Visualization of microtubules of cells in situ by indirect immunofluorescence.

Byers, H. Randolph; Fujiwara, Keigi; Porter, Keith R.

doi:10.1073/pnas.77.11.6657

Cited by 22 publications

(12 citation statements)

References 20 publications

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“…These data also support the growing information indicating that the presence of microfilament bundles in vitro may not be an artifact of cells grown in culture. Recently, Byers and Fujiwara 119821 and Rogers and Kalnins 119831 have demonstrated quite elegantly the presence of microfilament bundles or stress fibers in certain cells in situ; microtubules [Byers et al, 1980;Rogers and Kalnins, 19831 and intermediate filaments [Blose and Meltzer, 19811 have recently been documented also in situ. Furthermore, it is interesting to note that in the periodontal ligament in vivo, many fibroblast-like cells are highly elongated, exhibit an abundance of oriented microtubules and microfilaments, and both adhaerens-type and gap junctions [Beertsen et al, 1974;Garant and Cho, 1979;Shore and Berkowitz, 1979;Beertsen and Everts, 19801.…”

Section: Discussionmentioning

confidence: 99%

Microfilament rearrangements during fibroblast‐induced contraction of three‐dimensional hydrated collagen gels

Farsi¹,

Aubin²

1984

Cell Motility

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In vitro models have been developed recently to study the ability of fibroblasts to generate tensile force within collagen gels. The present study was initiated to assess the role of the cytoskeleton in the cell shape changes and force generation in one such model system. Porcine periodontal ligament fibroblasts ( PPLF ) were cultured within three-dimensional collagen gels attached to glass coverslips. Fluorescence microscopy, using nitrobenzooxadizole (NBD)- phallacidin labeling for microfilaments and tubulin antibody staining for microtubules, was combined with phase and Nomarski optics to determine the intra- and extracellular architecture of the cells and collagen fibers. Samples were observed from 30 minutes to 24 hours after initiation of cell attachment. During attachment and spreading, NBD- phallacidin staining changed dramatically until large microfilament bundles became prominent. Collagen fiber alignment, compaction, and finally tearing from the coverslip occurred during this time. After release of tension, microfilament bundles were no longer evident. The change in microtubule distribution during these processes was less dramatic, appearing to follow the change in cell shape. These results indicate that microfilaments play an essential role in generating force to align and compact collagen, while microtubules may have a secondary role only.

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Section: Discussionmentioning

confidence: 99%

Microfilament rearrangements during fibroblast‐induced contraction of three‐dimensional hydrated collagen gels

Farsi¹,

Aubin²

1984

Cell Motility

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“…The absorbed antigen and the immunological reagent for each disk are: disk 1, fish skeletal muscle actin and absorbed IgG (0.37 mg/ml) ; disk 2, fish skeletal muscle actin and absorbed IgG (0.037 mg/ml) ; disk 3, fish skeletal muscle actin and antiactin (0.25 mg/ml) ; disk 4, fish skeletal muscle actin and antiactin (0.10 mg/ml) ; disk 5, fish skeletal muscle actin and antiactin (0.01 mg/ml) ; disk 6, human platelet actin and antiactin (0.25 mg/ ml) ; disk 7, chicken gizzard tropomyosin and antiactin (0 .25 mg/ ml) . Disks [3][4][5][6] show reaction products, while disks 1, 2, and 7 do not. The antiactin made against fish skeletal muscle actin crossreacts with human platelet actin (disk 6) .…”

Section: Characterization and Purification Of The Antiactin Antiseramentioning

confidence: 99%

“…Indirect immunofluorescence procedures (6) were carried out on different scales with the following antibodies : affinity-purified antiactin (50 lag/ml), a 1 :20 dilution of serum containing antimyosin, and a 1 :20 dilution of anti-alpha-actinin serum .…”

Section: Preparation and Staining Of Cells In Situmentioning

confidence: 99%

“…To help evaluate the biological significance of the voluminous research on stress fibers in cultured cells, it is important to ask whether stress fibers are epiphenomena or artifacts of cells in tissue culture . To determine whether stress fibers actually exist in cells in situ, we used a tissue that has recently been amenable to irnmunofluorescent visualization of microtubules in situ (6) . In this tissue, fibroblast cells, called scleroblasts, reside as a thin monolayer on the surface of the 804 fibrillary plate of the fish scale (20,29) .…”

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Stress fibers in cells in situ: immunofluorescence visualization with antiactin, antimyosin, and anti-alpha-actinin.

