Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90)

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.     

Effect of ultrasound on the contraction of isolated myocardial cells of adult rats

Microtubules and microfilaments are major cytoskeletal elements in mammalian ova and are important modulators of many fertilization and post-fertilization events. In this study, the integrated distribution of microtubules and microfilaments in pig oocytes were examined under a laser scanning confocal microscope, and the requirements of their assembly during in vitro fertilization and parthenogenesis in in vitro matured pig oocytes were determined. After sperm penetration, an aster of microtubules was produced in the spermatozoon, and this microtubule aster filled the whole cytoplasm during pronuclear movement. During pronuclear formation after activation by insemination, microfilaments became concentrated at the male and female pronuclei and, after electrical stimulation, at the female pronucleus. At metaphase of cleavage, microtubules were detected in the spindle and microfilaments were found mainly in the cortex. At anaphase, microtubule asters assembled at each spindle pole. During cleavage, large asters filled each daughter blastomere and a microfilament-rich cleavage furrow was observed. Cytochalasin B, a microfilament inhibitor, inhibited microfilament polymerization but affected neither pronuclear formation nor movement. However, syngamy and cell division were inhibited in eggs treated with cytochalasin B. Treatment with nocodazole after sperm penetration inhibited microtubule assembly and prevented migration leading to pronuclear union and cell division. These results indicate that microtubule and microfilament assembly in pig oocytes are integrated during fertilization and are required for the union of sperm and egg nuclei and for subsequent cell division.     

Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells

The organization of LETS protein on the surface of NIL8 hamster cells has been examined by immunofluorescence staining. The distribution of LETS protein was found to depend on the culture conditions; in subconfluent, low-serum arrested cultures the LETS protein is predominantly located at the cell-substrate interface and also in regions of cell-cell contact, whereas in dense cultures the cells are surrounded by a network of LETS protein fibrils. Transformed derivatives of these cells exhibit only sporadic staining for LETS protein, in the form of short intercellular bridges. Agents that cause alterations in cell shape and cytoplasmic filaments have been used to explore the relationship of LETS protein to the internal cytoskeletal elements. Reciprocally, perturbations of the cell surface were examined for their effects on internal filaments. The arrangement of microtubules seems to be unrelated to the presence of LETS protein in the cells studied. Actin microfilament bundles and LETS protein respond in a coordinate fashion to some perturbants but independently with respect to others. The patterns of staining for LETS protein are consistent with an involvement in cell-to-cell and cell-to-substrate adhesion.     

Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos

In a number of embryonic systems, centrosomes that have lost their association with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules. To identify additional activities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently labeled tubulin. Our recordings follow multiple rounds of free centrosome duplication and separation during the cortical division. The rate and distance of free sister centrosome separation corresponds well with the initial phase of associated centrosome separation. However, the later phase of separation observed for centrosomes associated with a spindle (anaphase B) does not occur. Free centrosome separation regularly occurs on a plane parallel to the plasma membrane. While previous work demonstrated that centrosomes influence cytoskeletal dynamics, this observation suggests that the cortical cytoskeleton regulates the orientation of centrosome separation. Although free centrosomes do not form spindles, they display relatively normal cell cycle-dependent modulations of their astral microtubules. In addition, free centrosome duplication, separation, and modulation of microtubule dynamics often occur in synchrony with neighboring associated centrosomes. These observations suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with aphidicolin supports this conclusion; large numbers of abnormal nuclei recede into the interior while their centrosomes remain on the cortex. Following individual free centrosomes through multiple focal planes for 45 min after the injection of aphidicolin reveals that they do not undergo normal modulation of their astral dynamics nor do they undergo multiple rounds of duplication and separation. We conclude that in the absence of normally dividing cortical nuclei many centrosome activities are disrupted and centrosome duplication is extensively delayed. This indicates the presence of a feedback mechanism that creates a dependency relationship between the cortical nuclear cycles and the centrosome cycles.     

Germ-line mutation analysis in patients with multiple endocrine neoplasia type 1 and related disorders

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.     

