Phenotype modulation in primary cultures of arterial smooth-muscle cells. Dual effect of prostaglandin E1

The effects of prostaglandin E1 (PGE1) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0-2), PGE1 stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3-6), when the majority of the cells had entered synthetic state, PGE1 inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Caveolae and cholesterol distribution in vascular smooth muscle cells of different phenotypes.

Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ER-Golgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipin-sterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or beta-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipin-sterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs.     

Ultrastructure and differentiation of the larval esophageal muscle cells of the starfish Pisaster ochraceus

The muscle cells that cause constriction of the starfish larval esophagus (esophageal muscle cells) are one of the first cell types to express their differentiated morphological characteristics during development. Ultrastructurally these muscle cells resemble vertebrate and invertebrate smooth muscles. They contain a nucleus, a Golgi apparatus, contractile myofilaments, hemidesmosome-like structures, and what appears to be a simple sarcoplasmic reticulum. In asteroid embryos, this muscle layer originates during mouth formation when mesenchyme cells migrate from the tips of the coeloms to the esophagus. Once there, they elongate, forming processes. Over the next few days, the processes become filled with arrays of longitudinally arranged thick and thin myofilaments and thin sacs of smooth endoplasmic reticulum. The latter appear between the bundles of contractile filaments and the cell membranes. Contractile activity begins at approximately this time. The cisternae may represent a sarcoplasmic reticulum that is required for contraction. The majority of the esophageal muscle cell processes extend around the circumference of the developing esophagus, but occasional cells may be oriented in other directions. The latter cells are always farther away from the basal lamina and probably have little or no contact with it. Contact with basal lamina may serve to direct the migration of the cells and the orientation of the processes. J. Morphol. 237:1–18, 1998. © 1998 Wiley-Liss, Inc.     

Application of the optical Fourier transform for analysis of the spatial distribution of collagen fibers in normal and osteopetrotic bone tissue

Summary Optical Fourier analysis was applied for evaluation of the differences between normal and pathologically changed bone tissue. Collagen fibers were used as markers of bone structure. To prove the usefulness of this technique for objective mathematical analysis of these differences the spatial distribution of collagen fiber bundles was evaluated in normal and osteopetrotic bone. The variation in the spatial distribution of collagen fiber bundles in cross sections of femur diaphyses was evaluated quantitatively by optical diffraction in three groups of Fatty Orl-op strain rats, i.e. phenotypically normal animals, osteopetrotic (op/op) mutants and op/op-mutants cured by transplantation of normal syngenic bone marrow. The histological sections of decalcified bone were stained with Sirus-Red and then photographed under polarizing microscope. The Sirus-Red staining was used to enhance the natural birefringency of collagen fibers. Diffractograms obtained from microphotographs of selected bone section areas, i.e. outer and inner circumferential lamellae and haversian bone of normal and cured op/op animals as well as whole cortical bone and woven bone filling the medullary cavities in op/op mutants were analysed separately. Diffractograms contain summarized information on the size and relative position of histological structures. The radial distribution of light energy informs on the size and/or distances between the structures, while angular distribution gives the relative position of these structures in histological sections. The radial and angular distribution of light energy were evaluated for each diffractogram with an electronic detector. The obtained distributions were described by several sets of parameters concerning the position, level and shape of local maxima and minima. Out of these parameters five with the highest discriminant power were chosen for further mathematical analysis. This analysis was based on the calculation of the position of centroids in the multidimensional space described by the linear functions of the chosen parameters for each of the evaluated bone section areas. The centroids (mean values of discriminant scores of each group) represent the centers of gravity of the analysed groups, while the separation of the centroids tested by the F-test illustrates the differences between the respective groups of selected bone section areas. A high level of separation of centroids was found when osteopetrotic bone was compared with normal one, what means that the spatial distribution, size and interstructural distances between the collagen fiber bundles in bone tissue in these two groups of animals differ markedly. A similar situation was observed when osteopetrotic bone was compared with bone tissue obtained from op/op mutants cured by transplantation of normal syngenic bone marrow. On the other hand, the level of separation of centroids was low when bone tissue of cured op/op mutants was compared with the control one, a finding which corresponds to the less pronounced histological differences between these two groups of animals. Computer-aided classification on single microphotographs of selected bone section areas to the known a priori type of bone tissue was performed. The percentage of cases correctly classified to one of the eight groups of selected bone section areas equals 47. The probability of reaching such a result by chance was estimated as less than 10^–4. The percentage of cases classified correctly to one of any two statistically different groups was higher than 90. These observations demonstrate that optical diffractometry allows to describe in numerical terms the differences between normal and pathologically changed bone tissue, and therefore might be used for automatic evaluation of histopathological sections and interlaboratory comparative studies.     

Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique

Actin-immunoreactive sites have been localized at the electron microscope level by the protein A-gold technique in striated and smooth muscle cells as well as in epithelial and secretory cells. The combination of the highly sensitive protein A-gold technique with the good ultrastructural preservation and retention of antigenicity obtained using low-temperature embedding conditions has allowed a very precise identification of the labelled structures with high resolution. In striated muscle cells the labelling was obtained over the myofilaments and the Z-band, mainly at its periphery. Labelling was also observed at the edge of the intercalated discs of the cardiac muscle cells. In smooth muscle cells the labelling was present over the myofilaments; the dense plaques associated with the plasma membrane were labelled at their periphery where actin filaments have been reported to anchor. In epithelial cells of the duodenum and the renal convoluted proximal tubule, the labelling occurred over the filamentous core of the microvilli and over the cell web. Gold particles were often present over, or closely associated with, the cell membrane at the tip of the microvilli or of invaginations and vesicular structures. At the level of the junctional complexes the gold particles were aligned at the edge of the dense zones. In pancreatic endocrine and exocrine secretory cells, actin-immunoreactive sites were revealed over the Golgi apparatus, mainly at the level of the inner cisternae in the maturing face over or closely associated with the membranes of the condensing vacuoles and secretory granules, and also over the plasma membrane. Microvilli and cell web were also labelled. Finally, in fibroblasts, gold particles were associated with the membrane of vesicular structures. The consistent finding of actin-immunoreactive sites closely associated with membranes of secretory granules and vesicular structures brings support to the proposal that contractile proteins might play an important role in transcellular transport and protein secretion.     

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.     

Organelle distribution in chick neuroepithelial cells: effects of colchicine and cytochalasin B

The intracellular distribution of mitochondria, cytoplasmic inclusions and rough endoplasmic reticulum cisternae of chick neuroepithelial cells was investigated at neurulation stages 6, 8, 10 and 12. These neuroepithelial cells were subdivided into three zones: apical, median and basal and the distribution percentages of distribution of these organelles were obtained. Mitochondrial distribution was related to the energy supply that mitochondria provide for apical microfilament contraction. Cytoplasmic inclusions were distributed preferentially in the apical zone of the neuroepithelial cells during the four stages. Rough endoplasmic reticulum cisternae were homogeneously distributed in the three zones at stages 10 and 12, but at stages 6 and 8 there are more elevated percentages of rough endoplasmic reticulum in the apical zones than in the other zones. Experimental treatments with colchicine and cytochalasin B does not modify the patterns of mitochondria and rough endoplasmic reticulum cisternae but alters the distribution of cytoplasmic inclusions. Finally, there is a correlation in the normal neurulating neuroepithelial cells between the distributions of mitochondria and rough endoplasmic reticulum distribution and between the distributions of mitochondria and cytoplasmic inclusions distribution. This relationship is retained in the treated neuroepithelial cells.     

