Time-lapse Cinephotomicrography Research Articles

Myosin in hepatocytes is essential for bile canalicular contraction

Active dynamic contraction of bile canaliculi has been observed in cultured doublet hepatocytes using time-lapse cinephotomicrography. This contractile movement plays an important role in normal bile formation. The mechanism of bile canalicular contraction has been proved to involve the Ca(2+)-calmodulin system and pericanalicular actin filaments. However, the role of myosin in this system is still unknown. In this study, using the newly synthesized myosin light-chain kinase inhibitor ML-9, we found that the treatment of cultured doublet hepatocytes with ML-9 inhibited canalicular contraction. This inhibitory effect suggests that myosin is involved in this complex cellular function and that the integrity of the actin-myosin system, as well as the Ca(2+)-calmodulin system is essential for normal bile canalicular contraction.

Growth rate analysis of neurites extended from triploid spinal cord explants

Nerve fibers extending from spinal cord explants of triploid frog tadpoles were analyzed by time-lapse cinephotomicrography. The triploid neurites grew at a mean rate that seemed to reflect the increased chromosomal dosage and had maximal velocities that were several times greater than for diploid neurites. Concurrently, the time between alternating episodes of acceleration and deceleration in triploid neurite elongation phases was decreased. During a 24-h period, however, triploid neurites spent less time elongating with a net result of shorter travel distances than for diploid neurites. The data suggest that the genome is one level of nerve fiber growth control that can be manipulated by dosage alterations.

Bile canalicular contraction in the isolated hepatocyte doublet is related to an increase in cytosolic free calcium ion concentration

Dynamic contractions of bile canaliculi have been observed in cultured doublet hepatocytes by means of time-lapse cinephotomicrography, and this contractile movement plays an important role in bile secretion. Although details of the mechanism are still unknown, the Ca2+-calmodulin system is believed to play a main role in this mechanism. In this study we measured the intracellular Ca2+ concentration of individual doublet hepatocytes using the Ca2+ indicator "fura 2" and microscopic fluorometry. We also observed the effects of A23187, norepinephrine and epinephrine on bile canalicular contraction and intracellular Ca2+ concentration. After loading 1 mumol/l fura 2 in doublet cells, we added A23187, epinephrine or norepinephrine and then measured the Ca2+ concentration in a given small area in the cytoplasm of individual doublet cell. A23187, norepinephrine and epinephrine caused a prompt increase of the intracellular Ca2+ concentration and also caused bile canalicular contraction. The present study indicates that the sudden increase of intracellular Ca2+ concentration causes bile canalicular contraction through the Ca2+-calmodulin system.

単球/マクロファージの役割

The adherence of monocytes to the endothelium of large arteries and their subsequent migration to the intima, followed by modulation into lipidladen macrophages are early events in diet-induced atherogenesis. In this study we have examined the interaction of rat peritoneal macrophages with cultured vascular endothelial cells, as well as macrophage-derived chemotactic activity of macrophages. It was found that adhesion of macrophages to endothelial cells was dependent on the presence of divalent cations, and was inhibited at 4°C. Pretreatment of endothelial cells with LDL and VLDL significantly increased subsequent adhesion of macrophages to these endothelial cells. In addition, macrophages from hypercholesterolemic animals showed enhanced attachment to the endothelial cells; they also moved more quickly when examined by time-lapse cinephotomicrography. Macrophages in culture secreted potent chemotactic factors for macrophages. This production of chemotactic factors by macrophages and chemotactic properties of macrophages to the autoconditioned media were remarkably increased when animals were exposed to hypercholesterolemia. The results suggest that in hypercholesterolemia, macrophages are more adhesive to endothelial cells and tend to accumulate in the intima. It seems likely that macrophagederived chemotactic activity for macrophages leads to the recruitment and retention of monocytes/macrophages in the arterial wall.

Advantages of Combining Histopathology, Tissue Culture and Ultrastructure in Studying Disease Processes

AbstractSpecial tissue culture (histoculture) methodologv, when coibined with hematoxylin and eosin (H&E) stained tissue sections, ultrastructural studies, and time-lapse cinephotomicrography, can provide information in neoplastic tissue that cannot be obtained from H&E alone. This approach has unlimited applicability and is particularly useful inthe evaluation of mitotic cells and host response to the disease process. The multidiciplinary nature of this approach yields results superior to any single method.

Contraction of scar tissue in the rabbit vitreous.

