(Fig. 1C and movies S1 to S3). Notably, ESCRT-
laden material, presumably membrane frag-
ments, frequently detached from the target
cell and adhered to the surface of the CTL
(Fig. 1, D and E, and movie S2). We observed
this phenomenon in ~60% of conjugates im-
aged in which targets expressed EGFP-Tsg101
or EGFP-Chmp4b (17 of 27 and 20 of 33 con-
jugates, respectively; Fig. 1D). Shedding of
ESCRT-positive membrane from the cell after
repair occurs after laser-induced plasma mem-
brane wounding ( 6 , 7 ). Plasma membrane
fragments shed from the target cell into the
synaptic cleft likely contain ligands for CTL-
resident receptors. Target cell death would
separate the CTL and target, revealing target-
derived material on the CTL surface.3D cryo-SIM and FIB-SEM imaging of CTLs
caught in the act of killing target cells
Although live-cell imaging indicated that ESCRT
complexes were rapidly recruited at sites of
T cell–target cell contact, light microscopy
alone is of insufficient resolution to establish
that this event occurred at the immunological
synapse (IS). We thus sought to capture acomprehensive view of the IS in the moments
immediately after secretion of lytic granules.
We used cryo–fluorescence imaging followed
by correlative focused ion beam–scanning elec-
tron microscopy (FIB-SEM), which can achieve
isotropic three-dimensional (3D) imaging of
whole cells at 8-nm resolution or better ( 11 – 13 ).
To capture the immediate response of target
cells after perforin exposure, we developed a
strategy whereby cryo-fixed CTL-target con-
jugates were selected shortly after perforation,
indicated by the presence of a PI gradient
in the target (fig. S1A). In live-cell imaging378 22 APRIL 2022•VOL 376 ISSUE 6591 science.orgSCIENCE
BC DMergeEGFP-Tsg101Propidium IodideA -0:30 0:00 0:30 1:30-1 012 3 4 520003000400050002000300040005000Time (min)EGFP Fluorescence (AU)EGFP-Tsg101 recruitment vs. PI influxEGFP-Tsg101 (AU)
PI (AU)PI Fluorescence (AU)First frame of
PI InfluxEGFPEGFP-TSG101EGFP-Chmp4b020406080100% conjugates with EGFP accumulationat synapse after secretionEGFPEGFP-TSG101EGFP-Chmp4b020406080100% conjugates with EGFP+
fragments on CTL after killingEMergeEGFP-Chmp4b-0:30 0:00 0:30 1:30 3:30 6:00Fig. 1. Fluorescently tagged ESCRT proteins in targets localize to site of
CTL killing after perforin secretion.(A) Live-cell spinning disk confocal
imaging of a fluorescently labeled OT-I CTL (magenta) engaging an MC38 cancer
cell expressing EGFP-Tsg101 (green) in media containing 100mM PI (red). Yellow
arrowheads highlight ESCRT recruitment. T-0:00 is the first frame of PI influx
into the target cell (time in minutes:seconds). Scale bar, 10mm. (B) Graph
of EGFP-Tsg101 and PI fluorescence intensity at the IS within the target over
time, from example in (A). AU, arbitrary units. (CandD) Quantification of
CTL-target conjugates exhibiting accumulation of EGFP at the synapse after PI
influx (C) or detectable EGFP-labeled material associated with CTL after target
interaction (D) (EGFP condition:N= 22 conjugates in seven independent
experiments; EGFP-Tsg101 condition:N= 27 conjugates in nine independent
experiments; EGFP-Chmp4b condition:N= 33 conjugates in 24 independent
experiments). (E) Live-cell spinning disk confocal imaging of OT-I CTL (magenta)
killing MC38 expressing EGFP-Chmp4b (green), demonstrating the presence
of target-derived EGFP-Chmp4b material (yellow arrowheads) associated with
CTL membrane after a productive target encounter. T-0:00 is the first frame of PI
influx into the target cell. Scale bar, 10mm.RESEARCH | RESEARCH ARTICLES