Thick Microfilaments Research Articles

Meat texture, muscle histochemistry and protein composition of Eriocheir sinensis with different size traits

The main aim of the present work was to investigate the effect of the inherent differences in the raw muscle on the textural quality of the cooked meat from different sized Eriocheir sinensis. The content of entrapped water was 73.8–77.7 g/100 g in raw muscle. The density and diameter of muscle thick microfilaments ranged between 137 and 158/μm2 and 20.9–27.0 nm. These results demonstrated that the raw muscle from the tender group had a smaller density of larger diameter thick microfilaments and more entrapped water than other samples, which could explain the high tenderness of the cooked meat (P < 0.05). The potential structural proteins that indicated tenderness include myosin regulatory light chain 2, ancient ubiquitous protein 1, tubulin α-2 chain, and β-catenin, was determined using liquid chromatography-tandem mass spectrometry. The inherent attributes of raw muscle could explain the textural differences of the cooked meat from Eriocheir sinensis.

Ultrastructural study on the foot and the shell of the oyster Pinctada radiata (leach, 1814),(Bivalvia Petridae).

The shape and microscopic structure of the foot of the oyster were described by light and electron microscopy. The foot is wedge-shaped with vertical furrows on the surface; it is composed of an epithelial layer, a connective tissue layer and a muscular layer. The epithelial layer was composed mostly of ciliated columnar epithelia and secretory cells. The cilia and microvilli were observed on the free surface of epithelial cells. The muscle fibers were composed of thin and thick microfilaments. Thick and thin filaments in muscle cells were reported in the foot as well as lateral projections of the cytoplasm contained granules. The outer surface of the periostracum of the shell is reported to be microridge and microfringe were present as well. The simplicity of periostracum of the shell layers was observed as thin simple layer whereas the prismatic layer was the opposite. Wedge-shaped and elongated prisms were reported near the outer surface. Fine lamellae were representing the nacreous layer and were divided by laminar inclusion. Micro-fringes were considered to be helping the direction of bivalve

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae)

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.

Micromorphology and Ultrastructure of the Foot of the Equilateral Venus &amp;lt;i&amp;gt;Gomphina veneriformis&amp;lt;/i&amp;gt; (Bivalvia: Veneridae)

The shape and microscopic structure of the foot of the equilateral Venus, Gomphina veneriformis are described by light and electron microscopy along with the substrate conditions of their habitat. The habitat sediment of G. veneriformis is composed of sand (2 - 0.063 mm in diameter), mainly. The foot is wedge-shaped with multiple vertical furrows on the surface. Although the foot is composed of an epithelial layer, a connective tissue layer and a muscular layer, the boundary between the connective tissue and muscular layer is not clear. The epithelial layer was composed mostly of ciliated columnar epithelia and secretory cells. Epithelial cells forming the apical region of the fold were long columnar, while cells of the interfold were mostly short columnar. The cilia and microvilli were commonly observed on the free surface of epithelial cells, while tight junctions of apico-lateral aspect and membrane interdigitations were found between the epithelial cells. Secretory cells were found to contain acidic mucopolysaccharide, and were classified into two types in accordance with the shapes and ultrastructures of secretory granules. The muscle fibers were composed of thin and thick microfilaments, the proportions of which were 81.3% and 18.7%, respectively. It was determined that such morphology and structural characteristics of the foot of G. veneriformis would present advantageous conditions for borrowing into substrate and mobility.

Alterations of Spindle and Microfilament Assembly in Aged Cat Oocytes

To obtain insights into the cytoplasmic maturation status of cat oocytes recovered from cat ovaries following hormone treatment, we first examined microtubule and microfilament assembly in cat oocytes recovered from hormone-treated ovaries at various stages of maturation. Additionally, we determined the alteration of spindle and microfilament assembly, as well as mitogen-activated protein kinase (MAPK) activity, in cat oocytes at 0, 6, 12 and 18 h of further maturation in vitro. We then looked at pronuclear formation and cleavage of these oocytes following parthenogenetic activation. Similar to other species, microtubules are present in germinal vesicle (GV) stage cat oocytes, and following GV breakdown, microtubules encompassed condensed chromatin particles to form the meiotic metaphase spindle. Microfilaments were located in the cortex and around the GV. A microfilament-rich area, in which the chromatin is located, was observed in the oocytes during meiotic maturation. Maturation rates in aged oocytes (cultured for 18 h) were increased when compared with that in relatively fresh oocytes (<12 h culture), and the number of oocytes with abnormal spindle shapes was also increased in aged oocytes. Furthermore, in aged oocytes, the incidence of the metaphase plate observed outside the thick microfilament domain was higher compared with that of young oocytes, and this seemed to result in an increase in the number of oocytes with two pronuclei and one polar body following activation. Western blot analysis revealed a decrease in MAPK activity in aged cat oocytes. Taken collectively, these results suggest that the optimum time for improved cytoplasmic maturation is <12 h in cat oocytes recovered from hormone-treated ovaries.

