larger SPOTs that range up to 10mmindia-
meter, are also SNX9-dependent, and vary in
substructure and shape. What factors dictate
their morphology and the fate of their pro-
teins? Although MFN1 and MFN2 are targeted
for degradation, it remains possible that other
SPOT-localized proteins such as SAM50, TOM70,
and TOM20 are recycled into the mitochon-
drial network. SPOTs being oftentimes multi-
vesicular and containing proteins required forfusion (MFNs) and trafficking (MIROs) raises
the possibility that these structures might be
able to fuse with one another, or with mito-
chondria to reintegrate into the mitochondrial
network.Liet al.,Science 375 , eabi4343 (2022) 14 January 2022 8 of 10
Fig. 6. SAM50 loss of function
mediates SPOT formation during
infection.(A) Anti-HA IPs were
prepared from WT andTOM70 KO
HeLas infected withDmaf1:HA
orDmaf1:HA-MAF1 parasites and
analyzed for TgMAF1, ~60 kDa;
TOM70, ~72 kDa; SAM50,
~55 kDa; MIC60, ~88 kDa; MIC19,
~25 kDa; TOM40, ~40 kDa.
(B) Representative IF images
of uninf andToxoHFFs at 24 hours
after infection. (Inset) SPOT in
Toxo-infected cell contains SAM50
but not MIC60 or ATP51B (IMM).
Scale bars, 5mm and (inset) 1mm.
(C) Representative live-cell images
of the OMM (BFP) in uninf and
Toxo-infectedCTRL,TOM70-,
SAM50-, andMIC60-suppressed
(KD) HeLas labeled with mitoT.
(Insets) SPOTs are indicated with
arrowheads. Scale bars, 5mm and
(inset) 1mm. (D) Percentage (%)
of SPOT-positive cells in
experiments as in (C); data are
mean ± SEM of more than 100 cells
counted fromn= 3 replicates; P<
0.05, P< 0.01, **P< 0.0001
for uninf versus inf, ####P<
0.0001 forCTRL KDversusSAM50
KD,MIC60 KDby means of two-
way ANOVA analysis. (EandF)
Scatterplots with mean (E) number
and (F) diameter of SPOTs in
experiments as in (C) from more
than 30 infected cells from
three replicates. (G) Representa-
tive live-cell images of the
OMM (GFP) in uninf and
Toxoplasma(mCh)Ðinfected U2OS
cells expressing matrix-BFP
(matrix) or BFP fused to an
OMM-IMM tether (O-It). (H) Per-
cent of SPOT-positive cells in
experiments as in (G). Data are
mean ± SEM of more than 100 cells
counted fromn= 3 biological
replicates; **P< 0.001,
****P< 0.0001 for uninf versus inf,
####P< 0.0001 for matrix
versus O-Itby two-way ANOVA
analysis. (IandJ) Scatterplots with
mean (I) number and (J) diameter
of SPOTs in experiments as in (G) from more than 30 infected cells from three replicates. (K) Model of SPOT formation: TOM70 (70) mediates the interaction between MAF1 and
SAM50 and/or a hypothetical translocase (?), which induces a disassembly of the MIB complex (SAM50, MIC60, and MIC19) and SPOT formation. The constitutive shedding
of SPOTs depletes OMM proteins that restrict parasite growth (MFN1 and MFN2) and sequesters import machinery required for mitochondrial biogenesis on SPOTs.
TOM70 KD SAM50 KD%SPOT+ cells0306090
****####
ns** *
####MIC60 KDTOM70TOM40SAM50
MIC19input MAF1 IP (αHA)
Wt TOM70 KO
Δmaf1:
HA
Δmaf1:
HA-MAF1
Δmaf1:
HA
Δmaf1:
HA-MAF1
Δmaf1:
HA
Δmaf1:
HA-MAF1
Δmaf1:
HA
Δmaf1:
HA-MAF1Wt TOM70 KO+matrix (ctrl)OMMmatrix mitoT(^0) uninfToxouninfToxo
5
10
0
20
40
60
CTRL KD
mitoT
SAM50 KD
TOM70 KD
MIC60 KD
uninf
+OMM-IMM tether (O-It)
(^0) uninfToxouninfToxo
10
****^20
matrix
O-It
uninf Toxo uninf Toxouninf Toxo uninf ToxoToxo uninf ToxoOMM mergematrix
O-Itmatrix
O-ItmergeOMM mitoTmergeOMMmatrix mitoTmerge
uninfToxouninfToxo SPOT diameter (μm)%SPOT+ cellsSPOT # per cellMIC6070
OMMCytosolPVMSAM
50IMMIMS
MIC60PV? SPOT formation
?0510010203040SPOT # per cellHA (MAF1)+ -+ -+ - +ToxoToxoToxoOMM mitoTmergeOMM mitoTmergeOMM mitoTmergeOMM mitoTmerge OMM mitoTmergeOMM mitoTmergeOMMmatrix mitoTmergeOMMmatrix mitoTmergeSPOTuninf ToxoIIMMMM MMIC60IC 60 SAM50SAM 50 mergemerge IMMIMM MMIC60IC 60 SAM50SAM 50 mmergeergeCTRL KD***####TgMAF120
MIC19SPOT diameter (μm)ADFECG HI JKBRESEARCH | RESEARCH ARTICLE