How should the optical tweezers experiment be used to characterize the red blood cell membrane mechanics?

Stretching red blood cells using optical tweezers is a way to characterize the mechanical properties of their membrane by measuring the size of the cell in the direction of the stretching (axial diameter) and perpendicularly (transverse diameter). Recently, such data have been used in numerous publications to validate solvers dedicated to the computation of red blood cell dynamics under flow. In the present study, different mechanical models are used to simulate the stretching of red blood cells by optical tweezers. Results first show that the mechanical moduli of the membranes have to be adjusted as a function of the model used. In addition, by assessing the area dilation of the cells, the axial and transverse diameters measured in optical tweezers experiments are found to be insufficient to discriminate between models relevant to red blood cells or not. At last, it is shown that other quantities such as the height or the profile of the cell should be preferred for validation purposes since they are more sensitive to the membrane model.     

Cellular recognition by rat liver cells of neuraminidase-treated erythrocytes : Demonstration and analysis of cell contacts

Freshly isolated rat liver cells adhere firmly to neuraminidase-treated rat or mouse erythrocytes but not to untreated erythrocytes. Binding between cells occurs only in the presence of calcium and is specially inhibited by D-galactose. We therefore suggest that cell adherence is mediated by a galactose-specific hepatic membrane receptor. Ultrastructural analysis of contact regions revealed point-like interactions between hepatic microvilli and erythrocytes and no broad areas of membrane contact. When liver cells are cultivated in vitro they lose their ability to bind erythrocytes within 24 h.     

Detachment of agglutinin-bonded red blood cells. II. Mechanical energies to separate large contact areas.

As detailed in a companion paper (Berk, D., and E. Evans. 1991. Biophys. J. 59:861-872), a method was developed to quantitate the strength of adhesion between agglutinin-bonded membranes without ambiguity due to mechanical compliance of the cell body. The experimental method and analysis were formulated around controlled assembly and detachment of a pair of macroscopically smooth red blood cell surfaces. The approach provides precise measurement of the membrane tension applied at the perimeter of an adhesive contact and the contact angle theta c between membrane surfaces which defines the mechanical leverage factor (1-cos theta c) important in the definition of the work to separate a unit area of contact. Here, the method was applied to adhesion and detachment of red cells bound together by different monoclonal antibodies to red cell membrane glycophorin and the snail-helix pomatia-lectin. For these tests, one of the two red cells was chemically prefixed in the form of a smooth sphere then equilibrated with the agglutinin before the adhesion-detachment procedure. The other cell was not exposed to the agglutinin until it was forced into contact with the rigid cell surface by mechanical impingement. Large regions of agglutinin bonding were produced by impingement but no spontaneous spreading was observed beyond the forced contact. Measurements of suction force to detach the deformable cell yielded consistent behavior for all of the agglutinins: i.e., the strength of adhesion increased progressively with reduction in contact diameter throughout detachment. This tension-contact diameter behavior was not altered over a ten-fold range of separation rates. In special cases, contacts separated smoothly after critical tensions were reached; these were the highest values attained for tension. Based on measurements reported in another paper (Evans et al. 1991. Biophys. J. 59:838-848) of the forces required to rupture molecular-point attachments, the density of cross-bridges was estimated with the assumption that the tension was proportional to the discrete rupture force x the number of attachments per unit length. These estimates showed that only a small fraction of agglutinin formed cross-bridges at initial assembly and increased progressively with separation. When critical tension levels were reached, it appeared that nearly all local agglutinin was involved as cross-bridges. Because one cell surface was chemically fixed, receptor accumulation was unlikely; thus, microscopic "roughness" and steric repulsion probably modulated formation of cross-bridges on initial contact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contact formation and transfer of mannose BSA gold from macrophages to cocultured fibroblasts

When macrophages were cocultured with fibroblasts many of the cells formed firm contacts. In some of these contacts both cell types were closely apposed and in others they were more clearly separated with numerous pseudopodia extending from macrophages toward the fibroblasts. Many small vesicles similar in structure to caveoli were observed immediately beneath the plasma membrane of some fibroblasts in regions immediately adjacent to areas of contact with macrophages. The membrane integrity of both cell types was always maintained and no connecting cytoplasmic strands were observed between contacting cells. Junctions were freely permeable to ruthenium red and less permeable to the larger cationized ferritin. Gold conjugated to mannose BSA was taken up readily by macrophages but not by fibroblasts. When fibroblasts were cocultured with macrophages that had been labeled with endocytosed gold, increasing amounts were transferred to them. Gold was observed within gaps formed between cocultured cells and within recipient fibroblasts in vesicles anatomically similar to lysosomes. These points of contact thus appear to provide a series of specialized protected clefts into which directed exocytosis of ligands from donor cells can take place and from which endocytosis into recipient cells is facilitated.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Fission and refusion of red blood cells using a micropipette technique