Phenotype modulation in primary cultures of arterial smooth-muscle cells

Abstract. The effects of prostaglandin E₁ (PGE₁) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0–2), PGE₁, stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3–6), when the majority of the cells had entered synthetic state, PGE₁ inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Tubular-vesicular transformation in the contractile vacuole system of Dictyostelium

The contractile vacuole complex of Dictyostelium is the paradigm of a membrane system that undergoes tubular-vesicular transitions during its regular cycle of activities. This system acts as an osmoregulatory organelle in freshwater amoebae and protozoa. It collects fluid in a network of tubules and cisternae, and pumps it out of the cell through transient pores in the plasma membrane. Tubules and vacuoles are interconvertible. The tubular channels are associated with the cortical actin network and are capable of moving and fusing. The contractile vacuole complex is separate from vesicles of the endosomal pathway and preserves its identity in a dispersed state during cell division. We outline techniques to visualize the contractile vacuole system by electron and light microscopy. Emphasis is placed on GFP-fusion proteins that allow visualization of the dynamics of the contractile vacuole network in living cells. Proteins that control activities of this specialized organelle in Dictyostelium have been conserved during evolution and also regulate membrane trafficking in man.     

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. Instead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II-III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharose 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serum-containing medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myotube-like structures in cultures of rat liver cells

Branching and budding myotube-like structures developed in primary cultures of rat liver cells and in the JH1 cell line derived from them. Elongated uninuclear cells aligned in chains and fused into multinuclear tubes of varying length and thickness. The tubes contained thick and thin filaments running in all directions. The filaments were occasionally linked with M lines and formed incomplete hexagonal patterns resembling those of skeletal muscle myofilaments, but a regular arrangement of the filaments and organelles was lacking. Cross-striation and contractions were never observed. Both uninuclear cells and multinuclear tubes contained numerous lysosomes, myelin figures and lamellated bodies as well as electronlucent or content-filled vacuoles and cisternae of variable size, sometimes reminding the sarcoplasmic reticulum in early stages of its development. Endocytotic caveolae and vesicles were present in all elements. These features together with interdigitated cell processes and specialized cell contacts suggested a possible relationship of the cells to the reticuloendothelial system.     

Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens

Smooth muscle cells from the aortic media of adult pigs and monkeys have been grown in primary culture by plating cells enzymatically dissociated from the intact aorta. During the first 6 d these cells are in the "contractile" phenotype. That is, they contract slowly in response to angiotensin II and their cytoplasm is filled with both thick and thin myofilaments. In this state they do not incorporate [3H]thymidine into DNA or proliferate in response to normolipemic or hyperlipemic whole blood serum (WBS). After 7 d in culture the cells undergo a spontaneous modulation of phenotype to a "synthetic" state where they cannot be stimulated to contract and their cytoplasm is filled with organelles usually associated with synthesis of secretory protein. Thick myosin-containing filaments can no longer be demonstrated. When challenged with normolipemic or hyperlipemic WBS the cells incorporate [3H]thymidine into DNA and undergo logarithmic growth. It is suggested that when smooth muscle is the contractile phenotype (as normally exists for most cells in the aortic media of adult animals) it does not divide when challenged with serum mitogens but can undergo a change of phenotype to a synthetic state in which division can be stimulated.     

Effects of nicotine on phenotypic modulation and initiation of DNA synthesis in cultured arterial smooth muscle cells

During the early stages of atherogenesis, as well as during in vitro cultivation, smooth muscle cells modulate from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex; it leads to decreased contractility and the commencement of cell growth and secretion of extracellular matrix components. In this paper, the effects of nicotine on adult rat arterial smooth muscle cells cultivated in vitro were studied by transmission electron microscopy and 3H-thymidine autoradiography. The results show that the drug speeded the initial rate of transition of the cells from contractile to synthetic phenotype in primary culture. Further, it stimulated the initiation of DNA synthesis in growth-arrested secondary cultures. Its effect was independent of other mitogens and additive to that of serum. The influences of nicotine, both on the modulation of the smooth muscle phenotype and the initiation of DNA synthesis, occurred at concentrations lower than those obtained in the blood after smoking and could contribute to the role of smoking as a risk factor for atherosclerosis.     

