Biomedical subjects

P B Bell

Publications and source records attributed to P B Bell.

At least 19 recordsLinked to original sources

PubMed2001

Reorganization of the actin cytoskeleton in the protruding lamellae of human fibroblasts.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge.

Actins↗

PubMed1999

CD79a detected by ZL7.4 separates chronic lymphocytic leukemia from mantle cell lymphoma in the leukemic phase.

Both B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are characterized by a lymphoproliferation of neoplastic CD5+ B-cells, but an accurate differential diagnosis between these two malignancies is vitally important for guiding treatment options. Because CD79a has been identified as a pan-B marker, we intended to use it in place of CD19 to identify B-cells and to use CD23 to distinguish between CLL and MCL in the leukemic phase. Anti-CD79a (clone ZL7.4) was used to detect the Igalpha/mb1 protein in fresh CD5+ B-lymphocytes by dual-channel flow cytometry. Expression of CD19 and CD23 were similarly assessed. As expected, CD19 was expressed in all specimens, whereas CD23 expression was zero in 3/4 MCLs, weak in 1/4 MCLs, and 2/8 CLLs (10-19%) and stronger in 6/8 CLLs (> or =45%). However, although all the CD19+/CD5+ cells of MCL expressed high CD79a levels, CD79a expression was negligible or absent in 8/8 CLL specimens (mean positivity for CD79a = 2.41 +/- 2.71%). CD79a (ZL7.4) levels may provide a more reliable distinction than CD23 levels between CLL and MCL. If these results hold up in a larger series, we recommend that the ZL7.4 antibody should be considered in routine marker panels for CLL and low-grade lymphoma.

Aged↗

PubMed1998

Distribution of cytoskeletal proteins in neomycin-induced protrusions of human fibroblasts.

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15-20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.

Actins↗

PubMed1997

Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP.

There is a pressing need to reduce the time and cost of developing new cytotoxic agents and to accurately identify clinically active agents at an early stage. In this study, the differential staining cytotoxicity (DiSC) assay was used to assess the efficacy of the novel antitumour cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP) (and its metabolite 8-Cl-adenosine) against 107 fresh specimens of human neoplastic and normal cells. Diagnoses included chronic and acute leukaemias, myeloma, non-Hodgkin's lymphoma (NHL) and miscellaneous solid tumours. The aim was to identify targets for subsequent phase I, II and III trials. 8-Cl-cAMP was tested at 4-985 microM, along with standard chemotherapeutic drugs. 8-Cl-cAMP and its metabolite caused no morphologically observable cell differentiation but induced dose-dependent cytotoxicity. Compared with untreated patients, previously treated chronic lymphocytic leukaemia (CLL) patients showed no increase in ex vivo resistance to 8-Cl-cAMP (P = 0.878); minimal cross-resistance with other cytotoxic drugs was detected. Compared with normal cells (mean LC90 = 1803 microM), 8-Cl-cAMP showed significant ex vivo activity against CLL (117.0 microM; P < 0.0001) and NHL (140.0 microM; P < 0.0001), of which eight were mantle cell NHL (84.7 microM), and greatest activity against cells from patients with acute myeloid leukaemia (AML; mean LC90 = 24.3 microM; in vitro therapeutic index 74-fold, P < 0.0001). Solid tumour specimens were comparatively resistant to 8-Cl-cAMP. The results highlight the clinical potential of 8-Cl-cAMP, point to several new phase I, II and III trial possibilities and provide a rationale for the inclusion of ex vivo cytotoxic drug evaluation in the drug development process.

2-Chloroadenosine↗

PubMed1996

Novel ex vivo analysis of nonclassical, pleiotropic drug resistance and collateral sensitivity induced by therapy provides a rationale for treatment strategies in chronic lymphocytic leukemia.

Extensive research into mechanisms of cytotoxic drug resistance and subsequent clinical trials of drug resistance modifiers have produced few encouraging results. In this report, we analyze 4,400+ ex vivo Differential Staining Cytotoxicity (DiSC) assay drug response results from patients with chronic lymphocytic leukemia (CLL) to investigate the development of drug resistance during treatment. Patients were untreated (n = 216) or previously treated with various cytotoxic agents (n = 188). Data was processed to identify ex vivo resistance (or sensitivity) induced by treating patients with prednisolone, chlorambucil, cyclophosphamide, anthracycline, or fludarabine. Induced resistance was apparently not associated with any one known mechanism. Treatment with chlorambucil induced a 10-fold sensitivity to steroids; cyclophosphamide induced greater resistance to anthracyclines than alkylating agents; anthracyclines induced greatest resistance to chlorambucil, cisplatin, carboplatin, and cladribine. Patients previously treated with at least two regimens were only 2.16-fold more resistant to CLL drugs than untreated patients, but had significantly reduced survival (median survival, 7.9 months compared with 61.1 months for untreated patients). These results suggest that chlorambucil and/or an antimetabolite should be administered before cyclophosphamide or anthracyclines to delay the onset of extensive pleiotropic drug resistance. Because individual differences in drug sensitivity are considerable, specific guidance could be obtained from ex vivo assay results. Furthermore, as a model for investigating drug resistance mechanisms, fresh CLL lymphocytes represent a useful alternative to drug-resistant cell lines.

