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1.
The transverse stiffness of glycerinated and demembranated fibers from the soleus muscle of Wistar rats in different functional states was measured by atomic force microscopy. It was demonstrated that the transverse stiffness of relaxed fibers near the Z disk is approximately twofold higher as compared with the M-line region. However, the stiffness of glycerinated fibers in the Z-disk and M-line regions is considerably lower than that of demembranated fibers. The values of mechanical parameters of activated fibers are significantly higher as compared with the relaxed fibers. However, the stiffness of activated glycerinated fibers near the Z disk approximately doubled as compared with the relaxed state, whereas the stiffness of the Z-disk region in demembranated fibers increased more than fourfold. The stiffness of both glycerinated and demembranized fibers near the M-line increased approximately threefold. 相似文献
2.
以体外培养的大鼠关节炎模型的滑膜细胞为研究对象,用蛋白激酶C(PKC)激活剂佛波酯(phorbol 12-myristate 13-acetate, PMA)刺激细胞模拟炎症过程,应用原子力显微成像技术研究了大鼠滑膜细胞在PKC活化后细胞骨架变化情况．结果表明,利用原子力显微镜成像可以较好地表征PMA刺激引起滑膜细胞骨架的收缩隆起及细胞表面质地的变化,并揭示了PKC活化可能是引起滑膜细胞骨架改变的重要原因．这些结果为加深了解类风湿关节炎的发生机制提供了实验基础． 相似文献
3.
A. McPherson A. J. Malkin Yu. G. Kuznetsov M. Plomp 《Acta Crystallographica. Section D, Structural Biology》2001,57(8):1053-1060
Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non‐perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystal's diffraction pattern. 相似文献
4.
A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact
cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described.
The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin,
which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method
is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations.
Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and
the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the
spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes.
The changes are quantitatively described using the pixel height histogram and window size grain analysis. 相似文献
5.
The strength and nature of interactions between monomeric gliadin proteins involving alpha-alpha, omega-omega, and alpha-omega interactions in 0.01M acetic acid, and the effect of urea has been investigated. It was shown by means of nanomechanical force measurements that the stretching events in the separation curve after adhesive phenomena originated from proteins. These stretching events displayed different responses of the alpha- and omega-gliadins to urea. While 2M urea caused the more globular alpha-gliadins to unfold, the beta-turn-rich omega-gliadins remained fairly stable even in 8M urea. This suggests different roles for gliadins in the formation of dough; while the omega-gliadins are still in a compact structure being responsible for the viscous flow, the alpha-gliadins have already started to participate in forming the network in dough. 相似文献
6.
Regulation of melanosome movement in the cell cycle by reversible association with myosin V. 总被引：6，自引：0，他引：6
S L Rogers R L Karcher J T Roland A A Minin W Steffen V I Gelfand 《The Journal of cell biology》1999,146(6):1265-1276
Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis. 相似文献
7.
Eri Akita Yaxiaer Yalikun Kazunori Okano Yuki Yamasaki Misato Ohtani Yo Tanaka Taku Demura Yoichiroh Hosokawa 《Plant Biotechnology》2020,37(4):417
Atomic force microscopy (AFM) can measure the mechanical properties of plant tissue at the cellular level, but for in situ observations, the sample must be held in place on a rigid support and it is difficult to obtain accurate data for living plants without inhibiting their growth. To investigate the dynamics of root cell stiffness during seedling growth, we circumvented these problems by using an array of glass micropillars as a support to hold an Arabidopsis thaliana root for AFM measurements without inhibiting root growth. The root elongated in the gaps between the pillars and was supported by the pillars. The AFM cantilever could contact the root for repeated measurements over the course of root growth. The elasticity of the root epidermal cells was used as an index of the stiffness. By contrast, we were not able to reliably observe roots on a smooth glass substrate because it was difficult to retain contact between the root and the cantilever without the support of the pillars. Using adhesive to fix the root on the smooth glass plane overcame this issue, but prevented root growth. The glass micropillar support allowed reproducible measurement of the spatial and temporal changes in root cell elasticity, making it possible to perform detailed AFM observations of the dynamics of root cell stiffness. 相似文献
8.
