(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 a
comprehensive 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 imaging
378 22 APRIL 2022•VOL 376 ISSUE 6591 science.orgSCIENCE
B
C D
Merge
EGFP-Tsg101
Propidium Iodide
A -0:30 0:00 0:30 1:30
-1 012 3 4 5
2000
3000
4000
5000
2000
3000
4000
5000
Time (min)
EGFP Fluorescence (AU)
EGFP-Tsg101 recruitment vs. PI influx
EGFP-Tsg101 (AU)
PI (AU)
PI Fluorescence (AU)
First frame of
PI Influx
EGFP
EGFP-TSG101EGFP-Chmp4b
0
20
40
60
80
100
% conjugates with EGFP accumulation
at synapse after secretion
EGFP
EGFP-TSG101EGFP-Chmp4b
0
20
40
60
80
100
% conjugates with EGFP+
fragments on CTL after killing
E
Merge
EGFP-Chmp4b
-0:30 0:00 0:30 1:30 3:30 6:00
Fig. 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.
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