Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodeling, and the localization of atherosclerotic lesions. Major regulation of the blood vessel responses occurs by the action of hemodynamic shear stresses on the endothelium. The transmission of hemodynamic forces throughout the endothelium and the mechanotransduction mechanisms that lead to biophysical, biochemical, and gene regulatory responses of endothelial cells to hemodynamic shear stresses are reviewed.

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Flow-mediated endothelial mechanotransduction

INTRODUCTION 487 MORPHOLOGY AND OBSERVATION OF FOCAL ADHESIONS 488 FORMATION OF FOCAL ADHESIONS 490 DYNAMICS OF FOCAL ADHESION COMPONENTS ...... 491

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Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton

ROCKs, or Rho kinases, are serine/threonine kinases that are involved in many aspects of cell motility, from smooth-muscle contraction to cell migration and neurite outgrowth. Recent experiments have defined new functions of ROCKs in cells, including centrosome positioning and cell-size regulation, which might contribute to various physiological and pathological states.

ROCKs: multifunctional kinases in cell behaviour

THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Actin-Based Cell Motility and Cell Locomotion

The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system

https://rupress.org/jcb/article-pdf/103/4/1557/1053568/1557.pdf

Microtubules and the endoplasmic reticulum are highly interdependent structures.

We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.

https://rupress.org/jcb/article-pdf/71/3/848/1072191/848.pdf

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.

Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells

The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.

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Keigi Fujiwara

Papers

Microtubules and the endoplasmic reticulum are highly interdependent structures.

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.

Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells

Factors influencing the expression of stress fibers in vascular endothelial cells in situ.

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin, and myosin