The buckling instability of aggregating red blood cells

Plasma proteins such as fibrinogen induce the aggregation of red blood cells (RBC) into rouleaux, which are responsible for the pronounced shear thinning behavior of blood, control the erythro- cyte sedimentation rate (ESR) a common hematological test and are involved in many situations of physiological relevance such as structuration of blood in the microcirculation or clot formation in pathological situations. Confocal microscopy is used to characterize the shape of RBCs within rouleaux at equilibrium as a function of macromolecular concentration, revealing the diversity of contact zone morphology. Three different configurations that have only been partly predicted before are identified, namely parachute, male-female and sigmoid shapes, and quantitatively recovered by numerical simulations. A detailed experimental and theoretical analysis of clusters of two cells shows that the deformation increases nonlinearly with the interaction energy. Models indicate a forward bifurcation in which the contacting membrane undergoes a buckling instability from a flat to a de- formed contact zone at a critical value of the interaction energy. These results are not only relevant for the understanding of the morphology and stability of RBC aggregates, but also for a whole class of interacting soft deformable objects such as vesicles, capsules or cells in tissues.Comment: 22 pages, 12 figure

On the rheology of red blood cell suspensions with different amounts of dextran: separating the effect of aggregation and increase in viscosity of the suspending phase

We investigate the shear thinning of red blood cell - dextran suspensions. Microscopic images show that at low polymer concentration, aggregation increases with increasing concentration until it reaches a maximum and then decreases again to non-aggregation. This bell shape dependency is also deduced from the rheological measurements, if the data are correctly normalized by the viscosity of the suspending phase since a significant amount of polymers adsorb to the cell surfaces. We find that the position of the maximum of this shear rate dependent bell shape increases with increasing viscosity of the suspending phase, which indicates a that the dynamic process of aggregation and disaggregation is coupled via hydrodynamic interactions. This hydrodynamic coupling can be suppressed by characterizing a suspension of 80% hematrocrit which yields good agreement with the results from the microscopical images.Comment: acceptd for publication in Rheologica Act

Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane

Evidence of in vivo exogen protein uptake by red blood cells: a putative therapeutic concept

For some molecular players in red blood cells (RBCs), the functional indications and molecular evidence are discrepant. One such protein is transient receptor potential channel of canonical subfamily, member 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse RBCs and its absence in human RBCs. We transfused TRPC6 knockout RBCs into wild-type mice and performed functional tests. We observed the “rescue” of TRPC6 within 10 days; however, the “rescue” was slower in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of RBCs with the opposed surface at around 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to RBCs as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving RBCs, such as hereditary xerocytosis or Gardos channelopathy

Sparteine-Free, Highly Stereoselective Construction of Complex Allylic Alcohols Using 1,2-Metallate Rearrangements

Stereotriads bearing allylic alcohols are privileged structures in natural products, and new methods accessing these in a stereoselective fashion are highly sought after. Toward this goal, we found that the use of chiral polyketide fragments allows for performing the Hoppe-Matteson-Aggarwal rearrangement in the absence of sparteine with high yields and diastereoselectivities, rendering this protocol a highly valuable alternative to the Nozaki-Hiyama-Takai-Kishi reaction. The switch of directing groups in most cases resulted in the reversed stereochemical outcome, which could be explained by conformational analysis on density functional theory level and a Felkin-like model

A novel universal algorithm for filament network tracing and cytoskeleton analysis

The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software

A novel universal algorithm for filament network tracing and cytoskeleton analysis