MICROSCOPIC ANATOMY OF FOOT OF THE SPINY TOP SHELL, BATILLUS CORNUTUS (LIGHTFOOT, 1786) (GASTROPODA: TURBINIDAE)

Foot structures of the spiny top shell, Batillus cornutus were investigated by light and transmission electron microscopy. The foot was composed of epithelial layer, connective tissue layer, and muscular layer from outside inward. The epithelial layer was multiple-folded and simple and was composed mostly of columnar and mucous cells. Epithelial layer thickness was approximately 40 μm in the pedal body and approximately 30 μm in the opercular zone. Distribution of mucous cells in the epithelial layer was found to be higher in the opercular zone than those in the pedal body. Mucous cells contained acidic material abundant in sulfate and carboxylate group, from the results of AF-AB reaction. From TEM observation, the epithelial layer is composed of epithelial cell, ciliated cell, absorptive cell, and secretory cell. Secretory cells can be classified into six types (A, B, C, D, E, F) in accordance with the shapes and ultrastructural characteristics of secretory granule. Type A secretory cell is the most abundant cell of the six types of secretory cells. The type of muscle fibers was mostly smooth muscle fibers. Muscle fibers can be divided into two types, one that is composed only of thin microfilaments and the other composed of thin and thick microfilaments.

299 LEPTIN ACCELERATES PRONUCLEAR FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION OF PORCINE OOCYTES: POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE INACTIVATION

Although evidence suggests that leptin modulates oocyte maturation in vitro, few studies have examined the direct effect of leptin on cytoplasmic maturation and pronuclear formation following fertilization of porcine oocytes.The aim of this studywas to determine microtubule and microfilament assembly following oocyte maturation, and to assess blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) of leptin-treated oocytes.A symmetrical shape of the meiotic spindle was considered to be the normal microtubule assembly, and a clear, thick microfilament area containing a second metaphase chromatin was considered to be morphologically normal. Significant differences were determined using Tukey's multiple range test with P &amp;lt; 0.05 being considered significant. Addition of 10 ng mL-1 of leptin to the in vitro maturation (IVM) medium significantly increased the percentage of zygotes that developed to the blastocyst stage (26.1 � 2.1%; P &amp;lt; 0.05) compared with that of the control (0 ng mL-1: 16.3 � 2.8%) or 1 ng mL-1 (18.6 � 2.6%) leptin treatment groups following parthenogenetic activation. After IVM for 44 h, the percentage of oocytes with normal microtubules was higher in the leptin (10 ng mL-1) group than in the control group (89.1 � 4.3% vs. 78.5 � 4.0%, respectively; P &amp;lt; 0.05). However, the percentage of normal microfilament assembly was similar in the leptin-treated and control groups (85.6 � 4.3% and 82.9 � 5.0%, respectively). In oocytes matured in vitro in the presence of 10 ng mL-1 of leptin and subsequently induced by parthenogenetic activation via chemical stimulation, there was a significant increase in the formation of 2 pronuclei (2PN: 52.1 � 5.2%; P &amp;lt; 0.05) at 6 h, compared with the control non-leptin-treated oocytes (40.7 � 4.0%). Similarly, addition of 10 ng mL-1 of leptin to the IVM medium also increased 2PN formation at the same time point following ICSI (leptin treatment: 47.9 � 4.0; control: 36.2 � 3.8%; P &amp;lt; 0.05). The addition of 10 ng mL-1 of leptin to the IVM medium decreased the level of phospho-ERK1/2 at 6 and 9 h and was lower in the leptin-treated group compared with the control non-leptin-treated group (P &amp;lt; 0.01). These results suggest that exogenous leptin enhances cytoplasmic maturation and pronuclear formation following fertilization, possibly via the MAPK pathway.

Development of cellular polarity of hamster embryos during compaction.

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.

Development of cellular polarity of hamster embryos during compaction

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.

Cytoskeletal alteration in aged porcine oocytes and parthenogenesis.

