Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

Barak, Larry S.; Yocum, R. Rogers; Nothnagel, Eugene A.; Webb, Watt W.

doi:10.1073/pnas.77.2.980

Cited by 393 publications

(180 citation statements)

References 19 publications

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“…In this study, we have defined the state of cellular actin molecules based on their reactivities toward different probes. Filamentous actin was detected as those reacting positively with fluorescent phalloidin (Wulf et al, 1979;Barak et al, 1980). Actin antibodies and fluorescent analogs of actin were used to examine the overall distribution of both filamentous and nonfilamentous actin (Lessard, 1988).…”

Section: Discussionmentioning

confidence: 99%

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Cao¹

1993

The Journal of Cell Biology

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Abstract. Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.

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Section: Discussionmentioning

confidence: 99%

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Cao¹

1993

The Journal of Cell Biology

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“…Actin-polymerization assay was performed as described earlier (Barak et al, 1980;Schraufstatter et al, 2001). Briefly, NCI-H82 and NCI-H446 SCLC cells were seeded on Labtech-eightwell-chamber slides precoated with 0.01% poly-L-lysine Solution (Sigma, St Louis, MO, USA).…”

Section: Actin-polymerization Assaymentioning

confidence: 99%

Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells

et al. 2003

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Small-cell lung cancer (SCLC) is an aggressive, rapidly metastasizing neoplasm. The chemokine stromal cellderived factor-1 (SDF-1/CXCL12) is constitutively secreted by marrow stromal cells and plays a key role for homing of hematopoietic cells to the marrow. Here, we report that tumor cells from patients with SCLC express high levels of functional CXCR4 receptors for the chemokine CXCL12. Reverse transcriptase-polymerase chain reaction and flow cytometry demonstrated CXCR4 mRNA and CXCR4 surface expression in SCLC cell lines. Immunohistochemistry of primary tumor samples from SCLC patients revealed high expression of CXCR4. CXCL12 elicited CXCR4 receptor endocytosis, actin polymerization, and a robust activation of phospho-p44/ 42 mitogen-activated protein kinase in SCLC cells. Furthermore, CXCL12 induced SCLC cell invasion into extracellular matrix and firm adhesion to marrow stromal cells. Stromal cell adhesion of SCLC cells was significantly inhibited by the specific CXCR4 antagonist T140, pertussis toxin, antivascular cell adhesion molecule-1(VCAM-1) antibodies, and CS-1 peptide, demonstrating the importance of CXCR4 chemokine receptor activation and a4b1 integrin binding, respectively. In addition, CXCL12 enhanced the adhesion of SCLC cells to immobilized VCAM-1, demonstrating that CXCR4 chemokine receptors can induce integrin activation on SCLC cells. As SCLC has a high propensity for bone marrow involvement, our findings suggest that CXCR4 chemokine receptors and a4b1 integrins play a critical role in the interaction of SCLC cells with stromal cells in the tumor microenvironment.

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“…After the coverslips were washed in PBS, the cells were permeabilized with 0.1% Triton X-100 (Sigma Chemical Company, St. Louis, Missouri) in PBS for 3.5 minutes. After a thorough washing, the coverslips were incubated with rhodamine-labeled phalloidin (Molecular Probes, Junction City, Oregon) in PBS 28 for 30 minutes. The coverslips were then washed and mounted over glycerol-PBS (1:1).…”

Section: Visualization Of F-actln Mlcrofllament Bundlesmentioning

confidence: 99%

Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments.

Wong¹,

Gotlieb²

1986

Arteriosclerosis

104

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Although the endothelial cell (EC) cytoskeleton has been studied both in vitro and in vivo, little is known about its role in endothelial integrity. We have previously suggested that specific EC microfilament (MF) structure, which we have termed the "dense peripheral band (DPB)," may play a major role in this process. We have extended our studies to characterize this structure in pig aortic ECs in vitro. During the growth of EC cultures, the DPB appears only when the cultures have attained confluency. Using double fluorescent labeling, we found that alpha-actinin, myosin, and tropomyosin colocalized with the F-actin making up the DPB. Occasional microtubules were present in this region, although there was no preferred association between microtubules and the DPB. Colocalization studies revealed vinculin plaques at the cell-cell interface. Thin MFs extended from the DPB into the cytoplasmic side of these plaques. The DPB was completely disrupted by low dose cytochalasin B within 30 minutes, whereas many central MF bundles were still present at 24 hours. The results of this study suggest that the DPB is a distinct structure in the confluent EC monolayer and is closely associated with the ability of ECs to form and maintain the EC monolayer. The disruption of the DPB as an important initial event in the pathogenesis of vascular diseases such as atherosclerosis is discussed. (Arteriosclerosis 6:212-219, March/April 1986) C urrent theories of atherogenesis suggest that loss of endothelial integrity may be one of the important early events leading to the formation and growth of the atheromatous fibrofatty plaque.1^1 This loss of integrity is due to endothelial cell (EC) denudation from the monolayer 5 " 7 as well as to nondenuding EC dysfunction. 8 ' 9 We have previously proposed that the F-actin cytoskeleton may be important in the maintenance of this integrity. 10The endothelial cytoskeleton has been described in several in vitro 10 " 18 and in vivo 17 " 25 studies in both the normal and injured aortic wall. We have earlier shown the importance of microfilament (MF) bundles, microtubules, and centrosomes in the process of aortic reendothelialization after large 12 " 15 and small 10 denuding injuries made in the confluent in vitro monolayer. The studies suggested that these aortic endothelial structures play an important role in cell adhesion, spreading, and translocation.The F-actin MF bundles in EC are present in two areas of the EC: at the cell periphery as a dense peripheral band (DPB), and more centrally as ventrally located bundles in Received July 19, 1985; revision accepted October 21, 1985. what are generally described as stress fibers. 24 Although much is known about the central MF bundles with respect to structure and function, the EC peripheral actin MF bundles have not been well studied. Our previous in vitro studies have shown that the presence of the DPB was closely associated with the lamellipodia extrusion occurring during rapid closure of single cell wounds in a confluent monolayer.10 In addition...

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Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

Cited by 393 publications

References 19 publications

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells

Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments.

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