Sheets of vitreous membrane (scar tissue) and associated retinal detachment were produced in the right eye of 86 adult New Zealand white rabbits by intravitreal injection of cultured autologous skin fibroblasts. The membranes were examined by light and electron microscopy and time-lapse cinephotomicrography. Immunohistochemistry was used to demonstrate alterations in the distribution of cytoplasmic contractile proteins. While retinal detachment and membrane contraction were taking place, there was pronounced increase in the numbers of fibroblasts with an elongated spindle shape. These spindle-shaped cells had some similarities to myofibroblasts including the presence of 'stress cables'. However, the myofibroblast-like cells stained much less avidly for cytoplasmic (actin) microfilaments than migratory fibroblasts seen at early stages of membrane development. The significance of migrating fibroblasts and myofibroblasts in scar contraction is discussed.

Colchicine effect on bile canalicular motility: long-term study using isolated cultured hepatocytes and time-lapse cinephotomicrography.

The effect of colchicine on spontaneous in vitro contractions of bile canaliculi was assessed using time-lapse cinephotomicrography. Colchicine 2.5 microM and/or taurocholate 5 microM were added to the culture medium. The experiment was long-term, lasting for 13 h. Frame-by-frame analysis revealed that colchicine, after a lag period of 4 h, suppressed the number of contractions over the remainder of the 13-h experimental period, even in the presence of taurocholate (p less than 0.001). The number of contractions per hour decreased in both the colchicine and combined colchicine-plus-taurocholate-treated groups compared to untreated and taurocholate-treated controls. These results do not necessarily mean that colchicine has an effect on the contractile process per se, since the contractions, when they occurred, appeared normal. It is likely that the canalicular motility events are related to bile secretion. The reduction in contractions after a lag period likely indicates that secretion is impaired, and hence the need for contractions is reduced. These results support the view that for normal canalicular bile secretory function, microtubules as well as a normal microfilament system are required.

The cells of the bovine outflow system in tissue culture

Meshwork cells from the bovine outflow system were grown in culture and their growth characteristics and behaviour in vitro reported. The cells were studied by inverted phase microscopy and time-lapse cinephotomicrography. It was found that plating efficiency was highest for third and seventh passage cells. These cells reached confluence within 2 weeks when plated out at 7·5 × 10 3 cells cm −2 in a conventional culture medium containing 10% serum. Third passage cells were studied in greatest detail. These cells exhibited postconfluence contact inhibition of division. By 2 weeks postconfluence the division rate decreased 10-fold to a level of less than six division per thousand cells per day. The contact inhibition was not overcome by increasing the serum content of the medium. On the other hand, a 30-fold increased division rate could be produced after scratching the culture flask. Postconfluent contact-inhibited cultures were considered to be a better reflection of the behaviour and activities of meshwork cells in vivo than cultures which retain a high level of division.

Size Control in a Small-size Mutant of Saccharomyces cerevisiae

The kinetics of cell proliferation of a population of a small-size mutant of Saccharomyces cerevisiae were compared with those of a wild-type cell population, using time-lapse cinephotomicrography. The mean size of small-size mutant cells was approximately half that of wild-type cells at corresponding points in the cell cycle. The cycle times of small-size mutant cells were much more variable, especially for daughter cells, than those of wild-type cells. The difference in the variability of cycle times of the two strains was mainly due to the different degree of variability of their respective unbudded periods. Like wild-type cells, daughter cells of the small-size mutant were smaller and had a longer cycle time than parent cells. The small-size mutant retains a single size control over the cell cycle.

Taurocholate accelerates bile canalicular contractions in isolated rat hepatocytes

The influence of sodium taurocholate on bile canalicular contractions was examined by time-lapse cinephotomicrography of isolated rat hepatocytes in primary monolayer cultures. Frame-by-frame analyses indicates that as the dose of taurocholate increased, the number of contractions increased in a dose-dependent manner up to 5 μM. Because bile canaliculi are the smallest biliary passages across which bile secretion occurs, and because it is known that taurocholic acid within the dose range used in these studies causes a linear increase in bile secretion, the results suggest that canalicular contractions may be a function of canalicular bile secretion.

Human trabecular meshwork in primary culture: A morphological and autoradiographic study

The growth and behaviour of human trabecular meshwork cells in primary culture was studied by light microscopy, time-lapse cinephotomicrography, autoradiography and electron microscopy. In all 925 explants of trabecular tissue from 132 eyes (68 ± 14 years) were set up, and 20% produced extensive monolayers. Failure to produce primary outgrowths did not increase substantially until the eyes were over 5 days post-mortem. The latent period prior to growth was anything from a few days to 4 weeks; only then did meshwork cells label with [ 3H]-thymidine. The cells which grew incorporated large amounts of [ 3H]-leucine and this served as a useful marker to identify migratory cells. In addition they had the ultrastructural features of metabolically active cells including many mitochondria, a well-developed Golgi apparatus, abundant rough endoplasmic reticulum and prominent lysosomes. It was considered that the cells in primary culture had many features in common with ‘activated’ rather than normal meshwork cells.

