often having the character of rigid lamellae. The organelle aggregates mainly occupy apical cell portions subjacent to the terminal web as well as basal cellular regions close to the basolateral plasma membrane. Tubular-vesicular as well as cisternal organelles react strongly for thiamine pyrophosphatase (TPPase), inosine diphosphatase (IDPase), acid phosphatase (AcPase) and trimetaphosphatase (TMPase). The staining pattern of TMPase differs from that of the other phosphatases in that the reaction is restricted to the colchicine-induced tubular-vesicular and cisternal aggregates, whereas TPPase, IDPase, and AcPase, respectively, also appear over Golgi stacks, multivesiculated bodies and plasma membrane. This phosphatase reactivity indicates the lysosomal character of the organelle aggregates.     

Fine structure of the sweat glands of the antebrachial organ of Lemur catta

Summary The sweat glands of the antebrachial organ of the ring-tailed lemur are atypical apocrine glands which have some characteristics of eccrine sweat glands. The myoepithelial cells are large and consist of well-differentiated basal and apical regions. The secretory cells form a monolayer of tall, columnar cells filled with numerous secretory vacuoles and capped with differentiated apical blebs. The vacuoles are formed in the Golgi region and their contents are discharged into the lumen and into intercellular canaliculi. The blebs are pinched off at the luminal surface by a true apocrine mechanism. In addition to the usual organelles (abundant rough endoplasmic reticulum, prominent Golgi region, large mitochondria, pigment, secretory vacuoles), the secretory cells contain bundles of microtubules. Each microtubule is about 325–350 Å in diameter. The glands are larger and more active in the male. These sweat glands are distinctly different from the apocrine glands of the general body surface of L. catta.Publication No. 128 of the Oregon Regional Primate Research Center, supported in part by Grants FR 00163 and AM 08445 from the National Institutes of Health. The author expresses thanks to D. McLean for preparation of the diagram.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells

A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.     

Fine structure of the Golgi complex during mitosis of cartilaginous cells in vitro

Chondrocytes were isolated enzymatically from guinea-pig epiphyses and grown in vitro. The fate of the Golgi complex during mitosis in relation to changes in the cytoplasmic microtubules was then studied by transmission electron microscopy. Interphase cells were observed to be polarized, with the Golgi complex occupying a well-defined juxtanuclear area of the cell's cytoplasmic pole. During prophase the cytoplasmic microtubules were largely lost, the nucleus moved to the center of the cell and the Golgi complex dissolved into single dictyosomes spread diffusely throughout the cytoplasm. The distribution of other organelles also changed to a more random pattern. In telophase, i.e. after the completion of nuclear division, the mitotic spindle decomposed and cytoplasmic microtubules reappeared. Furthermore, the organization of the Golgi complex and other organelles returned to that characteristic of interphase cells. Previous studies on cells treated with colchicine have indicated that the polarized distribution of cell organelles is dependent on the presence of intact cytoplasmic micro-tubules. It is suggested that the disappearance of such tubules observed here to be coupled with the disorganization of cell interphase structure fulfills the double function of providing free tubulin units from which to build the mitotic spindle and ensuring an approximately equal distribution of dictyosomes and other organelles to the daughter cells during cytokinesis.     

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Distribution and content of microtubules in relation to the transport of lipid. An ultrastructural quantitative study of the absorptive cell of the small intestine

To determine whether microtubules are linked to intracellular transport in absorptive cells of the proximal intestine, quantitative ultrastructural studies were carried out in which microtubule distribution and content were determined in cells from fasting and fed animals. Rats were given a 1-h meal of standard chow, and tissue was taken from the mid-jejunum before, 1/2 h, and 6 h after the meal. The microtubule content of apical, Golgi, and basal regions of cells was quantitated by point-counting stereology. The results show) that microtubules are localized in intracellular regions of enterocytes (apical and Golgi areas) previously shown to be associated with lipid transport, and that the microtubule content within apical and Golgi regions is significantly (P less than 0.01) reduced during transport of foodstuffs. To determine the effect of inhibition of microtubule assembly on transport, colchicine or vinblastine sulfate was administered to postabsorptive rats, and the lipid and microtubule content of enterocytes determined 1 and 3 h later. After treatment with these agents, lipid was found to accumulate in apical regions of the cells; this event was associated with a significant reduction in microtubule content. In conclusion, the regional distribution of microtubules in enterocytes, the decrease in assembled microtubules after a fat-containing meal, and the accumulation of lipid after the administration of antimicrotubule agents suggest that microtubules are related to lipid transport in enterocytes.     

