Biochemical,electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions

Background

Filopodia, retraction fibers and microvilli, are fragile microextensions of the plasma membrane that are easily damaged by mechanical force during specimen preparation for microscopy. To preserve these structures for electron microscopy glutaraldehyde is generally used, but it often causes antigen masking. By contrast, formaldehyde is generally used for immunofluorescence light microscopy, but few studies have been concerned with the loss of microextensions.

Results

We demonstrate in biochemical experiments that cultured cells needed to be kept in 4% formaldehyde for at least 60 min at room temperature or for 20 min at 37°C to irreversibly crosslink most of the polypeptides. Also, fragmentation of fragile microextensions was observed after Triton X-100 extraction depending on concentration and extent of crosslinking. We also report on a novel fixation procedure that includes the cationic detergent dodecyltrimethylammonium chloride (DOTMAC). Treatment of NIH3T3 cells with DOTMAC resulted in complete removal of membrane lipids and in good preservation of the cytoskeleton in microextensions as well as preservation of ultramicroextensions of <0.05μm in diameter that have not been observed previously unless glutaraldehyde was used. Stress fibers and microextensions of DOTMAC-extracted cells were readily stained with anti-β-actin antibodies, and antibodies to vinculin and moesin stained focal contacts and microextensions, respectively.

Conclusions

Some microextensions were fragmented by the standard Triton X-100 permeabilization method. By contrast, DOTMAC completely extracted membrane lipids while maintaining the cytoskeleton of microextensions. Thus, DOTMAC treatment may provide a valuable new tool for the reliable visualization of previously undetectable or poorly detectable antigens while preserving the actin cytoskeleton of microextensions.

     

Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface

Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology.     

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols).

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.     

Oblique banding pattern in collagen fibrils reconstituted in vitro after trypsin treatment.

Collagen fibres from rat tail tendon suspended in small pieces in a solution (pH 7.8) containing 0.5 M CaCl2 were treated with purified bovine trypsin at 20 degrees C for 20 h. After the enzyme treatment collagen from this solution was precipitated out and reconstituted in vitro into native-type fibrils. The banding pattern in these reconstituted fibrils was found to be oblique. This is comparable to that observed recently in fibrils reconstituted from cartilage collagen. On the other hand, normal transverse banding pattern was observed in the fibrils reconstituted in vitro from collagen solution of rat tail tendon which was not pre-treated with trypsin. No significant change was, however, observed in the segment long spacing fibrils precipitated from the enzyme-treated collagen solution. It is possible that the enzyme might affect the mode of organization of tropocollagen molecules during in vitro fibrillogenesis into native-type fibrils either by interacting with the "telopeptide" regions or with the non-collagenous components associated with the native protein and this could probably result into the formation of fibrils with oblique banding pattern.     

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols)

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.Abbreviations CM Cytoplasmic membrane - PTA Phosphotungstic acid - UOx Uranyl oxalate - SDS sodium dodecyl sulfate This communication is Journal Acticle No. 7644 from the Michigan Agricultural Experiment Station     

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells were studied by utilizing the multivalent ligand, polycationic ferritin, as a visual probe. Our observations revealed that anionic sites are distributed over the entire cell surface, with the highest density of sites being located on cell surface microextensions. Following the initial binding of polycationic ferritin to the surface of unfixed cells, the ligand-bound anionic sites redistributed by migrating from the surface of microextensions to the surface of the cell body. In 20 min, this migration resulted in a total clearing of anionic sites from the surface of microextensions concomitant with the formation of patches of anionic sites on the surface of the cell body. Polycationic ferritin-induced migration and patch formation of anionic sites was not prevented by 2,4- dinitrophenol, N-ethylmaleimide, colchicine, or cytochalasin B. However, the ligand-induced redistribution of cell surface anionic sites was prevented by prefixation of cells with glutaraldehyde.     

Adhesion of culture cells to their substratum

The attachment of cells to culture dishes has been investigated by replica techniques and scanning electron microscopy. Cells were removed from their substratum either in a stream of medium or by micromanipulation. In the most usual case of interphase cells one finds many small points of attachment uniformly distributed over the whole cell underside. Attachment sites are also found under the lamellipodia and at the trailing edge of the cells. A large portion of the cell underside is sometimes left behind. This is probably because the sole plate is reinforced by cytoskeletal elements, and does not necessarily indicate the presence of large adhesion points. The distal ends of the retraction fibers formed as the cells round-up in trypsin, or in the cold, represent the attachment points of the cell to the substratum. Agents which tend to stabilize microtubules greatly slow cell detachment by proteolytic agents. The primary effect of trypsin is not on the glue which holds the cells to the substratum but rather on the cell shape itself, affecting the rigidity of cytoskeletal elements.     

Changes in cellular adhesiveness during the development of the slime mold Dictyostelium discoideum

A method was devised to measure the adhesiveness to the substratum of the amoebae of the cellular slime mold, Dictyostelium discoideum, and measurements were conducted with the cells at various stages of development. The adhesiveness of the vegetative amoebae was low, and remained unchanged as long as they fed on bacteria. During the transition from the vegetative stage to the interphase (due to the cessation of feeding), the adhesiveness increased rapidly, and afterwards continued to rise, as development proceeded. The adhesiveness of the interphase amoebae was greatly decreased by the treatment with proteolytic enzymes, lipase, and acid phosphatase. These indicate that accumulation of some substance(s) such as lipoprotein on the cell surface is responsible for the increase in adhesiveness during the interphase. EDTA and periodic acid had no noticeable effect on the adhesiveness of the interphase amoebae. EDTA, however, decreased the adhesiveness in co-operation with trypsin or lipase. The cells disaggregated from the anterior part of the migrating slug showed higher adhesiveness than those from the posterior part. The adhesiveness of either cells was higher than that of the interphase amoebae.     