A novel cell-to-cell interaction between mast cells and other cell types

Time-lapse cinephotomicrography and transmission electron microscopy (TEM) have been used to study the interactions between rat mast cells and different cell monolayers in culture (fibroblasts, vascular endothelial cells and cardiac muscle cells). This report documents a novel form of behavior between mast cells and certain other cell types. We have tentatively termed this cellular behavior ‘transgranulation’, which involves sequential changes not seen in control cells, including: (1) formation of a granule-containing mast cell pseudopod that becomes closely applied to an adjacent cell; (2) development of specialized plasma membrane interrelationships between apposing cells; (3) alteration of granules and perigranular membranes within the mast cell pseudopod; (4) occasional transfer of exocytosed mast cell granules to the cytoplasm of the adjacent cell; (5) presence of a specialized inclusion body in the mast cell; and finally, (6) either withdrawal of the pseudopod by the mast cell, or casting-off of the pseudopod from the mast cell, leaving it on the surface of the adjacent cell (pseudopod translocation). These mast cell interactions occur specifically with fibroblasts and endothelial cells in vitro and are never observed with cardiac muscle cells or non-cellular substrates. Our investigations of rat mesenteries in situ confirm that these cell-cell interactions also occur in vivo. We suggest it represents a form of cell-to-cell communication involving secretion from a mast cell pseudopod to another cell type. The significance of specialized contacts between mast cells and other cell types in vivo is discussed.

Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy

To obtain baseline data for our experimental investigations in vitro we established a pure cellline culture model of human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts, using an identical environment for each cell type. We studied the growth characteristics, morphology and phagocytic activity of live cultures by phase-contrast microscopy and time-lapse cinephotomicrography; fixed preparations were examined by light and scanning electron microscopy. From these observations, we were able to characterize the distinctive features of the cells. By correlated ultrastructural and tissue culture studies, we showed that human cadaver eyes cold-stored (4°C) up to 5 days can be used as a tissue source of trabecular endothelium, keratocytes and scleral fibroblasts. Corneal endothelium, however, rarely survived after 4 days of postmortem storage. Our observations revealed that, in primary confluent culture, the trabecular cells grew in a monolayer. Individual cells had flattened, elongated cell bodies, a centrally located, oval nucleus, and a generally smooth cell surface with occasional microvillous projections; in places, adjacent cells and their processes adhered closely one to another. Corneal endothelium at confluency presented a flattened hexagonal profile of cells arranged in a honeycomb pattern, interdigitating cell borders, a centrally located nucleus, prominent perinuclear microvillous projections, and numerous surface invaginations. The keratocytes and scleral fibroblasts had a thin and elongated, spindle-shaped profile with a remarkably smooth cell surface and an oval to oblong nucleus surrounded laterally by scanty cytoplasm; the cell surfaces and processes frequently overlapped but rarely formed firm attachments. In contrast to the monolayer growth of trabecular and corneal endothelium, these cells characteristically grew in superimposed layers. In primary culture and with latex spheres and carmine particles used as markers, the trabecularendothelial cells showed a greater phagocytic capability than did corneal endothelium, keratocytes and scleral fibroblasts. Our preliminary studies indicate that cytochalasin B markedly inhibits the phagocytic activity of the trabecular cells.

A study of bile canalicular contractions in isolated hepatocytes.

Living hepatocytes observed under standard methods of cell isolation and culture show frequent active contractions of bile canaliculi when viewed by time-lapse cinephotomicrography. This report gives details of the methods used to show the contractions. In addition, the characteristics of the contraction pattern are defined and analyzed. The contractions are regular, canalicular contraction time (systole) lasts 60 sec, and the most probably interval between contractions is 5 1/2 min. The finding of a regular pattern of contraction suggests an ordered, programmed, and integrated mechanism which entails a high degree of communication and functional coordination between neighboring hepatocytes.