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D₂O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.Abbreviations DMSO dimethylsulfoxide - CB cytochalasin B Dedicated to Prof. Dr. A. Pirson on the occasion of his 70. birthday     

Fine Structure of the Schizont and Merozoite of Isospora sp. (Sporozoa: Eimeriidae) Parasitic in Gehyra variegata (Dumeril and Bibron, 1836) (Reptilia: Gekkonidae)

SYNOPSIS. A study was made of the fine structure of some stages in the life cycle of an undesignated species of Isospora parasitic in a gecko. The merozoites which lay within a membrane-bound periparasitic vacuole in the host epithelial cell, had a striking similarity to Plasmodium, Lankesterella, Toxoplasma, Besnoitia, Sarcocystis, Eimeria and the M-organism. Each merozoite was invested with a triple-layered pellicle, the outer membrane of which was loosely applied. At the anterior end of the merozoite were conoid and apical rings; microtubules terminated in the posterior apical ring. Other organelles included nucleus, endoplasmic reticulum, mitochondria, micropyle, paired organelle, toxonemes and a variety of vacuoles. Although the sequence of development of the merozoite was not completely followed, some events in this process were recorded. The evidence suggests that anterior ends are formed early and that merozoites develop subsequently by a process of budding. The merozoite pellicle appears to be continuous with, altho structurally different from, the investing membrane of the parent cell.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.     

Recent progress in living cell imaging of plant cytoskeleton and vacuole using fluorescent-protein transgenic lines and three-dimensional imaging

Summary. In higher-plant cells, microtubules, actin microfilaments, and vacuoles play important roles in a variety of cellular events, including cell division, morphogenesis, and cell differentiation. These intracellular structures undergo dynamic changes in their shapes and functions during cell division and differentiation, and to analyse these sequential structural changes, the vital labelling technique, using the green-fluorescent protein or other fluorescent proteins, has commonly been used to follow the localisation and translocation of specific proteins. To visualise microtubules, actin filaments, and vacuoles, several strategies are available for selecting the appropriate fluorescent-protein fusion partner: microtubule-binding proteins, tubulin, and plus-end-tracking proteins are most suitable for microtubule labelling; the actin binding domain of mouse talin and plant fimbrin for actin microfilament visualisation; and the tonoplast-intrinsic proteins and syntaxin-related proteins for vacuolar imaging. In addition, three-dimensional reconstruction methods are indispensable for localising the widely distributed organelles within the cell. The maximum intensity projection method is suitable for cytoskeletal structures, while contour-based surface modelling possesses many advantages for vacuolar membranes. In this article, we summarise the recent progress in living cell imaging of the plant cytoskeleton and vacuoles using various fusions with green-fluorescent proteins and three-dimensional imaging techniques. Correspondence and reprints: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8562, Japan.     

The ultrastructure of mammary explants from virgin ovariectomized goats during hormone-induced lactogenesis

The effect of insulin (I), cortisol (F) and prolactin (P) on the ultrastructural morphology of epithelial cells of cultured mammary explants from virgin ovariectomized (OV-X) goats were studied. The epithelial cells showed little structural organization and were devoid of fat droplets and secretory protein granules at zero time of culture. The cytoplasm contained few profiles of smooth and rough endoplasmic reticulum and the Golgi apparatus was rudimentary. After being cultured in Waymouth's medium without added hormones the epithelial cells were indistinguishable from epithelial cells of uncultured explants. The addition of I induced changes mainly in the appearance of nucleoli. The nucleoli were enlarged and fibrillogranular areas with light spaces were observed. The most obvious cytological changes of epithelial cells of explants cultured in the presence of I and F are translocation of the nucleus into the basal cytoplasm, increase of rough endoplasmic reticulum, an increase in the size of the Golgi apparatus, presence of one or two lipid droplets and in some cells vacuoles with protein granules were present. Mitochondria were more abundant. The epithelial cells of explants cultured in the presence of I, F and P were characterized by the polarization of organelles within the cytoplasm and by the formation and release of protein granules and small and large fat droplets. The cell nucleus was in the basal cytoplasm, the Golgi apparatus was supranuclear. The rough endoplasmic reticulum was extensively developed and formed large sacs. Golgi vacuoles contained protein granules.(ABSTRACT TRUNCATED AT 250 WORDS)