Cellular compartmentalization of protein kinase activity during the cell cycle

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.     

Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad

To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of "intermediate vesicles" including similarly structured tubules which may be over 300 nm long and are referred to as "intermediate tubules." The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled. The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytoplasmic actomyosin fibrils in tissue culture cells

Summary A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chemical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle.These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.We thank Dr. W. Meier-Ruge and Mr. W. Bielser (Basic Medical Research Departments, Sandoz AG, Basle, Switzerland) for making the laser equipment available to us and for their kind cooperation during this investigation. Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

The fine structure of the parasitic dinoflagellateHaplozoon axiothellae

Summary The multicellular parasitic dinoflagellateHaplozoon axiothellae Siebert was studied with electron microscopy. The trophocyte (attachment cell) bears a suction apparatus with a movable protruding stylet that penetrates the epithelial cell of the host gut. The gonocytes are binucleate and divide frequently. Nuclear structure is similar to the mesokaryotic condition of other dinoflagellates although the chromosomes lack the helically coiled appearance of the DNA fibrils. During nuclear division the nucleus retains its envelope intact and cytoplasmic invaginations develop in which packets of parallel microtubules occur. The microtubules attach to the nuclear envelope opposite the site of chromosome attachment. The chromosomes remain condensed during interphase but the helically coiled DNA fibrils characteristic of the mesokaryotic condition are not evident.The theca which encloses all cells is composed of elements similar to those of typical free-living dinoflagellates, the outer cell membrane and flattened vesicles which contain either flat thin plates or larger spines. No subthecal microtubules are present. The theca grows inward following nuclear division and separates the daughter cells. Trichocysts, pusules, flagellar structures and chloroplasts are not present. The relationship ofHaplozoon to other free-living and parasitic dinoflagellates is discussed.     

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.     

The ultrastructure of human mitotic chromosomes and interphase nuclei treated by Giemsa banding techniques

Total preparations of mitotic chromosomes and interphase nuclei prepared as for Giemsa banding techniques were investigated by standard transmission electron microscopy and by a method of a three dimensional representation. Chromosomes as well as interphase nuclei appear to be composed of irregularely folded fibrils of at least 300 Å thickness. In the G-band regions the chromosomes are thicker containing more foldings of fibrils. Also the fibrils are darker stained in the G-band regions. Loops of fibrils stick out from chromosomes as well as from interphase nuclei. When chromosomes or interphase nuclei come to lie close enough, such loops may stick together and form fibrillar bridges between them. These as well as interchromatid bridges are considered to be artefacts. The fibrils seem to be built up either of one or of several finer fibrils. No further conclusions regarding the fine structure of the fibrils can be drawn.     

Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF-7.

A human breast cancer cell line (MCF-7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF-7 cells, suggesting that MCF-7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF-7 cells with 17β-estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF-7 cells. Considerably less activity was detected in the CM obtained from MCF-7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.     

Redistribution of motor proteins by Cytochalasin J treatment

In this study we extend our analysis of the effect of Cytochalasin J (CJ) on mitotic and interphase cells by the use of immunocytochemical techniques to localize antigens to anti-beta-tubulin, anti-dynein heavy chain (HC), anti-dynein intermediate chain (IC), and anti-kinesin antibodies following CJ treatment. Anti-dynein IC and HC staining of CJ treated cells showed a significant reduction in anti-dynein staining in the nuclear region of interphase cells. Monolayer cultures of PtK(1)cells treated with 10 microg/ml CJ for 10 min showed a significant reduction in pixel luminosity of fluorescence staining using anti-dynein IC and HC antibodies (P<0.05). Cytochalasin J treatment reorganized anti-dynein staining from a cytoplasmic punctate staining with greatest intensity in the perinuclear region, to a more uniform staining throughout the cytoplasm.     

Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria

Cultures of whole fetal rat sensory ganglia which had matured and myelinated in culture were treated for 1-3 h with a pulse of 0.2% trypsin. The tissue was observed during the period of treatment and during subsequent weeks using both light and electron microscopy. Within minutes after trypsin addition the matrix of the culture was altered and the nerve fascicles loosened. Progressive changes included the retraction of Schwann cell processes from the nodal region the detachment of the myelin-related paranodal Schwann cell loops from the axon, and lengthening of the nodal region as the axon was bared. The retraction of myelin from nodal stabilized several hours after trypsin withdrawal. Breakdown of the altered myelin segments was rare. There were no discernable changes in neurons or their processes after this exposure to trypsin. The partial repair which occured over a period of several weeks included the reattachment of paranodal Schwann cell loops to the axolemma and the insertion of new myelin segments where a substantial length of axolemma had been bared. The significance of these observations to the characterization of the Schwann cell-axolemmal junctions on myelinated nerve fibers is discussed. The dramatic degree of myelin change that can occur without concomitant myelin breakdown is particularly noted, as is the observation that these altered myelin segments are, in part, repaired.