Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM

Abstract

Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.

K Ca 2 channel localization and regulation in the axon initial segment

“…We additionally corroborated this finding through the use of single‐molecule AFM (18–21). An AFM‐based approach allows for the quantitative measurement of surface K Ca 2 channels in live neurons with nanometer resolution (20‐40 nm).…”

Nano-scientific Application of Atomic Force Microscopy in Pathology: from Molecules to Tissues

“…Moreover, metal ions such as zinc (II) and copper (II) are implicated with increased aggregation of the Aβ peptide into toxic oligomers, accelerating the pathogenesis of Alzheimer's disease [22,23]. Thus, the AFM-based studies attempted to investigate whether the disruption of the cell-cell/ substrate adhesions affects the progression of these diseases [24][25][26][27][28].…”

Microfluidic experimental setup for adhesion and recovery measurements of red blood cells in sickle cell disease

“…where the expression of active adhesion receptors was measured (Maciaszek et al, 2012;Zhang et al, 2017). Because of this, we expect that the same level of wall shear stress will not have the same effect on all RBCs in terms of detachment from the substrate.…”

“…It has been shown that the adhesive behavior and viscoelastic response of SS-RBCs are important factors in vaso-occlusion (Evans et al, 1984). SS-RBC adhesion to specific proteins, such as human integrin α β v 3 , expressed on the endothelium has been implicated as part of the mechanism that triggers vaso-occlusive crises (Zennadi et al, 2004;Zhang et al, 2017). During a vaso-occlusive crisis adherent SS-RBCs in the vasculature obstruct other SS-RBCs due to their viscoelastic and mechanical properties (Hillery and Panepinto, 2004;Quinn et al, 2011).…”

“…A number of experimental tools have been used to quantify SS-RBC adhesion to proteins expressed on the endothelium. To this end, atomic force microscopy (AFM) has been employed in single-molecule force spectroscopy (SMFM) and single-cell force spectroscopy (SCFS) assays (Maciaszek et al, 2012;Maciaszek et al, 2014;Zhang et al, 2017). These assays offer insight into molecular level interactions, but only a few RBCs can be tested this way under each condition.…”