Lectin-mediated agglutination of liposomes containing glycophorin: Effects of acyl chain length

Glycophorin from human erythrocytes has been incorporated into liposomes of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The thermal properties of unsonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42±22 (DMPC), 197±28 (DPPC) and 240±64 (DSPC). The initial rates of agglutination of sonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 by wheat germ agglutinin in the concentration range 0–7 μM have been measured over a range of temperature. Below the gel-to-lamellar phase transition (T_c) the rates of agglutination increase with acyl chain length in the sequence DMPC < DPPC < DSPC. Agglutination is found to be second order in liposome concentration and is completely reversed on saturation of the wheat germ agglutinin-binding sites by N-acetylglucosamine. Agglutination rates decrease with increasing temperature below T_c and are largely independent of temperature above T_c. The results are discussed in relation to the clustering of glycophorin in the phospholipid bilayers and its effect on binding and subsequent interliposomal bridge formation by wheat germ agglutinin.     

Membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study

Melittin-induced membrane fusion between neutral and acidic phospholipids was examined in liposome systems with a high-sensitivity differential scanning calorimeter. Membrane fusion could be detected by calorimetric measurement by observing thermograms of mixed liposomal lipids. The roles of hydrophobic and electrostatic interactions were investigated in membrane fusion induced by melittin. Melittin, a bee venom peptide, is composed of a hydrophobic region including hydrophobic amino acids and a positively charged region including basic amino acids. When phosphatidylcholine liposomes were prepared in the presence of melittin, reductions in the phase transition enthalpies were observed in the following order; dimyristoylphosphatidylcholine (DMPC) > dipalmitoylphosphatidylcholine (DPPC) > distearoylphosphatidylcholine (DSPC) > dielaidoylphosphatidylcholine (DEPC). The plase transition enthalpy of an acidic phospholipid, dipalmitoylphosphatidylserine (DPPS), was raised by melittin at low concentrations, then reduced at higher concentrations. DPPC liposomes prepared in melittin solution were fused with DPPS liposomes when the liposomal dispersions were mixed and incubated. Similar fusion was observed between dipalmitoylphosphatidylcholine and dimyristoylphosphatidic acid (DMPA) liposomes. These results indicate that a peptide including hydrophobic and basic regions can mediate membrane fusion between neutral and acidic liposomes by hydrophobic and electrostatic interactions.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca²⁺-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca²⁺ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca²⁺ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca²⁺ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca²⁺ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca²⁺, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca²⁺ concentration, and identical to that with pure phosphatidylserine in excess Ca²⁺. The results imply that Ca²⁺-induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

A raman study of the interaction of Mg2+, Ca2+, and Ba2+ ions with an acidic model membrane

The interaction of dipalmitoylphosphatidylgly cerol DPPG) liposomes with divalent ions of magnesium, calcium and barium has been investigated with laser-Raman spectroscopy over the temperature range of 0–60°C. The effect of Ca²⁺ ions was also investigated as a function of concentration. At a Ca²⁺/DPPG molar ratio of 0.1, the number of trans carbon to carbon bonds in the hydrocarbon domain of the phospholipid and the lateral order of the hydrocarbon chains was increased both below and above the gel to liquid crystal transition. At higher Ca²⁺ concentrations the number of trans bonds and the lateral order is further increased over the entire temperature range studied, while the transition disappears. Magnesium and barium ions have a much smaller ordering effect on the side-chain packing of DPPG liposomes. At a molar ratio of 0.3, the gel to liquid crystal transition is still discernible for DPPG liposomes in the presence of Ba²⁺ ions, but not in the presence of Mg²⁺ ions.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Kinetics of calcium-induced mixing of lipids and aqueous contents of large unilamellar phosphatidylserine vesicles

Calcium ion-induced fusion events in suspensions of large unilamellar phosphatidylserine (PS) liposomes were monitored by fluorescence methods. Mixing of vesicle contents was studied by measuring the increase in terbium emission intensity due to formation of a complex between Tb³⁺ ions and dipicolinic acid trapped in the liposomes. Lipid redistribution was determined with the aid of the resonance transfer of excitaton energy using dipalmitoylphosphatidylethanolamine labelled with the donor N-(7-nitro-2,1,3-benzoxadiazol-4-yl) or the acceptor tetramethylrhodamine at the free amino group. The two methods yielded significantly different results. While recombination of contents could not be detected at Ca²⁺ concentrations below 2.5 mM the threshold concentration for lipid mixing was 1 mM. For saturating Ca²⁺ concentrations (>5 mM Ca²⁺) initial rates were higher by almost an order of magnitude for lipid mixing than for recombination of liposome contents. These observations indicate that the observation of rapid lipid mixing phenomena does not allow one to draw conclusions as to the fate of the enclosed volumes.     

