Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

The cortical actin cytoskeleton of unactivated zebrafish eggs: Spatial organization and distribution of filamentous actin,nonfilamentous actin,and myosin-II

Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization. © 1996 Wiley-Liss, Inc.     

Dynamic polymorphism of single actin molecules in the actin filament

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.     

Arrangement of cytoskeletal filaments at the equator of chicken intrafusal muscle fibers

Summary The organization of the cytoskeleton at the equator of chicken intrafusal fibers was examined with immunofluorescence light microscopy, using monoclonal antibodies against myosin heavy chains, desmin, actin and tropomyosin. Actin was localized in the cytosol and in equatorial nuclei, while myosin heavy chains, desmin and tropomyosin were only observed in the cytosol. Although all four proteins were present at the equator and at the pole, the fluorescence produced after incubation with the different antibodies varied considerably between the two regions. Staining at the pole was in the form of striations, but at the equator it was non-striated and more uniform. The observed fluorescent patterns suggest that at the equator filaments are assembled into looser arrays than in the sarcomeres of the pole. A flexible cytoskeleton at the equator would be an appropriate substrate for distorting the affixed sensory endings during an applied stress.     

Arrangement of cytoskeletal filaments at the equator of chicken intrafusal muscle fibers

The organization of the cytoskeleton at the equator of chicken intrafusal fibers was examined with immunofluorescence light microscopy, using monoclonal antibodies against myosin heavy chains, desmin, actin and tropomyosin. Actin was localized in the cytosol and in equatorial nuclei, while myosin heavy chains, desmin and tropomyosin were only observed in the cytosol. Although all four proteins were present at the equator and at the pole, the fluorescence produced after incubation with the different antibodies varied considerably between the two regions. Staining at the pole was in the form of striations, but at the equator it was non-striated and more uniform. The observed fluorescent patterns suggest that at the equator filaments are assembled into looser arrays than in the sarcomeres of the pole. A flexible cytoskeleton at the equator would be an appropriate substrate for distorting the affixed sensory endings during an applied stress.     

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.     

Localization of myosin and actin in ocular nonmuscle cells

Summary Myosin and actin were localized by indirect immunofluorescence microscopy using specific antibodies prepared in rabbits against highly purified gizzard myosin and actin. A strong fluorescence staining with both antibodies was observed in rat corneal epithelial cells, anterior lens epithelial cells, rod inner segments, and in rat and frog pigment epithelial cells. The immunohistochemical localization of myosin in corneal epithelial cells was further supported by the electrophoretic and immunological identification of smooth muscle type myosin heavy chain in pure corneal epithelial abrasions. Electron-microscopic observations revealed a clear correlation between staining with actin antibodies and the presence of numerous thin cytoplasmic filaments (50–80 Å in diameter). The functional and biochemical nature of 90–110 Å filaments occurring in corneal and lens epithelial cells, as well as the ultrastructural localization of myosin in ocular nonmuscle cells under study remains obscure.     

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

Immunogold localization of basement membrane molecules in rat retinal capillaries

Vascular basement membrane contains laminin, fibronectin, proteoglycan and collagens. These molecules have been identified in various tissues by immunolabeling methods and biochemical analyses. We have previously localized laminin, fibronectin and type IV collagen to the basement membrane of rat retinal vessels at the ultrastructural level using an immunoperoxidase method. In this study, we use an immunogold method to re-examine the distribution of these molecules and also to study the localization of heparan sulfate proteoglycan and types I, III and V collagen in the retinal capillary basement membrane. Gold labeling for laminin, type IV collagen and proteoglycan were found diffusely on the basement membrane of the endothelium and pericyte, while that for fibronectin and type V collagen was spotty and variable and that for types I and III collagen was negligible. The segment of basement membrane between the endothelial cell and pericyte appeared less reactive to anti-laminin and anti-type IV collagen than the membrane between the pericyte and perivascular neuroretina. The immunogold method may be useful in quantitative studies of thickened basement membranes under abnormal conditions.     

Comparative maps of motion and assembly of filamentous actin and myosin II in migrating cells

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments.     

Studies on the organization and localization of actin and myosin in neurons

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.     