Byers

Fujiwara²

1982

The Journal of Cell Biology

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119

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Stress fiber-like patterns are visualized by indirect immunofluorescence in scleroblasts (fibroblasts) in situ on the scale of the common goldfish, Carassius auratus, using an affinity-purified antiactin, antimyosin, and anti-alpha-actinin . These fibers demonstrate the classical convergent and parallel patterns exhibited by stress fibers in tissue culture cells. Because the dimensions, the composition, and the pattern of distribution of these cytoplasmic fibers correspond well with those of stress fibers in cultured cells, we will call these fibers stress fibers also . The staining patterns with anti-alpha-actinin and antimyosin along the stress fibers often reveal a periodicity of 1-2 Am, identical to that found in cells in vitro. The majority of scleroblasts do not exhibit stress fiber staining and they are specifically located in the central regions of the scale. Stress fibers are present in scleroblasts residing on or near the edges or radical ridges of the scale . They are consistently orientated perpendicular to these structures ; however, unlike microtubules, stress fibers show no co-alignment with collagen fibers of the scale . The finding that stress fibers are Icoated in regions of the scale more subject to shearing forces may indicate their role in increased cellular adhesion to the substratum .

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“…When stained with antibodies to tubulin and viewed in situ, the microtubules are seen to extend out radially from the cells' organizing centers, and then curve abruptly to become aligned with the collagenous fibers of the underlying scale. It is impressive that microtubules in adjacent cells are similar in number, all oriented in the same manner, and all share a nearly identical pattern (Byers et al, 1980). It seems highly unlikely that the centrosome alone can induce changes in the direction of microtubule growth from a distance of many micrometers without the aid of other organizing components situated in peripheral cytoplasm.…”

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Organization of microtubules in centrosome-free cytoplasm.

McNiven

Porter

1988

The Journal of Cell Biology

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Abstract. Many different cell types possess microtubule patterns which appear to be polarized and oriented, in part, by cytoplasmic factors not directly associated with a centrosome. Recently, we demonstrated that cytoplasmic extensions ("arms") of teleost melanophores will reorganize their microtubule population outward from their centers after surgical isolation (McNiven, M. A., M. Wang, and K. R. Porter. 1984. Cell. 37:753-765). In the study reported here, we examine microtubule dynamics within the centrosomefree fragments and find that, after severing, microtubule reorganization is initiated at the proximal (cut) end of an arm and migrates distally with the aggregated pigment mass until it becomes permanently positioned at the middle of the arm. Computer-aided image analysis demonstrates that this middle position is located at the arm centroid, implicating the action of a cytoplasmic gel in this process. Morphological studies of arms devoid of pigment reveal that microtubules do not emanate from a single site or structure within the centroid area, but from a more generalized region. Taken together, these findings suggest that factors distributed throughout cytoplasm participate in microtubule assembly and organization.

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Visualization of microtubules of cells in situ by indirect immunofluorescence.

Cited by 22 publications

References 20 publications

Microfilament rearrangements during fibroblast‐induced contraction of three‐dimensional hydrated collagen gels

Microfilament rearrangements during fibroblast‐induced contraction of three‐dimensional hydrated collagen gels

Stress fibers in cells in situ: immunofluorescence visualization with antiactin, antimyosin, and anti-alpha-actinin.

Organization of microtubules in centrosome-free cytoplasm.

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