The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software

Physikalische Charakterisierung der Aggregation roter Blutzellen

In this work, five aspects of the red blood cell aggregation induced by macromolecules are investigated. A rheological approach focused on the normalization of viscosity as a function of the macromolecular adsorption rates using a commercial rheometer is proposed. Derived from that approach, the yield stress of aggregating red blood cell suspensions is investigated. The sedimentation rates of the utilized biological system are then studied. Microscopical investigations, including measurements of the microscopical aggregation index, lead to the conclusion that the C-reactive protein, a plasma protein, does not influence the aggregation behavior of red blood cells. Detailed microscopical studies on the morphology of the interaction zones of aggregated red blood cells show that these strongly depend on the macromolecular concentration in good agreement with numerical simulations that allow to derive an approximation of the interaction energies. The latter are also directly measured with single cell force spectroscopy using an atomic force microscope with the additional result that the viscosity of the surrounding medium can influence the results significantly. Finally, the physical origin of aggregation is discussed and supported by several additional measurements. This allows to combine two existing theories and explain the bell-shape of interaction energy versus macromolecular concentration curve in a new way.In dieser Arbeit werden fünf Aspekte der makromolekül-induzierten Aggregation roter Blutzellen untersucht. Ein rheologischer Ansatz unter Verwendung eines kommerziellen Rheometers behinhaltet die Viskositätsnormierung als Funktion der Adsorptionsraten von Makromolekülen. Hiervon abgeleitet werden die Fließspannung von Suspensionen aggregierter roter Blutzellen untersucht. Außerdem werden die Sedimentationsraten des verwendeten biologischen Systems analysiert. Mikroskopische Untersuchungen, einschließlich des mikroskopischen Aggregationsindexes, führen zu dem Schluss, dass das C-reaktive Protein (ein Plasmaprotein) die Aggregationseigenschaften roter Blutzellen nicht beeinflusst. Detaillierte mikroskopische Studien der Morphologie von Interaktionszonen aggregierter roter Blutzellen zeigen eine starke Abhängigkeit von der molekularen Konzentration, was gut mit numerischen Simulationen, die eine Annäherung an die Interaktionsenergien zulassen, übereinstimmt. Letztere wurden direkt mit Einzelzellkraftmikroskopie unter Verwendung eines Atomkraftmikroskops gemessen, was zu dem Resultat führt, dass die Viskosität des umgebenden Mediums einen signifikanten Einfluss auf die Ergebnisse haben kann. Am Ende wird der physikalische Ursprung der Aggregation diskutiert und durch verschiedenen Messungen ergänzt. Dies erlaubt es, beide existierenden Theorien zu vereinen und die Glockenkurve der Interaktionsenergie als Funktion der Makromolekülkonzentration auf eine neue Weise zu erklären

La caractérisation physique de l'agrégation des globules rouges

In this work, five aspects of the red blood cell aggregation induced by macromolecules are investigated. A rheological approach focused on the normalization of viscosity as a function of the macromolecular adsorption rates using a commercial rheometer is proposed. Derived from that approach, the yield stress of aggregating red blood cell suspensions is investigated. The sedimentation rates of the utilized biological system are then studied. Microscopical investigations, including measurements of the microscopical aggregation index, lead to the conclusion that the C-reactive protein, a plasma protein, does not influence the aggregation behavior of red blood cells. Detailed microscopical studies on the morphology of the interaction zones of aggregated red blood cells show that these strongly depend on the macromolecular concentration in good agreement with numerical simulations that allow to derive an approximation of the interaction energies. The latter are also directly measured with single cell force spectroscopy using an atomic force microscope with the additional result that the viscosity of the surrounding medium can influence the results significantly. Finally, the physical origin of aggregation is discussed and supported by several additional measurements. This allow to combine two existing theories and explain the bell-shape of interaction energy versus macromolecular concentration curve in a new way.Ce travail a été réalisé autour de cinq aspects de l’agrégation des globules rouges (RBCs) sanguins induite par des macromolécules. Une approche rhéologique, ciblée sur la normalisation de la viscosité en fonction du taux d’adsorption des macromolécules et mesurée par un rhéomètre commercial, est proposée. Par cette approche, la contrainte seuil de suspensions de cellules sanguines agrégées est aussi évaluée. De plus, les taux de sédimentation des solutions biologiques utilisées sont aussi mesurés. Nos données microscopiques, incluant des mesures d’indice d’agrégation microscopique, ont eu pour conclusion que la protéine C réactive, une protéine du plasma, n’a pas d’influence sur le phénomène d’agrégation des RBCs. Des mesures microscopiques détaillées de la morphologie des zones de contact des RBCs ont montrées que ces dernières dépendent fortement de la concentration de macromolécules, en accord avec des simulations numériques dont ont pu être extraites des valeurs d’énergie d’interaction. Ces dernières ont en outre pu être directement mesurées par microscopie à force atomique avec pour résultat supplémentaire que la viscosité du milieu peut influencer la mesure de manière significative. Enfin, l’origine physique de l’agrégation est discutée et confirmée par des mesures additionnelles. Ceci permet de concilier deux théories et permet d’expliquer la forme en cloche de l’énergie d’interaction en fonction de la concentration en macromolécules d’une nouvelle manière