ARTICLE |
Correspondence to: Jeffrey L. Salisbury, Tumor Biology Program, Guggenheim-14, Mayo Clinic, Rochester, MN 55905. E-mail: salisbury@mayo.edu
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Summary |
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We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development. (J Histochem Cytochem 47:12651273, 1999)
Key Words: cytoskeleton, polymer, MTOC, development, disease
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Introduction |
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Centrosomes and mitotic spindle poles are the major microtubule organizing centers (MTOCs) of interphase and mitotic cells (
Since the earliest reports of visualization of microtubules by indirect immunofluorescence (
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Materials and Methods |
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Sample Procurement and Cell Culture
Human breast tissues obtained from mastectomy and lumpectomy surgeries were collected according to an Institutional Review Board-approved protocol. Data from breast adenocarcinoma specimens and normal breast samples from reduction mammoplasties are reported in this study. All tumors were invasive and were designated by staff pathologists at the time of surgery as histological Grade 3 or 4 (Mayo grading system). Specimens were obtained from patients who had undergone no chemotherapy or radiation therapy before surgery. Tissue specimens were frozen in liquid nitrogen immediately after surgery and were stored at -70C until use. HeLa cells (
Microtubule Nucleation on Cryosections and in Detergent Extracted Cell Models
Cryosections of unfixed human breast tissue mounted on coverslips coated with Fro-Tissuer (Electron Microscopy Sciences; Ft Washington, PA) and cultured cells were permeabilized for 10 min with chilled microtubule stabilizing buffer (MTSB: 1% Triton X-100, 10 mM Pipes, pH 7.2, 2 mM EGTA, 1 mM MgSO4) and washed several times in this buffer without detergent. Cold reaction mixture (75 µl), consisting of cytostatic factor arrested Xenopus egg extract (see below;
Microtubule Localization on Tissue Sections and Cell Preparations
Tissue sections and cell preparations were stained for microtubules using the indirect immunofluorescence technique. Specimens were rehydrated in three changes of MTSB and then treated in blocking buffer, consisting of PBS, pH 7.2, with 5% normal goat serum, 1% glycerol, 0.1% bovine serum albumin (Fraction V; Sigma), 1% fish skin gelatin, and 0.04% sodium azide, for 5 minutes at room temperature (RT). Specimens were reacted for 1 hr at RT with primary antibody consisting of a mixture of anti--tubulin and anti-ß-tubulin monoclonal preparations (B512 and 2.1; Sigma) at 1:2000 and 1:200 dilution, respectively, in the blocking buffer. Some specimens were stained for centrosome localization using antibodies against centrin [26/14-1 (
Preparation of Xenopus Egg Extract
Eggs of the frog Xenopus laevis are laid arrested in metaphase II of meiosis (
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Results |
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Microtubule Nucleation by Centrosomes In Vitro
Figure 1 shows an "aster" of microtubules that was nucleated by the centrosome of a human breast tumor cell prepared using the touch method described above. Over 120 microtubules originate from a single focus and extend outward approximately 2025 µm in a uniform radial array. Individual microtubules are either relatively straight or show one or more gently curved regions. Under the conditions used here [8% extract concentration (v/v), 7-min incubation time, and 28C], few free microtubules are present that do not appear to originate from the centrosome region. Unattached microtubules may originate by spontaneous assembly, or some may represent broken ends of microtubules that originate at the centrosome. Varying the concentration of cytoplasmic extract from 5 to 20% produced greater numbers of centrosome-bound microtubules until a plateau was reached. This observation indicates that the number of nucleation sites at the centrosome is finite and can be saturated. However, when cytoplasmic extract was used at high concentration, many spontaneous polymerization events occurred, which resulted in many free microtubules that were not anchored at centrosomes. Because excess free microtubules made accurate counts difficult, each experiment was carried out with a series of cytoplasmic extract dilutions (extract concentration from 5 to 20%). Only the highest concentration of extract that gave centrosome nucleation and no or very little spontaneous free microtubule assembly was used for comparative analysis. Furthermore, because cells attached to the glass coverslip may cause steric limitations to microtubule growth from centrosomes, these methods may result in an underestimation of the actual microtubule nucleation capacity. Nevertheless, for identically treated samples, statistically significant differences in microtubule nucleation capacity were observed between normal and tumor tissue and for cells at different stages of the cell cycle (see below).
