Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography ³, that topography limits adhesive bond formation with other surfaces ⁹, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface ^{3, 7}. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface ⁶. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface ⁴. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo ². The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa ⁵. The thickness and structure of the glycocalyx have been studied using electron microscopy ⁸, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.     

The mechanical transduction of physiological strength electric fields

In this article it is proposed that electric fields of physiological strength (approximately 100 V/m) are transduced by the mechanical torque they exert on glycoproteins. The resulting mechanical signal is then transmitted to the cytoskeleton and propagated throughout the cell interior. This mechanical coupling is analyzed for transmembrane glycoproteins, such as integrins and the glycocalyx, and for glycoproteins in the extracellular matrix of cartilage. The applied torque is opposed by viscous fluid drag and restoring forces exerted by adjacent molecules in the membrane or cartilage. The resulting system represents a damped, driven harmonic oscillator. The amplitude of oscillation is constant at low frequencies, but falls off rapidly in the range 1-1000 Hz. The transition frequency depends on parameters such as the viscosity of the surrounding fluid and the restoring force exerted by the surrounding structure. The amplitude increases as the fourth power of the length of the transmembrane glycoproteins and as the square of the applied field. This process may operate in concert with other transduction mechanisms, such as the opening of voltage-gated channels and electrodiffusion/osmosis for DC fields.     

Hepatitis C virus infection propagates through interactions between Syndecan‐1 and CD81 and impacts the hepatocyte glycocalyx

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan‐1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan‐1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan‐1 or Xylosyltransferase 2, a key enzyme of Syndecan‐1 biosynthesis. Simultaneous knockdown of Syndecan‐1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan‐1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan‐1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan‐1 and Xylosyltransferase 2 was altered within days post‐infection, and the remaining Syndecan‐1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.     

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

Jejunal malabsorption in the rat infected by the nematode Nippostrongylus brasiliensis

Symons L. E. A., Gibbins J. R. and Jones W. O., 1970. Jejunal malabsorption in the rat infected by the nematode Nippostrongylus brasiliensis. International Journal for Parasitology, 1: 179–187. The rate of jejunal absorption of a range of substances absorbed actively or by diffusion was depressed in the rat infected by the nematode Nippostrongylus brasiliensis. The degree of malabsorption of actively absorbed substances was directly related to the severity of the infection.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.     

Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (³H₂DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of ³H₂DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the ³H₂DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ_½ = 360 min) component representing 33% of the radioactivity, and a fast (τ_½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ_½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither ³H₂DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.     

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg²⁺ or Ca²⁺, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.     

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement

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Derepressive Formation of l-Leucine-Pyruvate Transaminase under Nitrogen-Free Conditions in Gluconobacter suboxydans

l-Leucine-pyruvate transaminase activity increased 6- to 20-fold in 3 hr when Gluconobacter suboxydans cells grown on yeast extract-medium were transferred to and incubated in a nitrogen-free medium. The increase in enzyme activity was influenced remarkably by the age and concentration of cells used. The phenomenon depended upon de novo synthesis of enzyme protein.

The enzyme activity in cell-free extracts of cells incubated under a nitrogen-free condition decreased remarkably after heat treatment at 50°C (pH 6.0) or after freezing and thawing. The level of such enzyme inactivation was high in extracts of cells in the early stages of induction and low in later stages.