Regulation of cytoskeletal organization in tumor cells by protein phosphatases-1 and -2A

Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-1 and -2A) are inhibited by okadaic acid, attaining the same level of motility as metastatic LLC (LLC-LN7) variants. This stimulation of LLC-C8 motility was tempered when protein kinase A activity was inhibited. We examined whether the okadaic acid-stimulated LLC-C8 motility was associated with alterations in the cytoskeletal organization so that these non-metastatic cells acquire the rounded morphology and diffuse cytoskeletal organization previously described for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 are typically adherent during culture, achieving a spread morphology. Treatment of non-metastatic LLC-C8 cells with okadaic acid resulted in a contraction of most of their extended processes, formation of spikes and membrane blebs within 10 min, and complete cell rounding within 20 min for most of the cells. While the overall level of F-actin was minimally affected by the okadaic acid, its uniform distribution shifted to localization toward the periphery of the rounded cells, often concentrating at a single focus. Immunofluorescent staining for vimentin showed a similar shift to the cell periphery and similar capping. After okadaic acid treatment, the filamentous network of microtubules in non-metastatic LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta-tubulin. These results show that PP-1 and -2A maintain cytoskeletal organization and that inhibition of this control reduces cytoskeletal organization and increases tumor cell motility.     

Role of the endothelial cytoskeleton in blood-brain-barrier permeability to protein

The role of the cytoskeletal elements, microfilaments and microtubules in cerebral endothelial permeability to protein during steady states was investigated by studies of cerebrovascular permeability to horseradish peroxidase (HRP) in rats pretreated with cytochalasin B or colchicine, agents known to disrupt microfilaments and microtubules, respectively. In addition, the effect of colchicine pretreatment on the alterations in cerebrovascular permeability that occur in acute hypertension were studied. Rats infused with cytochalasin B showed increased cerebrovascular permeability to HRP in multifocal areas of the ipsilateral hemisphere. Most of the permeable vessels were arterioles; however, capillaries and venules also showed increased permeability. Ultrastructural studies of permeable vessels showed HRP in all layers of vessel walls and in endothelial and smooth muscle cell pinocytotic vesicles, which were increased in number. Although segments of interendothelial spaces were labeled by tracer, continuous labeling of interendothelial spaces from the luminal to the abluminal end was not seen and tight junctions were not disrupted. Normotensive rats pretreated with colchicine showed no alteration in cerebrovascular permeability to HRP. Colchicine pretreatment attenuated the permeability alterations that were observed in acutely hypertensive rats. This study demonstrates that integrity of endothelial actin filaments is important for maintenance of the blood-brain barrier to protein during steady states since increased permeability occurred in the presence of an actin disrupting agent. The microtubular network had no demonstrable role during steady states; however, disruption of the microtubular network had a protective effect and prevented the development of alterations in permeability to protein in acute hypertension.     

Spatial organization of microtubules and microfilaments in larval and adult salivary glands of Drosophila melanogaster

We examined the distribution of microtubules and microfilaments by conventional fluorescence microscopy and laser scanning confocal microscopy in larval and adult salivary glands of Drosophila melanogaster. The cells of the larval salivary gland epithelium were characterized by the same spatial distribution of microfilaments, whereas microfilament localization was more complex in adult salivary glands, showing some regional differentiation. Microtubules distributed throughout the cell cytoplasm of the larval salivary glands, whereas in adult glands they were mostly observed in the basal or apical cytoplasm of the cells. These observations were related to the secretory process and the mechanism of saliva discharge.     

Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a gamma-tubulin-green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of gamma-tubulin-green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.     

Differences in behavior among the chlorides of seven rare earth elements administered intravenously to rats

The aim of this study was to evaluate the effects of various cell culture conditions on cell morphology. Cell morphology was estimated by means of video recording and computer-assisted image analysis. Cell contours from the stored images of either live cells or fixed and stained cells were determined automatically, and cellular area, form factor, and average cell brightness were calculated. Using the mouse fibroblastoid L 929 cell line (L-cells) and the rat glioma BT4C cell line, it was found that a number of methodological parameters strongly affected cell morphology. These included confluency of cells before dissociation, dissociation procedure, cell seeding density, cultivation time, and culture substratum. The substratum, particularly collagen type I and fibronectin, profoundly affected cell morphology. Using drugs affecting cytoskeletal organization or cell substratum interactions, it was shown that average cell brightness was a valuable parameter for estimation of cellular attachment. Cytochalasin D, which impairs actin filaments, caused a dramatic increase in the average cell brightness in both cell lines. Nocodazole, which depolymerizes microtubules, mainly affected the L-cells, whereas the BT4C-cells were largely unaffected, indicating that microtubules were morphological determinants for the former cell line but not for the latter. When cells were grown on fibronectin, an RGD-peptide only affected L-cell attachment, indicating that BT4C-cells only expressed low (if any) amounts of RGD recognizing integrins. The interassay precision of the employed procedure depended on culture substratum; the coefficients of variation ranged from 7-24%. Lowest variations in area determination were found for cells grown on fibronectin. The coefficient of variation of form factor determinations was generally around 20%, independent of substrata and culture time.     

