title: Actin depletion initiates events leading to granule secretion at the immunological synapse.
abstract: Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK; National Institute of Child Health and Disease, NIH, Bethesda, MD 20892, USA.
authors.forename: Alex T
authors.initials: AT
authors.lastname: Ritter
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Asano
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Jane C
authors.initials: JC
authors.lastname: Stinchcombe
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: N M G
authors.initials: NM
authors.lastname: Dieckmann
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Bi-Chang
authors.initials: BC
authors.lastname: Chen
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: C
authors.initials: C
authors.lastname: Gawden-Bone
authors.affiliation: National Institute of Child Health and Disease, NIH, Bethesda, MD 20892, USA.
authors.forename: Schuyler
authors.initials: S
authors.lastname: van Engelenburg
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Wesley
authors.initials: W
authors.lastname: Legant
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Gao
authors.affiliation: National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Betzig
authors.affiliation: National Institute of Child Health and Disease, NIH, Bethesda, MD 20892, USA. Electronic address: lippincj@mail.nih.gov.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK. Electronic address: gg305@cam.ac.uk.
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Griffiths
mesh_terms: D000199:Actins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D054992:Immunological Synapses / Q000378:metabolism*; D018448:Models, Immunological; D010743:Phospholipids / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000378:metabolism
chemical_list: D000199:Actins; D010743:Phospholipids