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Microtubule Nucleation by Centrosomes of Tissue Sections
Figure 2A2E illustrate centrosome location and microtubule nucleation on frozen-sectioned normal tissue from human breast specimens. Centrosomes were localized using antibodies directed against centrin, a protein found in centrosomes of all eukaryotic cells (
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Figure 2F2H illustrate centrosome staining and microtubule nucleation capacity on frozen-sectioned tissue of human breast adenocarcinoma specimens. Centrosomes were stained using indirect immunofluorescence for centrin (Figure 2F). Unlike normal tissue, tumor cells show unusually large and irregular masses of centrin staining, with many cells displaying more than one concentrated region of centrin localization. A previous study of 35 high-grade tumors demonstrated that centrosomes of human breast adenocarcinoma cells characteristically display abnormal structure, aberrant protein phosphorylation, and supernumerary centrioles (
Microtubule Nucleation by Centrosomes of Touch Preparations
To more accurately quantify microtubule nucleation capacity, we have exploited the nucleation assay using touch preparations of normal and tumor tissue. Figure 3 shows individual normal breast (Figure 3A and Figure 3B) and breast tumor (Figure 3C and Figure 3D) cell touch preparations that were employed in the microtubule nucleation assay as described in Materials and Methods. The low cell density and lack of interference from extracellular matrix material in touch preparations allows accurate counts of microtubules to be made. Touch preparations made from normal breast tissue (Figure 3A and Figure 3B) show a single distinctly focused microtubule aster of less than 30 microtubules for each cell. On the other hand, breast tumor touch preparations (Figure 3C and Figure 3D) show large numbers of microtubules that emanate from one or more centrosomes. These observations corroborated our findings on frozen whole-mounts of sectioned tissue. Both touch preparations and sections from normal tissue consistently nucleated fewer microtubules, which were focused at a single centrosome, and tumor preparations nucleated many more microtubules, often emanating from multiple foci in individual cells. Table 1 shows quantitative analysis of these differences.
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Microtubule Nucleation by Centrosomes of Cell Models
The distribution of endogenous microtubules seen in an interphase HeLa cell is shown in Figure 3E, and microtubule nucleation by centrosomes of detergent-extracted interphase, prophase, and mitotic HeLa cell models is shown in Figure 3F3H, respectively. Microtubules course through the cytoplasm and do not necessarily focus at a single juxtanuclear centrosome in interphase HeLa cells, although many individual microtubules converge near the nucleus (Figure 3E). Although this microtubule distribution is characteristic for many HeLa cells in interphase, mitotic cells exhibit distinct foci of microtubules at the spindle poles (not shown). Nevertheless, when interphase cells are extracted with detergent and then assayed for microtubule-nucleation capacity, most microtubules nucleate and grow from a single juxtanuclear centrosome (Figure 3F). In the example shown here, approximately 20 microtubules originate at the centrosome and extend outward beyond the original margins of the cell boundary. Individual microtubules are quite straight, and their overall distribution is not comparable to that seen in interphase cells that were otherwise untreated (compare the distribution of microtubules in Figure 3E to those in Figure 3F). This observation has implications for the dynamics and redistribution of microtubules in vivo (see below). The microtubule-nucleating capacity of centrosomes has been estimated to be approximately fivefold greater in mitotic compared to interphase cells (
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Discussion |
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Late in the nineteenth century, Van Beneden recognized that the position of the centrosome relative to the nucleus defines a structural "cell axis" that indicates the overall functional polarity of the cell (see
Microtubules typically originate and remain anchored at the centrosome. However, in some cells individual microtubules may not appear to be associated with the centrosome (-tubulin and associated proteins (
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Acknowledgments |
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Supported by the National Cancer Institute of the NIH CA 72836, Race for the Cure/Twin Cities, Breast Cancer Research Foundation, Fraternal Order of Eagles Cancer Research Fund, Sitt Foundation, and the Mayo Foundation.
Received for publication January 15, 1999; accepted June 15, 1999.
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