In micropipette experiments with small capillaries and moderate high pressure difference (approximately 1000 Pa) cell fragmentation (fission) of human red blood cells without hemolysis was observed by TV-system for a large number of fresh red blood cells of different donors. After separation, the fragment moves away from the residual cell. In seven cases this process was evaluated quantitatively and was shown that the rate of the fragment was constant in time. Two mechanisms for this phenomenon are discussed. In particular cases a spontaneous re-fusion with the residual cell body in the capillary can be observed. In our opinion probably protein-depleted membrane surfaces arise and membrane fusion is possible simply by mechanical contact without additional electric fields and/or fusion agents.     

A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis.

Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.     

Sequential translocation of an artificial precursor protein across the two mitochondrial membranes.

We have constructed a chimeric mitochondrial precursor protein consisting of a mutant bovine pancreatic trypsin inhibitor coupled to the C terminus of a purified artificial precursor protein. This construct fails to complete its import into isolated mitochondria and becomes stuck across sites of close contact between the two mitochondrial membranes. When the mitochondria are then depleted of ATP and the intramolecular disulfide bridges of the trypsin inhibitor are cleaved by dithiothreitol, the trypsin inhibitor moiety is transported across the outer membrane into the intermembrane space. This translocation intermediate can be chased across the inner membrane by restoring the ATP levels in the matrix. These results show that translocation of pancreatic trypsin inhibitor across a biological membrane is prevented by its intramolecular disulfide bridges, that import into the matrix involves two distinct translocation system operating in tandem, and that ATP is required for protein translocation across the inner but not the outer membrane.     

Decoration with myosin subfragment-1 disrupts contacts between microfilaments and the cell membrane in isolated Dictyostelium cortices

We used isolated cortices from ameboid cells of Dictyostelium discoideum to examine the structural nature of attachments between microfilaments and the cell membrane and to determine the effect of myosin subfragment-1 (S-1) on such contacts. By varying several parameters in our previously described isolation procedure (Condeelis, J., 1979. J. Cell Biol., 80:751-758), we have improved this procedure and have been able to isolate stable cortices. In this paper we identify two types of contact sites between microfilaments and the cell membrane similar to those seen in the brush border of intestinal epithelial cells: (a) an end-on attachment between the barbed end of actin filaments and the cell membrane; and (b) a lateral attachment mediated by rod-shaped bridges measuring approximately 6 X 15 nm. The spacing between bridges averages 36 nm, which suggests that the helical twist of the actin filament influences bridge location. Together these contacts account for an average of approximately 25,000 attachments per cell. Incubation of cortices with concentrations of S-1 sufficient to saturate binding sites on the microfilaments caused disruption of the contacts. This observation was confirmed by quantitative morphometry to show a threefold loss in the number of contact sites following S-1 decoration. These results indicate that S-1 decoration should be used with caution when information about the precise location of microfilaments and their attachment to the membrane is required.     

A quantitative ultramorphological approach for systematic assessment of sperm head regions: an example in rams

Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.     

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells

We detected cell-to-cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 microm in diameter, and from a few microns to at least 50-100 microm in length. Time-lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1-3 microm in diameter) between adjacent cells. Immunofluorescence detected alpha-tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anti-cancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.     

Affinity of red blood cell membrane for particle surfaces measured by the extent of particle encapsulation.

