Paul B. Bell

Identification of a protein associated with both membrane and cytoskeleton fractions from branched but not unbranched microvilli of 13762 rat mammary adenocarcinoma ascites tumor sublines

Abstract MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma exhibit striking differences in cell surface morphology and receptor mobility. MAT-C1 cells are covered with highly branched microvilli and have essentially immobile receptors, while the MAT-B1 cells have predominantly unbranched microvilli and highly mobile receptors. Microvilli have been isolated from the two sublines by gently shearing the microvilli from the cells and purifying them by differential centrifugation. By electron microscopy, plasma membrane marker enzyme analyses, metabolic labeling with glucosamine and polypeptide analysis by SDS-polyacrylamide gel electrophoresis the microvilli preparations from the two sublines were shown to be very similar except for their morphology and the presence of a 58 000 dalton polypeptide (58 kDa) in MAT-C1 microvilli which was undetected in MAT-B1 microvilli. Extraction of MAT-C1 microvilli with Triton X-100 in phosphate-buffered saline left a cytoskeletal residue composed predominantly of 58 kDa polypeptide and actin. Membranes were prepared from MAT-B1 and MAT-C1 microvilli by homogenization in glycine-EDTA-mercaptoethanol to facilitate removal of cytoskeletal proteins and characterized as noted above. MAT-C1 membranes were enriched in 58 kDa polypeptide, suggesting its association with the microvillus membrane. Additional evidence for membrane association was provided by reconstitution from deoxycholate, in which 58 kDa polypeptide is soluble. The polypeptide appears to be associated with only the microvilli of the MAT-C1 cells; it is absent from cell bodies from which microvilli have been removed. We suggest that 58 kDa polypeptide may be involved in the stabilization of the branched microvilli, possibly serving as a linking element between the microvillus membrane and actin.

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.

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°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.

PREPARING WHOLE MOUNTS OF BIOLOGICAL SPECIMENS FOR IMAGING MACROMOLECULAR STRUCTURES BY LIGHT AND ELECTRON MICROSCOPY

In this article we critically review the procedures involved in preparing whole mounts of biological samples for microscopic observation, particularly at high resolution. We discuss practical methods for optimizing specimen preservation to achieve the two principal goals of biological specimen preparation: (a) preserving biological structures as close to their living configuration as possible, and (b) rendering them visible with the desired imaging method. Drawing on examples taken from our own work, and using fluorescence light microscopy and scanning electron microscopy to study the cytoskeleton of vertebrate cells in culture, we show that obtaining optimal results is often a compromise between maximum preservation and the clear visualization of structures. We have found that fixing cells with bifunctional protein crosslinking reagents, in combination with detergent extraction, allows internal cell structures to be preserved, labeled with specific probes, and visualized with microscopic methods. We also review and discuss the relative merits of different procedures for fixing (chemical fixation and cryofixation), drying (air‐drying, critical point‐drying, and freeze‐drying) and coating biological specimens with metals to facilitate visualization in the electron microscope.

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.