Actin Depletion Initiates Events Leading to Granule Secretion at the Immunological Synapse

Alex T. Ritter8

Yukako Asano8

Jane C. Stinchcombe

N.M.G. Dieckmann

Bi-Chang Chen5

C. Gawden-Bone

Schuyler van Engelenburg6

Wesley Legant

Liang Gao7

Michael W. Davidson

Eric Betzig

Jennifer Lippincott-Schwartz9

Gillian M. Griffiths9

5Present address: Research Center for Applied Science, Academia Sinica, Taipei 11529, Taiwan

6Present address: Department of Biological Sciences, University of Denver, Denver, CO 80208-9010, USA

7Present address: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA

8Co-first author

9Co-senior author

Open Access

DOI: http://dx.doi.org/10.1016/j.immuni.2015.04.013

Open access funded by Wellcome Trust

Highlights

•4D imaging elucidates the order of events leading to secretion
•Actin depletion initiates events leading to centrosome polarization and secretion
•Lattice light-sheet imaging reveals a rearward flow of actin away from the synapse
•Both centrosome and granules are delivered to an area of membrane depleted of actin

Summary

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.

Received: October 1, 2014; Received in revised form: December 20, 2014; Accepted: March 15, 2015;