Adult↗

PubMed1996

Enhanced ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology.

Ex vivo drug sensitivity testing is of considerable benefit in aiding the choice of optimum chemotherapy for leukaemia patients, especially when several therapeutic options exist, e.g. for relapsed chronic lymphocytic leukaemia (CLL). We have used the Differential Staining Cytotoxicity (DiSC) assay to assess drug sensitivity in CLL for over a decade and here present many methodological improvements, including depositing multiple samples per microscope slide and performing a rapid LC90 evaluation. Using these improvements, 412/450 specimens were successfully tested. Failures were mainly due to extended specimen transit time. All 38 drugs tested exhibited dose-dependent cell kill and broad ranges of resultant LC90S were observed. Comparison of 2- and 4-day incubations underscored a requirement for 4-day incubation with pentostatin and steroids. The rapid, simple and streamlined DiSC assay presented here can aid choice of optimum therapy, identify novel anticancer agents and be used to study drug resistance.

Antineoplastic Agents↗

PubMed1996

Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy.

Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.

Actins↗

PubMed1996

Correlation of bcl-2 with P-glycoprotein expression in chronic lymphocytic leukaemia and other haematological neoplasms but of neither marker with ex vivo chemosensitivity or patient survival.

We compared bcl-2 with P-glycoprotein expression (C494 and JSB1), and both with ex vivo chemosensitivity by Differential Staining Cytotoxicity (DiSC) assay (25 cytotoxic drugs), in 76 fresh haematological specimens, including 51 chronic lymphocytic leukaemias (CLL). Strong correlations were seen between bcl-2 and Pgp expression in both CLL (r = 0.5; p < 0.001) and AML (r = 0.9; p < 0.001) although bcl-2 expression was only raised in Pgp positive cells. However, there was no correlation between high or low marker levels and either ex vivo drug sensitivity (-0.30 < r < 0.37; p all > 0.1) or patient survival (chi 2 < or = 0.1; p > 0.7). One B-CLL, one PLL and one hairy cell leukaemia were negative for both bcl-2 and Pgp, whilst 3 T-cell specimens were bcl-2 negative but strongly positive for Pgp. These results suggest that the expression of Pgp and bcl-2 may be interlinked and related to immunophenotype and that clinical sensitivity to MDR-inducing and/or apoptosis-inducing drugs is best determined by ex vivo chemosensitivity testing rather than measurement of Pgp or bcl-2 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.

Actins↗

PubMed1995

The effect of neomycin on PDGF-induced mitogenic response and actin organization in cultured human fibroblasts.

Actin organization and DNA synthesis were studied in cultured, serum-starved, subconfluent human fibroblasts in response to human recombinant platelet-derived growth factor (PDGF)-BB and neomycin, an inhibitor of phosphoinositide (PI) turnover. The labeling of F-actin with TRITC-phalloidin showed that PDGF (10 ng/ml) and neomycin (3.5 mM) induced, within minutes, similar changes in the actin cytoskeleton and when used together potentiated each other's effects. PDGF stimulated a twofold increase in DNA synthesis, as shown by [3H]thymidine incorporation, 48 h after the beginning of incubation. Neomycin did not induce DNA synthesis but reduced the mitogenic response to PDGF to control levels. Stimulation of DNA synthesis was correlated with an intense increase in lamellipodia formation and ruffling, whereas inhibition of the DNA synthesis was correlated with the development of elongated cell shape with few ruffles and lamellipodia, similarly to untreated control cells. Our findings support the conclusion that early changes in the actin cytoskeleton and induction of DNA synthesis are separable biological processes that are mediated by different pathways. The inhibitory effect of neomycin on PDGF-induced DNA synthesis was dose-dependent during the first 24 h of PDGF incubation, after which the cells were irreversibly and maximally stimulated to synthesize DNA. Results of experimental treatments with neomycin at various times during the incubation with PDGF indicate that the critical period during which PDGF acts to stimulate DNA synthesis is after the 1st h but before the 12th h of incubation.

Actins↗

PubMed1995

Improved methods for preserving macromolecular structures and visualizing them by fluorescence and scanning electron microscopy.

To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% paraformaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bifunctional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes.

Actins↗

PubMed1993

PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts.

The addition of platelet-derived growth factor (PDGF) to serum-starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI-cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI-cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent-extracted cells was labelled with TRITC-phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF- and neomycin-induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF-receptors. In conclusion, the present study indicates that an increased turnover of the PI-cycle is not essential for the changes in actin organization induced by PDGF.

3T3 Cells↗

PubMed1992

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.

