Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation
The immune synapse (IS) enables cell-cell communication between immune cells through close contacts, as well as T-cell activation and vesicle secretion. It is sustained by fine-tuned molecular interactions of receptors at both cell sides of the IS and int
- PDF / 708,615 Bytes
- 16 Pages / 504.567 x 720 pts Page_size
- 69 Downloads / 199 Views
Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation Noa B. Martı´n-Co´freces, Amelia Rojas-Gomez, Sara G. Dosil, Irene Fernandez-Delgado, and Francisco Sa´nchez-Madrid Abstract The immune synapse (IS) enables cell-cell communication between immune cells through close contacts, as well as T-cell activation and vesicle secretion. It is sustained by fine-tuned molecular interactions of receptors at both cell sides of the IS and intracellular cytoskeletal components. The resulting intracellular polarization of different organelles, through cytoskeleton-guided vesicular traffic, is a key player in IS formation and signaling. We describe herein a method to analyze rapid changes of vesicle localization through microscopy analysis upon polarization toward the IS. These vesicles are monitored using the centrosome and its associated microtubular network or the actin-based structures as spatial references during the organization of the IS. Keywords Immune synapse, Cytoskeleton, Signaling, T-cell receptor, Vesicles, Endosomes, Actin
1
Introduction The immune synapse (IS) is a specialized cell-to-cell contact that allows the communication between the T cell and the antigenpresenting cell (APC). This highly organized structure enables the relocation of different membrane receptors such as the T-cell receptor (TCR), integrin receptors, and the activation of associated downstream signaling pathways [1, 2]. The actin- and tubulinbased cytoskeletons undergo dramatic changes, helping the narrowing of the extracellular space into the synaptic cleft and cell internal rearrangement. The actin and microtubular networks reorganize toward the IS and regulate the polarization of several organelles around the centrosome, such as the Golgi apparatus (GA); the endolysosomal system, including multivesicular bodies (MVB); and mitochondria [3, 4]. The intense vesicular traffic observed at the IS is mainly driven by the microtubules beneath the plasma membrane, while actin-related structures mediate the
Sa´nchez-Madrid and Martı´n-Co´freces contributed equally to this work.
Noa B. Martı´n-Co´freces et al.
reorganization of the receptors on the cell surface. Actin cytoskeleton also regulates exocytosis sites at the IS [1, 5–8]. Here, we describe several microscopy-based approaches to assess dynamic changes in the cell during the IS. These include the distribution of vesicles and vesicle-associated structures related to actin dynamics, cell adhesion, and T-cell activation in response to specific antigen stimulation. To carry out this assessment, we take advantage of different protein markers and methodologies that allow the study of TCR activation and IS formation.
2
Materials
2.1 Primary Cells and Cell Lines
1. Primary T lymphocytes from human healthy donors (purified CD4+, SEE- or PHA-specific blasts). 2. Primary CD4+T lymphocytes from mouse lymph nodes and spleen. 3. Jurkat lymphoblastoid cell lines; E1–6 (Vαl.2Vβ8+TCR) or CH7C17 cells (HA1.7Vβ3+transgenic αβTCR, specific for HA peptide).
2.2
Reagents
1. Fl
Data Loading...