Inhibition by Liposomal Cholesterol of Streptococcus Pneumoniae Induced Lung Epithelial Cell Damage

Abstract

Streptococcus pneumoniae was shown to be capable of lysing A549 cells in culture. Membrane damage to cells as assessed by trypan blue exclusion increased with increasing concentration of bacteria. After 45 min of incubation with 7.5 × 10⁸ bacteria/ml less than 20% of A549 cells excluded trypan blue. The lytic activity of S. pneumoniae was inhibited by phosphatidylcholine liposomes containing cholesterol. Using an haemolysis assay and S. pneumoniae's culture filtrates, the efficiency of the anti-lytic activity of liposomes was found to be distearoylphosphatidylcholine (DSPC) > dipalmitoylphosphatidylcho-line (DPPC) > dimyristoylphosphatidylcholine (DMPC). Furthermore, the anti-lytic activity also depended on the cholesterol content in a non-trivial manner. There was no protection against haemolytic activity at cholesterol content of less than 20% for DSPC and 35 mole% for DPPC and DMPC liposomes respectively. Above these threshold values inhibition of lytic activity increased sharply. In agreement with the haemolysis results, A549 cells were protected by liposomes against the lytic activity of S. pneumoniae with the efficiency also being DSPC > DPPC > DMPC. Clearly the efficiency of liposomal cholesterol is increased with increasing gel to liquid crystalline phase transition temperature of the lipid matrix. The results suggest that liposomal cholesterol may be used to protect the host against cell damage caused by S. pneumoniae.     

The interaction of mequitazine with phospholipid model membranes

The effect of increasing concentrations of mequitazine, a quinuclidinylmethyl-phenothiazine, on the phase transition temperature (T_c), the broadening of the transition peak, the enthalpy and entropy of transition of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholine (DPPC) liposomes was studied. Pest critical micelle concentrations of mequitazine (CMC = 5.23 X 10^?2M), caused broadening of the transition peak and lowering of the T_c of pure liposomes. The ratio of peak heights from the nuclear magnetic resonance (NMR) spectra of egg phosphatidycholine liposomes was used as a criterion for assessing the interaction of the drug with phospholipid membranes. Mequitazine interacts with both the polar head groups and hydrophobic membrane interior.     

Ethanol induces interdigitated gel phase (L beta I) between lamellar gel phase (L beta') and ripple phase (P beta') in phosphatidylcholine membranes: a scanning density meter study

Effects of ethanol on dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) dispersions were investigated with an automated scanning density meter and a differential scanning calorimeter (DSC). The temperature-dependent profile of specific volume measured by the density meter clearly exhibited phase transitions of the DPPC and the DSPC dispersions as drastic changes in the thermal expansion coefficients. On increasing the ethanol concentration in the DPPC dispersions, the pretransition temperature was reduced faster than the main transition temperature was. An interdigitated gel phase (L beta I) appeared as a region of lower specific volume at the pretransition temperature when the ethanol concentration reached 40 mg/ml. The L beta I phase spread both its ends in an ethanol-dependent fashion, and the high-temperature end merged to the main transition at 50 mg/ml of ethanol. The temperature-ethanol phase diagram has been determined for DPPC. The transitions L beta' to L beta I and from L beta I to P beta' were also observed on the thermograms of DSC measurements. In the DSPC dispersions, the L beta I phase was induced between the L beta' and the P beta' phases by a lower ethanol concentration (about 20 mg/ml).     

Control of membrane fusion by phospholid head groups I. Phosphatidate/phosphatidylinositol specificity

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca²⁺ or Mg²⁺ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90° light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca²⁺ and Mg²⁺ at threshold concentration ranges of 0.03–0.1 mM (Ca²⁺) and 0.07–0.15 mM (Mg²⁺) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca²⁺ or Mg²⁺ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5–10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.     

The gelation kinetics of platelet extracts

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca²⁺ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca²⁺ concentration up to about 2 μM. At 4–15 μM free Ca²⁺, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca²⁺ concentrations. Trifluoperazine (50 μM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca²⁺ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K⁺, ATP, cytochalasin E and colchicine. K⁺ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelations, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 μM) and colchicine (1 mM) interfered with the gelation process.     