The objective of this study was to examine the changes in microtubule and microfilament assembly in aged porcine oocytes and to determine their developmental pattern after parthenogenetic activation. Porcine oocytes were cultured in Whitten's medium containing 10% follicular fluid with hormonal supplements (eCG and hCG) for 22 hr and 40 hr additional culture without hormonal supplements. At 40, 50, and 60 hr of culture, the oocytes were fixed for immunocytochemistry or activated by electrical pulse. In metaphase II stage oocytes, microtubules were detected only in the meiotic spindle. Two microfilament domains existed in the egg cortex, a thick and a thin microfilament domain. In aged oocytes (50 and 60 hr of culture), the incidence of metaphase II plates observed outside of the thick microfilament domain was higher (P < 0.05) than in young oocytes (40 hr of culture). After activation, a polar body was usually emitted from the chromatin at the microfilament rich domain or two pronuclei were formed outside of the microfilament rich domain. The percentage of activated oocytes with one female pronucleus was higher (P < 0.05) in oocytes at 40 hr of maturation than at 50 and 60 hr of culture. At 24 and 30 hr after stimulation the incidence of cleavage to the 3- to 4-cell stage was higher (P < 0.05) in aged oocytes (50 and 60 hr of maturation) than that in oocytes at 40 hr of culture. These results suggested that a role of microfilaments is to retain the chromatin at the proper position in the oocyte cortex, and that aging results in a disruption of the microfilaments such that atypical development results after parthenogenetic activation.

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro-tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley-Liss, Inc.

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both microtubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

The Cytological and Immunohistochemical Study of Septal Cells in the Rat Lung.

Septal cells in the alveolar interstitium of the lung have been assumed to be contractile and involved in the regulation of ventilation/perfusion ratio. We studied two cytoskeletal components of the septal cell in the rat lung. One contains thick microfilament bundles: by decoration with heavy meromyosin, the bundle was shown to be composed of actin filaments of opposite polarity. The other contains intermediate filaments: by pre-and post-embedding immunogold electron microscopy, they were shown to contain both desmin and vimentin. Double labeling immunoelectron microscopy further revealed that some intermediate filaments are co-polymers of desmin and vimentin. The features revealed in the present study support the hypothesis that septal cells can contract and change the architecture of the air-blood barrier.

Effects of phalloidin and colchicine on diethylmaleate‐induced choleresis and ultrastructural appearance of rat hepatocytes

Diethylmaleate is used as a model compound whose glutathione conjugates are secreted into bile, and which induce choleresis and the formation of Golgi-derived vesicles in hepatocytes. This study was performed to test the assumption that these vesicles are involved in the bile canalicular secretion of diethylmaleate. We reasoned that phalloidin and colchicine, two drugs acting on microfilaments and microtubules, respectively, can modify the movements of diethylmaleate-induced vesicles towards the bile canaliculus. Phalloidin induced the formation of a thick microfilamentous network around the bile canalicular plasma membrane domain. A significant decrease in diethylmaleate-stimulated choleresis was observed, associated with a striking accumulation of pericanalicular vesicles, which were confirmed by morphometric analysis. In contrast, in rats pretreated with colchicine, after diethylmaleate administration, only a few vesicles were observed around the bile canaliculus, while diethylmaleate-induced choleresis also decreased. These results suggest that: a) the thick microfilament network induced by phalloidin prevents diethylmaleate-associated vesicles reaching the bile canalicular plasma membrane; and b) colchicine produces a dispersion of these vesicles in the cytoplasm of hepatocytes by inhibiting the polymerization of microtubules. These observations support a role of vesicles in the transport of diethylmaleate by hepatocyte into bile, and are consistent with the existence of a vesicular pathway for the biliary secretion of diethylmaleate and possibly other organic anions.

Cultured microvascular endothelial cells (MVEC) differ in cytoskeleton, expression of cadherins and fibronectin matrix. A study under the influence of interferon-gamma.

Endothelial cells are known to undergo transitions in cell shape during long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovine corpus luteum represent constitutively different cell strains cannot automatically be made. For this reason, particular morphological qualities from four of five reported MVEC types were studied. Confluent cultures of MVEC types 1, 3, 4 and 5 were either left untreated or exposed to recombinant bovine interferon-gamma (IFN-gamma; 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were permeabilized with Triton X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filaments, microtubules, E- and N-cadherins as molecules of cell adhesion plaques, and fibronectin filaments were localized by the application of specific antibodies in combination with epifluorescence microscopy. Cells from untreated single cultures uniformly and reproducibly showed an actin cytoskeleton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin filaments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominent circular band of thick microfilament bundles from which short filaments radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vimentin filaments occurred as a dense network constricted to the central area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent in MVEC type 5. Fibronectin assembled into two differently shaped layers at the basal cell side of each MVEC type. Under IFN-gamma treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cadherin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear contour. The upregulation of E-cadherin by IFN-gamma treatment could also be demonstrated by western blotting, which revealed a 135 kDa full-sized molecule and a 95 kDa tryptic fragment characteristic of cadherins. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.

