Spicule movement on RBCs during echinocyte formation and possible segregation in the RBC membrane

Melzak, Kathryn A.; Moreno‐Flores, Susana

doi:10.1016/j.bbamem.2020.183338

Cited by 3 publications

(12 citation statements)

References 79 publications

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“…6B, bottom graph). In line with the NM2 mechanism proposed here, spicules emerging at the initial stage of echinocytosis, i.e., when NM2 heads are still able to detach from and reattach to actin junctions, exhibit random movement over the RBC surface (Melzak et al, 2020). Since the RBC volume decreases during echinocytosis from 100 to ~ 75 fL (Chen et al, 2002), the large excess (~ 40%) of membrane area allows the formation of up to ~ 50-60 spicules per cell (Brecher and Bessis, 1972;Reinhart and Chien, 1986).…”

Section: Discussionsupporting

confidence: 69%

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Shirakashi

Sisario

Taban

et al. 2022

Biomech Model Mechanobiol

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Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.

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Section: Discussionsupporting

confidence: 69%

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Shirakashi

Sisario

Taban

et al. 2022

Biomech Model Mechanobiol

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“…Our findings highlight the dynamic formation and movement of single spicules on the membrane of ibuprofen-treated RBCs in real-time and provide evidence for a dose-dependent reversibility of RBC morphological alterations. The shape of the RBC is dependent on the interplay between the two main membrane components, which are the lipid bilayer and the spectrin cytoskeleton (44, 45). Thus, when the membrane asymmetry between the inner and the outer layers increases in favor of the outer layers, spicule formation is triggered as a natural response to the expansion of the outer leaflet coupled with the resistance of the cytoskeleton to the morphological distortion (44).…”

Section: Discussionmentioning

confidence: 99%

“…The theoretical elastic membrane energy model and available experimental data support the preferential initial spicule formation on the RBC contour due to the highest curvature of the cytoskeleton (44). Driven by the continuous expansion of the outer monolayer, specular structures tend to move from the rim of the cell towards regions with a lower curvature, including the central area where the distinctive dimple is lost following the progression of echinocytosis, and finally distribute uniformly around the cell membrane (45). RBCs treated with higher ibuprofen concentrations showed that specular structures are more likely to steadily stay in place towards the later stages of echinocytosis, when a sphero-echinocyte morphology prevails.…”

Section: Discussionmentioning

confidence: 99%

“…RBCs treated with higher ibuprofen concentrations showed that specular structures are more likely to steadily stay in place towards the later stages of echinocytosis, when a sphero-echinocyte morphology prevails. Before this occurs, the dynamic movement of spicules associated with increased membrane tension can induce spontaneous spicule splitting (45, 46). Here, a singular specular structure separates into two smaller daughter spicules as observed between 7:13 min and 7:36 min time points in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…5 N-P ) as the RBC shape returns to its discocyte morphology and the asymmetry between the two membrane leaflets is restored. Therefore, spicule motion tracking can provide real-time information on RBC nano-mechanics and it can act as a potential indicator for membrane bilayer defects (45). We suggest that in low ibuprofen concentration conditions, the rate of ibuprofen molecules interacting with the RBC membrane bilayer decreases over time, resulting in the transition back to a normocyte.…”

Section: Discussionmentioning

confidence: 99%

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Label-free digital holotomography reveals ibuprofen-induced morphological changes to red blood cells

Bergaglio

Bhattacharya²,

Thompson³

et al. 2022

Preprint

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Understanding the dose-dependent effect of over-the-counter drugs on red blood cells (RBCs) is crucial for hematology and digital pathology. Yet, it is challenging to continuously record the real-time, drug-induced nanoscopic shape changes of RBCs in a label-free manner. Here, we demonstrate digital holotomography (DHTM) enabled real-time, label-free concentration-dependent and time-dependent monitoring of ibuprofen on RBCs from a healthy donor. The RBCs are segmented based on 3D and 4D refractive index tomograms and their morphological and chemical parameters are retrieved with their shapes classified using machine learning. We directly observed the formation and motion of spicules on the RBC membranes when aqueous solutions of ibuprofen were drop cast on wet blood, creating rough-membraned echinocyte forms. At low concentrations of 0.25-0.50 mM, the ibuprofen-induced morphological change was transient but at high concentrations (1.5-3 mM) the spiculated RBC remained over a period of up to 1.5 hours. Molecular simulations confirmed that aggregates of ibuprofen molecules at high concentrations significantly disrupted the RBC membrane structural integrity and lipid order, but produced negligible effect at low ibuprofen concentrations. Control experiments on the effect of urea, hydrogen peroxide and aqueous solutions on RBCs showed zero spicule formation. Our work elucidates the dose-dependent chemical effects on RBCs using label-free microscopes that can be deployed for the rapid detection of overdosage of over-the-counter and prescribed drugs.

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Label-Free Digital Holotomography Reveals Ibuprofen-Induced Morphological Changes to Red Blood Cells

et al. 2023

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Understanding the dose-dependent effect of overthe-counter drugs on red blood cells (RBCs) is crucial for hematology and digital pathology. Yet, it is challenging to continuously record the real-time, drug-induced shape changes of RBCs in a label-free manner. Here, we demonstrate digital holotomography (DHTM)-enabled real-time, label-free concentration-dependent and time-dependent monitoring of ibuprofen on RBCs from a healthy donor. The RBCs are segmented based on three-dimensional (3D) and four-dimensional (4D) refractive index tomograms, and their morphological and chemical parameters are retrieved with their shapes classified using machine learning. We directly observed the formation and motion of spicules on the RBC membrane when aqueous solutions of ibuprofen were drop-cast on wet blood, creating rough-membraned echinocyte forms. At low concentrations of 0.25−0.50 mM, the ibuprofen-induced morphological change was transient, but at high concentrations (1−3 mM) the spiculated RBC remained over a period of up to 1.5 h. Molecular simulations confirmed that aggregates of ibuprofen molecules at high concentrations significantly disrupted the RBC membrane structural integrity and lipid order but produced negligible effect at low ibuprofen concentrations. Control experiments on the effect of urea, hydrogen peroxide, and aqueous solutions on RBCs showed zero spicule formation. Our work clarifies the dose-dependent chemical effects on RBCs using label-free microscopes that can be deployed for the rapid detection of overdosage of over-the-counter and prescribed drugs.

show abstract

Spicule movement on RBCs during echinocyte formation and possible segregation in the RBC membrane

Cited by 3 publications

References 79 publications

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Label-free digital holotomography reveals ibuprofen-induced morphological changes to red blood cells

Label-Free Digital Holotomography Reveals Ibuprofen-Induced Morphological Changes to Red Blood Cells

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