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Mechanical properties have been proven to be a pivotal parameter to enhance our understanding of living systems. While research during the last decades focused on cells and tissues, little is known about the role of organelle mechanics in cell function. Here, mitochondria are of specific interest due to their involvement in numerous physiological and pathological processes, e.g., in the production and homeostasis of reactive oxygen species (ROS). Using real-time fluorescence and deformability cytometry, we present a microfluidic technology that is capable to determine the mechanical properties of individual mitochondria at a throughput exceeding 100 organelles per second. Our data on several thousands of viable mitochondria isolated from rat C6 glial cells yield a homogenous population with a median deformation that scales with the applied hydrodynamic stress. In two proof-of-principle studies, we investigated the impact of exogenously and endogenously produced ROS on mitochondria mechanics. Exposing C6 cells to hydrogen peroxide (H2O2) triggers superoxide production and leads to a reduction in mitochondria size while deformation is increased. In a second study, we focused on the knockout of tafazzin, which has been associated with impaired remodeling of the mitochondrial membrane and elevated levels of ROS. Interestingly, our results reveal the same mechanical alterations as observed after the exposure to H2O2, which points to a unified biophysical mechanism of how mitochondria respond to the presence of oxidative stress. In summary, we introduce high-throughput mechanical phenotyping into the field of organelle biology with potential applications for understanding sub-cellular dynamics that have not been accessible before.
The capability to parameterize shapes is of essential importance in biomechanics to identify cells, to track their motion, and to quantify deformation. While various shape descriptors have already been investigated to study the morphology and migration of adherent cells, little is known of how the mathematical definition of a contour impacts the outcome of rheological experiments on cells in suspension. In microfluidic systems, hydrodynamic stress distributions induce time-dependent cell deformation that needs to be quantified to determine viscoelastic properties. Here, we compared nine different shape descriptors to characterize the deformation of suspended cells in an extensional as well as shear flow using dynamic real-time deformability cytometry. While stress relaxation depends on the amplitude and duration of stress, our results demonstrate that steady-state deformation can be predicted from single cell traces even for translocation times shorter than their characteristic time. Implementing an analytical simulation, performing experiments, and testing various data analysis strategies, we compared single cell and ensemble studies to address the question of computational costs vs experimental accuracy. Results indicate that high-throughput viscoelastic measurements of cells in suspension can be performed on an ensemble scale as long as the characteristic time matches the dimensions of the microfluidic system. Finally, we introduced a score to evaluate the shape descriptor-dependent effect size for cell deformation after cytoskeletal modifications. We provide evidence that single cell analysis in an extensional flow provides the highest sensitivity independent of shape parametrization, while inverse Haralick's circularity is mostly applicable to study cells in shear flow.
Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.
Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell–cell and cell‐matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F‐actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F‐actin density. Instead, we observe changes in the organization of F‐actin indicated by the appearance of blebs in the semi‐adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.
Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.
Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates
(2022)
Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.
Abstract
Differentiation of cardiac progenitor cells (CPC) into cardiomyocytes is a fundamental step in cardiogenesis, which is marked by changes in gene expression responsible for remodeling of the cytoskeleton and in altering the mechanical properties of cells. Here we have induced the differentiation of CPC derived from human pluripotent stem cells into immature cardiomyocytes (iCM) which we compare with more differentiated cardiomyocytes (mCM). Using atomic force microscopy and real‐time deformability cytometry, we describe the mechanodynamic changes that occur during the differentiation process and link our findings to protein expression data of cytoskeletal proteins. Increased levels of cardiac‐specific markers as well as evolution of cytoskeletal morphology and contractility parameters correlated with the expected extent of cell differentiation that was accompanied by hypertrophic growth of cells. These changes were associated with switching in the balance of the different actin isoforms where β‐actin is predominantly found in CPC, smooth muscle α‐actin is dominant in iCM cells and sarcomeric α‐actin is found in significantly higher levels in mCM. We link these cytoskeletal changes to differences in mechano‐dynamic behavior of cells that translate to changes in Young's modulus that depend on the cell adherence. Our results demonstrate that the intracellular balance of actin isoform expression can be used as a sensitive ruler to determine the stage of differentiation during early phases of cardiomyocyte differentiation that correlates with an increased expression of sarcomeric proteins and is accompanied by changes in cellular elasticity.
Objective
Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life –threatening organ complications, such as respiratory and renal failure, is unknown.
Design
Organ dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification.
Results
Similar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation.
Conclusion
In AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.
Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1
−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1
−/−
BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1
−/−
BOECs which was observed in CCM1
+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.