Animals↗

PubMed1991

Cryosputtering--a combined freeze-drying and sputtering method for high-resolution electron microscopy.

Preparing cellular structures for visualization by high-resolution scanning electron microscopy (SEM) is a multi-step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high-resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze-dryer to accommodate a magnetron sputtering head, in order to sputter-coat the frozen-dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1.5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent-extracted human glioma cells or fibroblasts and the specimens were examined by high-resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two-stage rotary vane pump was connected to the drying-coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at -90 degrees C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat formed during sputtering.

Cell Line↗

PubMed1990

Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation.

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.

Actins↗

PubMed1989

Problems associated with the preparation of whole mounts of cytoskeletons for high resolution electron microscopy.

The resolution currently available in both transmission and scanning electron microscopes is theoretically adequate to visualize the organization of the cytoskeleton at the supramolecular and macromolecular levels. However, achieving this resolution in practice requires that the methods used to prepare the specimens both preserve the structures of interest and render them visible for observation in the microscope without obscuring or altering them. In this paper we discuss our own and others efforts to develop methods to overcome several problems associated with preparing whole mounts of cytoskeletons for observation by electron microscopy. These problems include: controlling the degree to which cellular components are extracted; the effects of osmium tetroxide on the cytoskeleton; controlling and recognizing shrinkage and drying artifacts; the choice of a method of visualization; deposition of grain-free ultrathin films of metal; and interpreting the results. The standard procedure which we currently use consists of the following steps: growing cells on carbon-stabilized Formvar-coated gold electron microscope grids; extracting in 0.5% Triton X-100 detergent in a microtubule stabilizing buffer; postfixing in 2.5% glutaraldehyde in stabilizing buffer; freeze-drying; magnetron sputter-coating with 1.5 nm of tungsten; and observation by TEM, SEM, or STEM. Cytoskeletons prepared in this manner contain over 100 polypeptides and are composed of a complex three dimensional meshwork of clean, uniform filaments, the smallest of which are 7 nm in diameter. A structure resembling the microtrabecular lattice is present only if the cells are prefixed with a relatively long bifunctional protein crosslinking reagent prior to extraction with detergent.

Animals↗

PubMed1988

Interaction of B16 malignant melanoma tumor cells with the murine mesentery in vitro.

We have used organs cultures of murine mesentery as a model system to investigate the mechanisms by which B16-F10 melanoma cells invade normal tissues. The mesentery has the advantage of being a real tissue, consisting of a loose connective tissue, containing a normal complement of stromal cells and extracellular matrix, covered by a continuous epithelium of squamous mesothelial cells which are separated from the connective tissue by a laminin-containing basement membrane. B16-F10 cells seeded onto the mesentery in vitro cause a local retraction of the mesothelial cells exposing the underlying basement membrane onto which the tumor cells spread. Direct contact between the tumor cells and the margins of the mesothelial cells is required to induce retraction. Most of the B16 cells remain on the surface of the mesentery where they spread on the basement membrane without disrupting it. A few B16 cells penetrate the basement membrane and invade the connective tissue interior of the mesentery where they flatten out and assume a fibroblastic morphology. Tumor cells within the connective tissue may continue to translocate and they adhere to and move along the fibers of the connective tissue extracellular matrix without appearing to destroy or disrupt them.

Animals↗

PubMed1988

Comparison of the effects of critical point-drying and freeze-drying on cytoskeletons and microtubules.

We have compared the effects of critical point-drying (CPD) and freeze-drying (FD) on the morphology of Triton-resistant cytoskeletons and microtubules by scanning (SEM) and transmission electron microscopy (TEM). In general, cytoskeletons attached to Formvar films suffer less structural damage than cells or cytoskeletons attached to glass, because the Formvar film absorbs some of the stress associated with shrinkage during drying. However, as seen in stereo-pair electron micrographs, the three-dimensional structure of cytoskeletons prepared by FD is better preserved and shows fewer artefacts than those prepared by CPD. CPD specimens are flatter, often have a concave and apparently collapsed nuclear matrix and show large cracks both in the perinuclear zone and through the cytoskeleton. At least some of the damage appears to be due to residual water in the CO2 used as the substitution fluid, because cytoskeletons dried with a water filter attached to the CPD apparatus show substantially less damage than those dried without the filter. Freeze-dried cytoskeletons consist mostly of unbroken, smooth filaments and have no perinuclear open space. Comparison of the effects of drying on the diameters of in vitro polymerized microtubules showed that the diameter of microtubules is reduced after drying, but that FD causes significantly less shrinkage than CPD. Addition of 0.2% tannic acid to the glutaraldehyde fixative significantly reduces the shrinkage of CPD microtubules, but has no effect on FD microtubules. The observations on microtubules support the hypothesis that drying-induced shrinkage is the result of both pressure and solvent evaporation and they indicate that tannic acid stabilizes samples against the former but not the latter.

Cytoskeleton↗