A structural scaffolding of intermediate filaments in health and disease

The cytoplasm of animal cells is structured by a scaffolding composed of actin microfilaments, microtubules, and intermediate filaments. Intermediate filaments, so named because their 10-nanometer diameter is intermediate between that of microfilaments (6 nanometers) and microtubules (23 nanometers), assemble into an anastomosed network within the cytoplasm. In combination with a recently identified class of cross-linking proteins that mediate interactions between intermediate filaments and the other cytoskeletal networks, evidence is reviewed here that intermediate filaments provide a flexible intracellular scaffolding whose function is to structure cytoplasm and to resist stresses externally applied to the cell. Mutations that weaken this structural framework increase the risk of cell rupture and cause a variety of human disorders.     

Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic microtubules

Glutamate can both facilitate and inhibit dendrite outgrowth in vitro. The major effects of low levels of glutamate occur only on the dendrites (not the axon) of pyramidal neurons and may be important for modulating dendrite outgrowth during neuronal development in vivo. Cytoskeletal changes resulting from glutamate exposure must underlie these changes in dendrite outgrowth. In the present study, hippocampal neuron cultures were used to measure the outgrowth of both axons and immature dendrites in the presence or absence of 50 microM glutamate. Subsequently, neurons were extracted and fixed for immunofluorescent labeling of microtubules and rhodamine phalloidin labeling of microfilaments. Additionally, neurons were prepared for electron microscopy to examine dendritic microtubules at the ultrastructural level. Glutamate led to increased dendrite outgrowth in the short term (4 hr) and dendrite retraction in the long term (8 hr). After short-term glutamate exposures, no obvious morphological changes occur in either the microtubules or microfilaments. However, longer glutamate exposure causes a decrease in the number of microtubules in the distal region of retracting dendrites, and causes an increase in microtubule number in the dendritic shaft of both retracting and growing dendrites. Thus, the microtubule cytoskeleton may be involved in producing the changes in dendrite outgrowth caused by glutamate exposure.     

Case report: right-sided periadrenal metastasis supplied by the hepatic artery. Clue to the genesis of pedunculated hepatocellular carcinoma

To demonstrate that cells both perceive and respond to external force, a strain/relaxation regimen was applied to normal human fetal and aged dermal fibroblasts cultured as monolayers on flexible membranes. The precisely controlled protocol of stretch (20% elongation of the culture membrane) at 6.67 cycles/min caused a progressive change in the monolayers, such that the original randomly distributed pattern of cells became a symmetric, radial distribution as the cell bodies aligned parallel to the applied force. High cell density interfered with the success of re-alignment in the fetal cell cultures observed, which may reflect a preference in this cell strain for cell-cell over cell-matrix contacts. The chronologically aged cells observed did not demonstrate this feature, aligning efficiently at all seeding densities examined. The role of microfilaments in force perception and transmission was investigated through the addition of cytochalasin D in graded doses. Both intercellular interactions and cytoskeletal integrity mediate the morphological response to mechanical strain.     

Microtubule nucleation by gamma-tubulin-containing rings in the centrosome

The microtubule cytoskeleton of animal cells does not assemble spontaneously, but instead requires the centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation. The minus, or slow-growing, ends of microtubules are embedded in the PCM and the plus, or fast-growing, ends project outwards into the cytoplasm during interphase, or into the spindle apparatus during mitosis. gamma-Tubulin is the only component of the PCM that is so far implicated in microtubule nucleation. Here we use immuno-electron microscopic tomography to show that gamma-tubulin is localized in ring structures in the PCM of purified centrosomes without microtubules. When these centrosomes are used to nucleate microtubule growth, gamma-tubulin is localized at the minus ends of the microtubules. We conclude that microtubule-nucleating sites within the PCM are ring-shaped templates that contain multiple copies of gamma-tubulin.     