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

眼科常用实验动物视网膜血管的比较

目的比较眼科常用实验动物视网膜血管尤其是视网膜毛细血管的情况,为实验时正确选择动物模型提供基础。方法取猕猴、家猪、新西兰大白兔、犬、猫、SD大鼠、C57小鼠以及豚鼠的正常眼球数个,完整剥离整个视网膜,用ADPase法进行血管染色,对视网膜血管进行形态学的比较。结果猕猴视网膜大血管从视盘穿出,分成四支分别供应视网膜四个象限,每条血管逐级分支最后成为毛细血管,其毛细血管呈网状分布,在赤道处分成两层,至周边变成一层,且有发育良好的黄斑区毛细血管拱环结构。家猪视网膜大血管由视盘发出后放射状走行,毛细血管也呈网状分布,无黄斑拱环结构。兔仅视盘两侧部分视网膜可见血管,毛细血管网状不明显。犬的视网膜血管也放射状走行,但迂曲明显,毛细血管不成网状。猫、大鼠、小鼠的视网膜大血管均由视盘发出,猫的分成上、鼻下、颞下三支,大鼠、小鼠的各方向均有,区域性不明显,三者的毛细血管网均发育良好,至周边部仍很密集,呈两层分布。豚鼠视网膜无可见的血管。结论用于研究人视网膜血管尤其是毛细血管时,可选用猕猴、家猪、猫、大鼠和小鼠作为动物模型;但要研究人黄斑区血管时,仅可选用猕猴等灵长类动物。     

The morphological basis of proestrus endometrial bleeding in canines

Endometrial bleeding during proestrus is a well-known phenomenon in the bitch. However, the exact events on the cellular level have not been studied. In the present investigation, immunohistochemical methods and transmission electron microscopy were employed to obtain more information about this cyclic event in canines. Long, stretched blood vessels were seen in H&E stained sections during proestrus. These vessels showed mitotic activity, as evidenced by Ki67 immunostaining. Although the endothelial lining and basement membrane of endometrial blood vessels seemed continuous, as indicated by immunohistochemical staining for laminin and Von Willebrand factor, transmission electron microscopy showed an extreme thinning and even interruption of the vascular wall in endometrial venules. Platelets were frequently seen in those areas, and also detected by immunohistochemistry. Interestingly, all endometrial capillaries examined by electron microscopy had an intact wall. We therefore postulate the endometrial venules to be the blood vessels that are mainly responsible for proestrus endometrial bleeding, rather than subepithelial capillaries.     

Flexibility in actin-myosin motility system revealed by in vitro motility assay

Flexibility of myosin molecule was studied by in vitro motility assay in terms of the direction of actin movement. Electron microscopy showed that HMM scattered on a nitrocellulose surface can bind actin filaments and form arrowhead-like patterns. Actin filaments can move in both directions on tracks of HMM made on a nitrocellulose surface. Further, actin filaments can move bidirectionally along native thick filaments over their central bare zone. These observations indicate that there is considerable flexibility in a myosin molecule and that the direction of the movement is determined by the polarity of actin filaments.     

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.     

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1.

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (S1). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.     

Correlative immuno-electron-microscopic and immunofluorescent localization of actin in sensory and supporting cells of the inner ear by use of a low-temperature embedding resin

Summary The cochleas from chinchilla inner ears were processed in the cold through Lowicryl K4M, and cured by UV light. Thick (2 m) sections were reacted with primary antibodies raised against actin, and anti-actin antibodies localized by FITC epifluorescence. On thin sections from the same blocks anti-actin antibodies were localized ultrastructurally with secondary antibodies coupled to colloidal gold.In the hair cells, actin was present in the stereocilia and cuticular plate, regions where thin filaments were observed by electron microscopy. Colloidal gold was uniformly distributed over these regions and over the stereocilia rootlets demonstrating that actin was present in this region although previously in permeabilized cells, the rootlet was not decorated with myosin subfragment S-1. Actin was present in the pillar and Deiters supporting cells at the reticular lamina and at the basilar membrane, where a meshwork of thin filaments was seen by electron microscopy. Colloidal gold particles were also localized over the thin processes of the pillar and Deiters cells, and over the region of the Deiters cell which envelops the base of the outer hair cell. In these regions actin co-localized with microtubules along the entire length of the supporting cells.     

Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction.

By decreasing ionic strength slowly, thick filaments of several micrometers in length were obtained from purified rabbit skeletal muscle myosin. Dark-field observation showed these filaments with their center scattering light extensively. Active movement of actin filaments complexed with tetramethyl rhodamine-phalloidin along the reconstituted myosin filaments was observed. Actin filaments moved towards the center of myosin filaments at a speed of 3.9 +/- 1.6 microns s-1 (mean +/- SD, n = 40) and often continued to move beyond the center towards the tip of the opposite side at a lower speed. The speed of the movement away from the center was 1.0 +/- 0.6 microns s-1 (n = 59). Thus, the functional bipolarity in terms of the movement speed which was first found in native thick filaments of molluscan smooth muscle is also seen in reconstituted filaments from purified rabbit skeletal muscle myosin. The difference of the speed between the two directions is considered to be due to properties of myosin molecules themselves.