Previous findings indicate that spatial restriction of intracellular calcium levels within growth cones can regulate growth cone behavior at many levels, ranging from filopodial disposition to neurite extension. By combining techniques for focal stimulation of growth cones with those for measurement of filopodia and for capturing low intensity calcium signals, we demonstrate that filopodia on individual growth cones can respond to imposed stimuli independently from one another. Moreover, filopodia and their parent growth cones appear to represent functionally and morphologically distinct domains of calcium regulation, possessing distinct calcium sources and sinks. Both are sensitive to calcium influx; however, application of the calcium ionophore A23187 to cells in calcium-free medium demonstrated the presence of potential intracellular calcium pools in the growth cone proper, but not in isolated filopodia. Thapsigargin significantly reduced the rise in growth cone calcium levels associated with excitatory neurotransmitters, further implicating release from calcium pools as one component of growth cone calcium regulation. The relative contributions of these pools were examined in response to excitatory neurotransmitters by quantitative calcium measurements made in both growth cones and isolated filopodia. Striking differences were observed; filopodia were sensitive to a low concentration of dopamine and serotonin, while growth cones displayed an amplified rise at a higher concentration. The spatial distribution of organelles that could serve as morphological correlates to such calcium amplification was examined using confocal microscopy. While the majority of organelles were located in the central core of the growth cone proper, peripheral organelles were detected at the base of a subset of filopodia. The distinctive distribution of calcium regulation within motile growth cones suggests one mechanism by which growth cones may regulate their complex behavior. © 1996 John Wiley & Sons, Inc. 相似文献
9.
The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system. 相似文献
10.
Kouji Kita Kunihiro Asanuma Takayuki Okamoto Eiji Kawamoto Koichi Nakamura Tomohito Hagi Tomoki Nakamura Motomu Shimaoka Akihiro Sudo 《Current issues in molecular biology》2021,43(3):1255
Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young’s modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis. 相似文献
11.
Stephen Estes Lei Ray Zhong Liana Artinian Karine Tornieri Vincent Rehder 《Developmental neurobiology》2015,75(5):435-451
The electrical activity in developing and mature neurons determines the intracellular calcium concentration ([Ca2+]i), which in turn is translated into biochemical activities through various signaling cascades. Electrical activity is under control of neuromodulators, which can alter neuronal responses to incoming signals and increase the fidelity of neuronal communication. Conversely, the effects of neuromodulators can depend on the ongoing electrical activity within target neurons; however, these activity‐dependent effects of neuromodulators are less well understood. Here, we present evidence that the neuronal firing frequency and intrinsic properties of the action potential (AP) waveform set the [Ca2+]i in growth cones and determine how neurons respond to the neuromodulator nitric oxide (NO). We used two well‐characterized neurons from the freshwater snail Helisoma trivolvis that show different growth cone morphological responses to NO: B5 neurons elongate filopodia, while those of B19 neurons do not. Combining whole‐cell patch clamp recordings with simultaneous calcium imaging, we show that the duration of an AP contributes to neuron‐specific differences in [Ca2+]i, with shorter APs in B19 neurons yielding lower growth cone [Ca2+]i. Through the partial inhibition of voltage‐gated K+ channels, we increased the B19 AP duration resulting in a significant increase in [Ca2+]i that was then sufficient to cause filopodial elongation following NO treatment. Our results demonstrate a neuron‐type specific correlation between AP shape, [Ca2+]i, and growth cone motility, providing an explanation to how growth cone responses to guidance cues depend on intrinsic electrical properties and helping explain the diverse effects of NO across neuronal populations. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 435–451, 2015 相似文献
12.
S Scheuring P Ringler M Borgnia H Stahlberg D J Müller P Agre A Engel 《The EMBO journal》1999,18(18):4981-4987
Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change. 相似文献
13.
原子力显微技术在酶学研究中的应用 总被引：1，自引：0，他引：1
酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。 相似文献
14.