Fine structure of macronuclei and micronuclei of the lower marine ciliate Trachelocerca obscura (Karyorelictida)

Trachelocerca obscura Raikov et Volkonitin (order Karyorelictida) is a large pigmented (dark brown) ciliate from the mesopsammal of the Japan sea. The nuclear apparatus of T. obscura is represented by a single loose group of 7–14 macronuclei (usually including two developing ones) and 2 micronuclei. The micronuclei are filled with condensed chromatin, the lacunae of which enclose curved laminae (chromosomal kinetochores persisting during interphase). These nuclei regularly contain also needle-shaped crystalloids with an oblique periodicity. The micronuclear envelope has practically no pores and is coated externally with a multi-layered capsule of flattened agranular cisternae. The macronuclear envelope, on the contrary, carries numerous pore complexes and lacks multimembranous capsule. Adult macronuclei have large peripheral chromocenters which sometimes fuse into a continuous layer of condensed chromatin. One or two aggregates of nucleoli, micronucleoli, and granular lamellae which are derivatives of the nucleolar cortex, occur nearer to the centre of the nucleus. Many macronuclei contain a microfibrillar sphere which contacts the nucleoli and is often coated with nucleolar granular lamellae. The sphere is resistant to pronase digestion. Some macronuclei also contain bundles of 5–6 nm thick microfilaments which contact the condensed chromatin. Developing macronuclear primordia are filled with heterochromatin bodies and lack nucleoli which first appear only in young macronuclei.

Patterns of endothelial microfilament distribution in the rabbit aorta in situ.

The available data on F-actin microfilament distribution in vascular endothelial cells in vivo is limited. In this study, the appearance and distribution of endothelial cell microfilaments in the rabbit thoracic aorta, the abdominal aorta and its major arterial branch points, and the aortic bifurcation were examined. Perfusion fixed rabbit aortas were stained in situ for F-actin by infusing rhodamine phalloidin via a peristaltic pump into the aortas at a slow flow rate. This new technique resulted in excellent visualization of branch points and allowed for a precise description of the actin microfilament bundles in endothelial cells along flow dividers. In the thoracic and abdominal aorta, away from branch ostia, actin microfilaments were localized in two regions of the endothelial cells, as a prominent band that completely outlined the cell periphery, and also as short central stress fibers. The central stress fibers were more frequent and prominent in cells of the abdominal aorta. At branch sites and at the aortic bifurcation, long, thick microfilament bundles were present in endothelial cells extending from the tip of the flow divider to a few millimeters along the branch arteries, the aorta, and the iliac arteries. Peripheral actin, however, no longer completely surrounded the cells. The thick bundles were not prominent in endothelial cells located adjacent to the proximal lip of branches or at the iliac arteries opposite the flow divider. This study shows that endothelial cell F-actin microfilament distribution in vivo is well defined along the aortic-arterial system. The prominent central microfilament bundles and the reduced peripheral microfilaments seen at localized regions may reflect an adaptive response to elevated shear stress at these sites.

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro.

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.

On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study.

In avian embryos the formation of ventrolateral abdominal muscles was studied by (1) heterospecific grafting experiments between chick and quail embryos and (2) ultrastructural examinations of cells having part in this process. The results demonstrate that the muscle cells are of somitic origin while the connective tissue derives from the somatopleure. Somatopleural cells do not differentiate into myocytes, and somite cells which have entered the ventrolateral abdominal wall, do not contribute to the connective tissue. It is concluded that both dermatome and myotome cells undergo muscular differentiation. The formation of muscles is found to take place in four characteristic steps. During the 4th day of development, epithelially structured ventral somite buds enter the somatopleure. The light cells of the inner myotome layer are elongated in a cranio-caudal direction and contain randomly distributed microfilaments. On the 5th day, the buds lose their epithelial arrangement and change into compact processes in which cells intermingle. The myotome cells show short bundles of thin and thick microfilaments. The third step can be characterized by the appearance of intercellular spaces and the disaggregation of processes becoming invaded by somatopleural cells. Thus, subdivision in single muscle blastemata begins to occur. In 7-day embryos, the muscle anlagen are distinctly separated and the first myotubes containing regularly arranged myofibrils are found. Coincidentally, signs of cell death are observed. Up to the 10th day, the tendons being of somatopleural origin become plainly outlined and the muscle anlagen move to their definitive positions. It is assumed that the formation of muscle pattern is controlled by the somatopleure.