Ionic requirements of impaled bull spermatozoa driven by external ADP and ATP

The flagellar motion of impaled bull spermatozoa can be maintained by external ADP or ATP. The post-impalement flagellar frequency depends sharply on the external Mg²⁺ concentration. 0.3 mM Mg²⁺ is required for half-optimal activity with ADP, 0.05 mM Mg²⁺ with ATP as external power source. Mn²⁺ can substitute partially for Mg²⁺ as ionic co-factor. Ca²⁺ cannot substitute for Mg²⁺, and at concentrations above 0.5 mM it inhibits motility slightly. Zn²⁺ acts only as inhibitor of post-impalement flagellar activity, reducing it to zero at concentrations above 1 mM.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Liposomes for cryopreservation of bovine sperm

In this study, the effect of various unilamellar liposomes on cryopreservation of bovine spermatozoa has been investigated. Liposomes were composed of saturated lipids with various acyl chain lengths: DSPC (18:0), DPPC (16:0), DMPC (14:0), or DLPC (12:0). Alternatively, liposomes were prepared using unsaturated egg phosphatidylcholine (EPC) or DOPC (18:1, neutral), alone or in combination with lipids with various head groups: DOPS (negatively charged), DOPG (negatively charged), and DOPE (neutral). Fourier transform infrared spectroscopy studies showed that bovine sperm membranes display a gradual phase transition from 10 to 24 ^oC. Phase transition temperatures of the liposomes varied from −20 to +53 ^oC. Sperm was incubated in the presence of liposomes for either 6 or 24 h at 4 °C prior to freezing. Postfreeze survival rates were determined based on the percentage of progressively motile cells as well as the percentage of acrosome- and plasma membrane-intact cells. With DOPC liposomes a postthaw progressive motility of 43% was obtained compared with 59% using standard egg yolk freezing extender. Postthaw progressive motility increased up to 52% using DOPC:DOPG (9:1) liposomes, whereas DOPC:DOPS or DOPC:DOPE liposomes did not increase survival compared with DOPC liposomes. Among the saturated lipids, only DMPC was found to increase cryosurvival, up to 44% based on progressive motility. DLPC liposomes caused a complete loss in cell viability, already prior to freezing, whereas DPPC and DSPC liposomes neither positively nor negatively affected cryosurvival. Taken together, the higher postthaw survival obtained with DOPC:DOPG liposomes as compared with DOPC liposomes can likely be attributed to increased liposome-sperm interactions between the charged phosphatidylglycerol groups and charged regions in the sperm membranes. Interestingly, the lipid phase state of the liposomes during preincubation is not the decisive factor for their cryoprotective action.     

CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES

The binding of glutamate decarboxylase (GAD), to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca²⁺ and Mg²⁺ concentrations was studied. Phosphatidylcho-line-phosphatidylserine (4:1) liposomes are capable of binding GAD in a Ca²⁺-dependent manner. The per cent of GAD bound increased from 5 to 65°., in a sigmoid shape with Ca²⁺ concentrations in the 0.2-4 mm range. Mg²⁺ also induces GAD binding but is less effective than Ca²⁺ The Ca²⁺ -dependent binding of GAD is not the result of unspecific association of protein, since Ca²⁺ did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore, the relative specific activity (^o_o enzyme activity/% protein) of GAD associated to liposomes increases 4-fold from 0 to 2 mm Ca²⁺. The per cent of GAD bound attains a plateau at a ratio phospholipid/protein of about 1.5. and decreases when the pH increases from 6.5 or 6.8 to 7 or 7.25. Na⁺ or K⁺ at a 100mm concentration also induce binding of GAD to liposomes. Phosphatidylcholine liposomes (without phosphatidylserine) practically did not bind GAD at any Ca²⁺ concentration. The Ca²⁺-dependent association of GAD to phosphatidylcholine-phosphatidylserine liposomes is very similar to that previously reported using brain membranes, and it correlates also well with the reported Ca²⁺-dependent aggregation of phosphatidylserine molecules in phospholipid membranes of similar composition. It is concluded that phosphatidylserine is probably involved in the Ca²⁺-dependent binding of GAD to brain membranes. Phospholipid vesicles seem to be a useful experimental model for studying the mechanisms of this GAD association to membranes and the possible physiological implications of the GAD-Ca²⁺-membrane interaction regarding the release of newly synthesized GABA from nerve endings.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Calcium uptake by placental plasma membrane vesicles

The Placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca²⁺. This is 24-fold higher than the external Ca²⁺ concentration.This process is dependent on ATP hydrolysis by the placental Ca²⁺-ATPase.The $\text{P}{}_{\mathrm{i}}\text{Ca}$ ratio is dependent on the external Ca²⁺ concentration, and reaches the value of 2 at 10 mM Ca²⁺.Phosphate (5 mM) can double Ca²⁺ uptake when measured in the presence of 5 mM Ca²⁺.Mg²⁺; increased Ca²⁺ uptake only at low Ca²⁺ concentrations, and had no significant effect at 5 mM Ca²⁺.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.