Fig 1.
Schematic of the immunological synapse (IS) and representative protein interactions in the synaptic space.
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.
Fig 2.
Spectrin translocates together with actin to the IS upon T-cell activation.
(A-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. (C-D) 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.
Fig 3.
Spectrin depletion impairs lamellipodia formation and cell adhesion.
(A) 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) 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. (C) 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. (D) 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.
Fig 4.
Spectrin depletion leads to impaired IS formation.
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.