Differential regulation of maternal vs. paternal centrosomes

Centrosomes are the main microtubule-organizing centers in animal cells. During meiosis and mitosis, two centrosomes form the poles that direct the assembly of a bipolar spindle, thus ensuring the accurate segregation of chromosomes. Cells cannot tolerate the presence of more than two active centrosomes during meiosis or mitosis because doing so results in the formation of multipolar spindles, infidelity in chromosome segregation, and aneuploidy. Here, we show that fertilization of Spisula solidissima oocytes results in cells that contain three active centrosomes, two maternal and one paternal. During meiosis I, the paternal centrosome's ability to nucleate microtubules is selectively shut off while maternal centrosomes remain competent to nucleate microtubules and assemble asters in the same cytoplasm. We propose that embryos can identify paternal vs. maternal centrosomes and can control them differentially.     

Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells

The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (Pi) was investigated in opossum kidney (OK) cells. Up-regulation of Na/Pi cotransport was achieved by incubating OK cells in a medium containing 0.1 mM Pi; down-regulation of Na/Pi cotransport was provoked by refeeding adapted cells with 2 mM Pi. Up-regulation of Na/Pi cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/Pi cotransport similar to that observed after refeeding adapted cells with 2 mM Pi. No effects of the microtubule disrupting drugs were observed on Na/L-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/Pi cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/Pi cotransport systems and that microtubules are not involved in the internalization of Na/Pi cotransport systems.     

The ultrastructure of the cell center in the enterocytes of mouse embryos and newborn mice

The centrosome structure was studied in differentiating small intestine enterocytes of mouse embryos (day 16 to 17 of gestation) and newborn mice. It is shown that fine structure of the centrosome in embryonic enterocytes differs from that found in cells of the adult mice. The centrosome of the crypt cells is more active: a greater number of cytoplasmic microtubules terminates there; mother centriole has two types of satellites; both centrioles are surrounded by fine fibrillar material. In 20% of crypt cells, replication of centrioles is observed. In the upper part of the villus of embryonic intestine, there still are two centrioles per cell, but they are located 3-7 um apart and devoid of any cytoplasmic microtubules attached to or directed toward them. In newborn mice, centrosomes of cells located at the bottom of the crypt are less active, as compared with centrosomes of embryonic cells. There are 1-3 satellites on mother centriole and few cytoplasmic microtubules closing to the centrosome. In 20% of cells from the bottom of the crypt centrioles are replicating. In the lateral part of the crypt, centrosome is inactive: centrioles are located 1-3 um apart and do not replicate. In the villus, centrioles undergo changes similar to those observed in the villus of adult mice. Centrioles loose portions of microtubule triplets. Near the top of the villus, only one centriole was found in two of 25 studied cells. In the enterocytes of the murine small intestine, centrioles are always located far from nuclei and close to the apical cell surface (1-3 um from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane.     

The fine structure of a microplate-microtubule array, microfilaments and polyhedral body associated microtubules in several species of Anabaena

A microplate-microtubule array was observed in Anabaena sp. (B-378). This structure consists of an arched plate, about 8 nm thick, and various microtubules, 12 nm in diameter and 50 nm long, arranged in rows. The microtubules project at right angles from one side of the plate into the cytoplasm or towards the plasma membrane. Up to twelve microplate-microtubule arrays were observed in a single section of a cell. Microfilaments, about 2.8 nm in diameter and of undetermined length, were observed in four isolates of Anabaena. The microfilaments were always found in bundles, nwhich varied in size, up to 0.63 mum across and 0.91 mum long. Microtubules, 10 nm in diameter and about 150 nm in length, were observed associated with one facet of polyhedral bodies in 8 out of 20 isolates of Anabaena. The microtubules occurred in groups of up to 20 or more, and were always oriented with the long axis parallel to the facet of a polyhedral body. In cross section, the microtubules had an electron transparent lumen 5 nm wide and a wall 2.5 nm thick. These structures are compared to previously described microtubules and microfilaments.     

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.