Three dimensional visualization of the Golgi apparatus and endoplasmic reticulum in the subcommissural organ cells with high voltage electron microscopy

Fine structure and stereo-images of the Golgi apparatus and endoplasmic reticulum (ER) in the subcommissural organ (SCO) cells were visualized by the application of zinc-iodide osmium tetroxide (ZIO) impregnation, conventional electron microscopy and high voltage electron microscopy (HVEM). The Golgi apparatus in the SCO cells of rats, gerbils and hamsters consisted of flattened saccules stacked in parallel array. It showed a selective staining toward ZIO mixture and might form a complex network of tubular structures because of the presence of numerous fenestrations in the flattened Golgi saccules. The cytoplasm of the SCO cells in the rat and gerbil was crowded by dilated cisternae of the ER with a few flattened profiles. In the hamster SCO cells, however, the dilated cisternae of the ER were not observed. Flattened cisternae of ER in all species studied showed a positivity for ZIO impregnation and formed a complex tubular network, whereas dilated cisternae of the ER in the rats and gerbils did not show any reactivity. It was thus determined that the observation of thin and thick sections selectively stained with appropriate reagent for defined cellular organelles under conventional electron microscopy and HVEM offered valuable information about three-dimensional organization of the cell. A definite species-specific variation of SCO ultrastructure and cytochemistry was also demonstrated.     

The fine structure of differentiating muscle in the salamander tail

Summary Thin methacrylate sections of developing tails of Amblystoma opacum larvae were examined in the electron microscope and a series of stages in the differentiation of the myotome musculature was reconstructed from electron micrographs and earlier light microscopic studies of living muscle. The earliest muscle cell precursor that can be clearly identified is a round or oval cell with abundant cytoplasm containing scattered myofilaments and free ribonucleoprotein granules, but little endoplasmic reticulum. These cells sometimes form a syncytium and they may also be fused with adjacent formed muscle fibers by lateral processes. Nuclei are large and nucleoli are prominent. This cell, called a myoblast here, is distinctly different in its appearance from the adjacent mesenchymal cells which have abundant granular endoplasmic reticulum. The earliest myofilaments are of both the thick and thin varieties and are distributed in a disorganized fashion in the cytoplasm. These filaments are similar to the actin and myosin filaments described by Huxley and they are present in the cytoplasm at an earlier stage of differentiation than heretofore suspected from light microscopy studies. The first myofibrils are a heterogeneous combination of thick and thin filaments and dense Z bands and are not homogeneous as so many light microscopists have contended. As development progresses, cross striations become more orderly and definitive sarcomeres are formed. Thereafter, new myofilaments and Z bands seem to be added to the lateral surfaces and distal ends of existing myofibrils.Free ribonucleoprotein granules are a prominent part of the myoblast cytoplasm and are found in close association with the differentiating myofilaments in all stages of development. In early muscle fibers and some of the formed fibers, similar granules are often concentrated in the I bands. A theory of myofilament differentiation based on current concepts of the role of ribonucleoprotein in protein synthesis is presented in the discussion. Stages in myofibril formation and possible relationships of the filaments in developing muscle cells to other types of cytoplasmic filaments are also discussed.Supported by grant C-5196 from the United States Public Health Service.     

THE NORMAL FINE STRUCTURE OF OPOSSUM TESTICULAR INTERSTITIAL CELLS

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.     

A freeze-fracture study of muscle fibres infected with Trichinella spiralis

The muscle fibres of mice containing the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The larva lies in what appears to be a fluid-filled cavity within the cytoplasm of an altered muscle fibre. There is no membrane separating the cytoplasm of the nurse cell from the cavity surrounding the larva which is therefore truly intracellular, unlike many parasites that reside within a membrane-lined parasitophorous vacuole within the host cell. This altered muscle fibre, known as a nurse cell, lacks myofilaments but does contain extensive cisternae of endoplasmic reticulum; membrane-bound vesicles are budded off from the endoplasmic reticulum and traverse the cytoplasm towards the cavity containing the nematode where they apparently pass into the cavity. It is suggested that the contents of these vesicles are used to sustain the nematode. Attention is drawn to the similarity to giant cells that have been induced by the plant-parasitic nematode Meloidogyne in the roots of host plants and which sustain the nematode. The conversion of the muscle fibre into a nurse cell is probably brought about by the presence of a metabolic sink, the larval nematode, within the cell. This take-over of the control of a metazoan cell by another metazoan organism is most unusual and warrants further study.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.