An experimental technique and a simple analysis are presented that can be used to quantitate the affinity of red blood cell membrane for surfaces of small beads or microsomal particles up to 3 micrometers Diam. The technique is demonstrated with an example of dextran-mediated adhesion of small spherical red cell fragments to normal red blood cells. Cells and particles are positioned for contact by manipulation with glass micropipets. The mechanical equilibrium of the adhesive contact is represented by the variational expression that the decrease in interfacial free energy due to a virtual increase in contact area is balanced by the increase in elastic energy of the membrane due to virtual deformation. The surface affinity is the reduction in free energy per unit area of the interface associated with the formation of adhesive contact. From numerical computations of equilibrium configurations, the surface affinity is derived as a function of the fractional extent of particle encapsulation. The range of surface affinities for which the results are applicable is increased over previous techniques to several times the value of the elastic shear modulus. It is shown that bending rigidity of the membrane has little effect on the analytical results for particles 1--3 micrometers Diam and that results are essentially the same for both cup- and disk-shaped red cells. A simple analytical model is shown to give a good approximation for surface affinity (normalized by the elastic shear modulus) as a function of the fractional extent of particle encapsulation. The model predicts that a particle would be almost completely vacuolized for surface affinities greater than or equal to 10 times the elastic shear modulus. Based on an elastic shear modulus of 6.6 x 10(-3) dyn/cm, the range for the red cell-particle surface affinity as measured by this technique is from approximately 7 x 10(-4) to 7 x 10(-2) erg/cm2. Also, an approximate relation is derived for the level of surface affinity necessary to produce particle vacuolization by a phospholipid bilayer surface which possesses bending rigidity and a fixed tension.     

Cross bonding and stiffening of the red cell membrane

Cross bonding and stiffening of the human red cell membrane was studied using treatments with SH, amino, and carboxyl reagents, oxidizing and denaturing treatments and acidification. Membrane cross bonding was initiated when, after red cell treatment, opposite areas of the cytoplasmic face of the red cell membrane were brought into contact by cell shrinking. Membrane cross bonding was detected by light microscopy when this contact persisted upon swelling the cells in a hypotonic medium. Membrane stiffening was recorded as a decrease in elongation of red cells in the shear field of a viscous dextran solution. No correlation was found between membrane cross bonding and membrane stiffening. The results are explained by the existence of two modifications of spectrin, type I causing solely membrane stiffening, type II causing membrane cross bonding as well as membrane stiffening. The amino and carboxyl reagents caused only type I modification. The other treatments caused both types of modification although with varying proportions. The results support the previously suggested mechanism of membrane cross bonding which involves a rearrangement of spectrin similar to denaturation by heat or urea, a decrease in associations within the membrane skeletal network, and a lateral aggregation of membrane proteins. These changes are proposed to occur by the type II modification. The data further substantiate the membrane stiffening effect of inter- and intra-molecular cross linking of spectrin which is identified with the type I modification. Finally, hypotheses are presented concerning the mechanism of membrane stiffening due to type II modifications of spectrin.     

Ultrastructural characterization of capsulated Haemophilus influenzae type b and two spontaneous nontypable mutants.

Capsulated Haemophilus influenzae type b and two spontaneous mutants (classes I and II variants) were characterized by transmission and scanning electron microscopy. When cells were treated with type b-specific antiserum prior to manipulations for electron microscopy, sectioned capsulated cells had electron-dense, fibrous capsular antigen-antibody complexes around them. In negatively stained preparations, the complexes appeared as electron-transparent zones surrounding cells. In contrast, only residual electron-dense, extracellular material was seen in sectioned, untreated, capsulated cells, and electron-dense "bridges" connected adjacent cells in negatively stained preparations. No extracellular capsular material was seen around the class I and II variants. Characteristic electron-translucent regions were always observed within the cytosol of the class I cells, both in thin sections and by negative staining. These areas were located adjacent to the cell envelope separating the plasma membrane from the dense cytoplasmic matrix. At times, electron-dense, thread-like material extended from the dense cytoplasmic matrix to the plasma membrane. No such regions were seen in the capsulated and class II cells. Class I cells fixed with methanol or suspended in NaCl or phosphate-buffered saline prior to treatment with fluorescein-tagged type b-specific antiserum (FTA reagent) exhibited, by immunofluorescence, patches of capsular antigen along their sides. However, when fixed with glutaraldehyde or OsO4 or suspended in tris-(hydroxymethyl)aminomethane plus Ca2+ buffer prior to treatment with FTA reagent, no patches of capsular antigen were seen. Subsequent exposure of the latter cells to methanol followed by treatment with FTA reagent resulted in the reappearance of the patches of capsular antigen. Thus, in the class I variant the capsular antigen is unlikely to be surface located. Scanning electron microscopy revealed that class I and II variant cells within undisturbed colonies were regularly aligned side-by-side, whereas cells within colonies of the capsulated strain were randomly distributed.     

The kinetics of haemolysis of spherocytic erythrocytes

Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.     

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.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.