Variability in individual cell cycles of Saccharomyces cerevisiae

The kinetics of cell proliferation of Saccharomyces cerevisiae were studied at 4 growth rates using time-lapse cinephotomicrography. Cells were grown on media with a high refractive index to reveal greater intracellular detail under the phase-contrast microscope. The morphological cell-cycle events scored were: bud emergence, nuclear migration, nuclear division, onset of cytokinesis and cell separation. Cell size was measured at cell separation and at bud emergence. The daughter-cycle time was always longer than the parent-cycle time mainly due to the large difference in the lengths of the unbudded phases. Parent cells had a shorter budded period than daughter cells. The large variance in daughter-cycle times was accounted for by the large variance in the lengths of the unbudded phase of daughter cells. The duration and variability of the periods in the cyclc from nuclear migration onwards were equivalent for parent and daughter cells. Daughter cells were always smaller than parent cells at division. There was wide variation in cell size at both division and bud emergence. The results indicated that a modified deterministic model could best explain cell proliferation kinetics in yeast. The data were used to evaluate 2 different models. The 'sloppy size control' model of Wheals (1981 a) was more consistent with the data than the 'tandem' model of Shilo, Shilo & Simchen (1976). The distribution of unbudded periods of daughter cells suggested that there was an additional incompressible period not present in parent cells.

Contractility of bile canaliculi: implications for liver function.

Dynamic contractions of bile canaliculi were observed in groups of cultured hepatocytes by time-lapse cinephotomicrography during the early stages of monolayer formation. The contractions, which were forceful and appeared to have a pumping action, may facilitate the flow of bile in the liver's canalicular system.

Preliminary observations on human trabecular meshwork cells in vitro.

This report presents our preliminary observations on the trabecular meshwork from human eyes up to 5 days post-mortem in tissue culture. Satisfactory primary cultures were obtained from about 20% of the 423 explants which were investigated. The period prior to growth was from 4 days to 4 weeks and from the appearance of the initial outgrowth it took 25 to 30 days to reach maximum cellular spread within the culture chambers. The progress of the explant and the spreading of the trabecular meshwork cells was monitored by phase-contrast microscopy, time-lapse cinephotomicrography, light microscopy, transmission electron microscopy, scanning electron microscopy and autoradiography (using tritiated thymidine). On the basis of their ultrastructural appearance the cultured meshwork cells seems to be metabolically active. Their cytoplasm contained abundant rough endoplasmic reticulum, many mitochondria, a well developed Golgi apparatus and many coated and uncoated micropinosomes. However, even in short-term culture the trabecular meshwork cells had adapted to the artificial environment of our system and no longer resembled "normal" trabecular meshwork cells as seen in vivo. Since trabecular meshwork cells can quickly adapt their morphology in a culture environment and because the adult human meshwork contains a significant population of non-trabecular cells, the value of long term culture as a means of investigating the cellular activity of the normal and glaucomatous outflow system must be open to question.

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. VIII. Cinephotomicrographic studies.

T cells from immune guinea-pigs produce clusters in vitro with macrophages pulsed with soluble protein antigens. The formation of clusters is antigen-specific. Time-lapse cinephotomicrography was used to study the sequence of events leading to cluster formation and the kinetics of already formed clusters. The central lymphocyte attaches through a broad base to the macrophage during the first few hours of incubation. After several hours, during which the central lymphocyte is situated alone on the surface of the macrophage, free lymphocytes begin to interact for shorter or longer periods of time through their uropods with the central lymphocyte, thus acting as peripheral lymphocytes. In spite of a frequent traffic of peripheral lymphocytes in and out, the cluster is a relatively stable structure. Occasionally the entire complex of lymphocytes leaves the macrophage, moves randomly in the culutre and attaches to a new macrophage or returns to the same macrophage.

Simple agar block slide culture suitable for varying the environment of bacteria during time-lapse cinephotomicrography.

A simple paper wick agar block slide culture is described for use in experiments which vary the chemical environment of bacteria during time-lapse cinephotomicrography.

Cloned pigmented retinal epiehtlium. The role of microfilaments in the differentiation of cell shape.

3-wk-old clones of pigmented epithelial cells from chick retina can be divided into four zones on the basis of cellular morphology and pigmentation. These zones appear to represent different stages in the re-expression of differentiation: those cells with essentially no differentiated characteristics are at the outer edge and those with the greatest number are at the center. Cells of the colony exhibit three different types of movement when analyzed by time-lapse cinephotomicrography: focal contractions, extension and retraction of apical protrusions, and undulations of the lateral membranes. All the cells of the colony contain microfilaments, 4--7 nm in Diam, which are primarily arranged as apical and basal webs. In addition, less well defined filamentous networks are found in the apical protrusions and lateral interdigitations. When colonies are treated with 10 micrograms/ml of the drug cytochalasin B (CCB), the apical microfilament arrays are disrupted and movement stops. Both phenomena are reversible upon removal of the drug. During the process of redifferentiation, the cells change their shape from squamous to cuboidal, and the greatest change is found where the colony exhibits the greatest number of focal contractions. The evidence suggests that the apical microfilament arrays are directly responsible for the observed movements, particularly the focal contractions, and that focal contractions contribute to the development of the differentiated cellular shape. Possible roles for the other movements are discussed.