Spherocytosis-Related L1340P Mutation in Ankyrin Affects Its Interactions with Spectrin

Previously, we reported a new missense mutation in the ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human β-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches—the study of association and dissociation responses of the spectrin–ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS

Spectrin translocates together with actin to the IS upon T-cell activation.

<p><b>(A-B)</b> Distribution of spectrin (red, 1) and actin (green, 2) in primary T lymphocytes from PBMCs population in the absence (naive) (A) or presence (activated) (B) of Dynabeads coated with anti-CD3 and anti-CD28, along with a merged (3) and a TL(4) image. The white rectangle marks the contact site. The scale bar = 5μm. The results are representative of at least three independent experiments. <b>(C-D)</b> Transmission electron micrographs of non-activated (C) and activated with Dynabeads coated with anti-CD3 and anti-CD28 (D) Jurkat T-cells labeled with antibodies directed against spectrin, followed by gold-conjugated secondary antibodies. The right column shows enlarged images of the marked areas. Arrows point to gold particles (spectrin). Almost all of the gold in panel D accumulate at the point of cell-bead contacts. Abbreviations: APC-antigen presenting cell simulated by Dynabeads coated with anti-CD3 and anti-CD28; N-nuclei, PM-plasma membrane, M-mitochondria. Magnification 30 000x, scale bar = 1 μm (200 000x, scale bar = 200 nm), indicated on micrographs.</p

Schematic of the immunological synapse (IS) and representative protein interactions in the synaptic space.

<p>Distribution of receptors and adhesion molecules in individual clusters in the immune synapse. The T-cell receptor (TCR) / CD3 complex interacts with MHC-peptide. The adhesion molecules on the surface of both cells (LFA-1—ICAM- 1 are responsible for the formation and stabilization of the IS, as well as for initiating signal transduction pathways activated by TCR. The distal ring of IS is rich in proteins, such as CD45 and F-actin controls lamellipodia and filopodia formation.</p

Spectrin depletion impairs lamellipodia formation and cell adhesion.

<p><b>(A)</b> Adhesion capability of spectrin-depleted Jurkat T-cells. The data are expressed as the percentage of adherent cells compared with the number of adherent control cells. Data are presented as mean±SD for n = 3, asterisk marks statistically significant (p<0.05), Student’s t test. Abbreviations: WT-Wild type Jurkat T-cells; SC–stable cell line treated with scrambled shRNA; KD–stable cell line treated with anti-spectrin shRNA <b>(B)</b> Scanning electron microscopy of contact sites formed by Jurkat T-cells and Dynabeads coated with anti-CD3 and anti-CD28 simulating the APC activation process. Arrowheads indicate lamellipodia formed by T-cells. Magnification 20 000x, scale bar = 1 μm. <b>(C)</b> Confocal microscopy of morphological changes and actin (yellow) distribution in T-cells upon IS formation on coverslips coated with anti-CD3 and anti-CD28 antibodies. Nuclei are stained with DAPI (blue). Arrowheads indicate lamellipodia formed by T-cells. Scale bar = 5μm. <b>(D)</b> Effect of spectrin knockdown (method A of transfection) on frequency of lamellipodia formation by live-imaging of Jurkat T-cells before and after activation with Dynabeads coated with anti-CD3 and anti-CD28. Cells were co-transfected with Ruby-Life Act plasmid to visualize actin. The frequency of lamellipodia formation and actin dynamics were analyzed in the Biostation system.</p

Spectrin depletion leads to impaired IS formation.

<p>Confocal images (a single z-plane) of actin (A, green), LFA-1 (B, yellow) and CD45 (C, red) distribution in wild type cells (WT) Jurkat T-cells treated with scrambled shRNA (SC) and cells treated with anti-spectrin shRNA (KD) after activation with Dynabeads coated with anti-CD3 and CD28. Fixed cells were stained with phalloidin-568, anti-LFA-1, or anti-CD45 antibodies. Asterisks indicate the location of the Dynabeads (simulating APC) in the conjugates. The scale bar = 5 μm.</p