At the distal most aspect of motile extending axons and dendrites lies the growth cone, a hand like macroorganelle of membrane bound cytoskeleton, packed with receptors, adhesion molecules, molecular motors, and an army of regulatory and signaling proteins. Splayed out along the substratum in vitro, the growth cone resembles an open hand with bundles of filamentous actin, barbed ends outstretched, as if fingers extending from a central domain of dynamic microtubule plus ends. The growth cone acts first as a sensory platform, analyzing the environment ahead for the presence of guidance cues, secondly as a mechanical dynamo establishing focal contact with the extracellular matrix to drive processive forward outgrowth, and thirdly as a forward biochemical command center where signals are interrogated to inform turning, extension, retraction, or branching. During his career, Paul Letourneau has made major contributions to our understanding of how growth cones respond to their environment. Here, we will summarize some of these major advances in their historical context. Letourneau's contributions have provided insights into cytoskeletal organization, growth cone dynamics, and signaling pathways. His recent work has described some important molecules and molecular mechanisms involved in growth cone turning. Although much remains to be understood about this important and intriguing structure, Letourneau's contributions have provided us with \"growth cone guidance.\" 相似文献
15.
Michael W. Baker Brent Kauffman Eduardo R. Macagno Birgit Zipser 《Developmental neurobiology》2003,56(1):41-53
In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality‐specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality‐specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye‐filled sensory afferents as a function of time. Using 2‐photon laser‐scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time‐lapse series of images revealed a highly dynamic and maturation‐state‐dependent pattern of growth. Upon entering the CNS, the growth cone‐tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior‐posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher‐order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en‐passant specializations accompanying the developmental onset of synapse formation. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 41–53, 2003 相似文献
16.
Tzu-Ping Ko John Day Alexander J. Malkin Alexander McPherson 《Acta Crystallographica. Section D, Structural Biology》1999,55(8):1383-1394
The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the 〈001〉 direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the `bottoms' and `tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM. 相似文献
17.
Abel Moreno Margarita Rivera 《Acta Crystallographica. Section D, Structural Biology》2005,61(12):1678-1681
The role of electrochemical processes on Fe and CdSO4 in the crystallization of horse spleen ferritin has been investigated using the cyclic voltammetry technique. It was found that although both species exhibit important redox properties in the presence of an external applied potential, CdSO4 played a leading role not only in the nucleation process but also in the growth behaviour and morphology of ferritin crystals. 相似文献
18.
Cell spreading is correlated with changes in important cell functions including DNA synthesis, motility, and differentiation. Spreading is accompanied by a complex reorganization of the cytoskeleton that can be related to changes in cell stiffness. While cytoskeletal organization and the resulting cell stiffness have been studied in motile cells such as fibroblasts, less is known of these events in nonmigratory, epithelial cells. Hence, we examined the relationship between cell function, spreading, and stiffness, as measured by atomic force microscopy. Cell stiffness increased with spreading on a high density of fibronectin (1000 ng/cm(2)) but remained low in cells that stayed rounded on a low fibronectin density (1 ng/cm(2)). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting f-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrate the relationship between cell stiffness and spreading in hepatocytes. They specifically show that normal actin and myosin function is required for hepatocyte spreading and DNA synthesis and demonstrate opposing effects on cell stiffness upon disruption of actin and myosin. 相似文献
19.
20.
Porosomes are the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the environment during cell secretion. In neurons, 12- to 17-nm cup-shaped lipoprotein structures possessing a central plug are present at the presynaptic membrane, where 40-50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. The neuronal porosome complex has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membranes. Earlier studies using AFM (atomic force microscopy), EM (electron microscopy), electron density and 3D contour mapping provide the structure and assembly of proteins within the neuronal porosome complex at the nanoscale level. A set of eight protein units lining the neuronal porosome cup is present, each connected via spoke-like elements to a central plug, hypothesized for the rapid opening and closing of the structure to the outside. In the present study, ultrahigh-resolution imaging of the presynaptic membrane of isolated synaptosome preparations demonstrate, for the first time, the presence of neuronal porosomes in both their open and close conformations. The results suggests that the central plug is retracted into the porosome cup in its open conformation and pushed outward to seal the porosome opening, supporting the hypothesis that it operates as the opening-closing device of the complex. 相似文献