Science - USA (2022-04-15)

B4GALT5encoding lactosylceramide synthase
(LCS) reduced toxin binding (fig. S2, B and C)
without inducing significant toxicity (fig. S2D),
indicating that toxins are a faithful proxy for
dHF sphingolipid composition in our setting.

As further validation, dHFs were first fixed and stained with toxins and then imaged by MALDI-MSI (Fig. 3B). ShTxB1a staining correlated best with Gb3 levels, and ShTxB2e staining correlated well with Gb3 and Gb4

levels, whereas neither correlated with SM levels. ChTxB staining, our proxy for the levels of GM1 ( 42 ), a sphingolipid not detected by MALDI-MSI in positive-ion mode, did not cor- relate with any of the sphingolipids detected

Capolupoet al.,Science 376 , eabh1623 (2022) 15 April 2022 4 of 12

Fig. 3. Identification of dHF lipotypes
by MALDI-MSI and toxin staining.
(A) Confocal micrographs showing cells
stained with bacterial toxins ShTxB1a
(green), ShTxB2e (red), ChTxB (blue), and
Hoechst (gray) for nuclei. Scale bar,
50 mm. (B) Side-by-side comparison of
toxin staining and MALDI-MSI acquisition
on the same cells. First, cells were stained
with bacterial toxins as in (A), and images
were acquired by confocal microscopy
(left panel). Then, MALDI-MSI (25mm^2 /
pixel) was performed on the same cells
(center panel). Mass images (320 ×
320 pixels) of complex sphingolipids
[SM (42:1), Gb3 (42:1), and Gb4 (42:1)]
are shown. Scale bar, 200mm. (C) Cells
were stained with bacterial toxins as in (A)
and with antibodies against Beta-COP
(COPI vesicles) or EEA1 [early endosomes
(EEs)], and images were acquired by
confocal microscopy. Normalized fluores-
cence intensities of toxin and organelle
marker stainings of single cells were
used to analyze the correlation between
lipotypes and cell area and with exo/
endocytic organelles. Data are shown
as violin plots. P< 0.05, P< 0.01,
P< 0.001, ordinary one-way ANOVA.
(D) Representative cells stained as for
EEA1 or Beta-COP and classified according
to their lipotypes. (E) Schematic repre-
sentation of dHF cell lineage tracking.
(F) Representative confocal micrograph of
toxin-stained dHFs before (left) and after
(right) segmentation with Cellpose. Seg-
mented cell colors correspond to the
different lipotypes. (G) Lineage recon-
struction for the cells illustrated in
(F) as inferred using TrackMate ( 37 ).
(H) Correlation plots of normalized
ChTxB, ShTxB1a, and ShTxB2e intensities
between daughter cells at the time
course end point. Dots are colored by the
number of hours after mother cell division.
(I) Heatmap of frequencies for two
lipotypes occurring in two sister cells
colored byz-score. Positive deviation from
zero indicates an increased observed fre-
quency of the sister-lipotype combination
compared with random chance, and negative
deviation from zero indicates a decreased
observed frequency of the sister-lipotype
combination compared with random chance.Pvalues were calculated using the bootstrap pairwisettest ( 37 ). (J) Probability of a lipotype state transition occurring in a
cell over a 21-hour time period as estimated using CELLMA ( 37 ). Probabilities are located at the corresponding arrow tails. (K) Markov modelÐsimulated evolution of a pure
ChTxB+(left) or pure ShTxB1a+/2e+(right) cell population over 7 days. (L) Line plots displaying the evolution of the state predictability of a cell (or its progeny) after a certain
time from an original state measurement. Differently colored tracks correspond to a different original lipotype measurement (t= 0). Kullback-Leibler divergence is evaluated
between the probability distribution vector obtained using the Markov transition matrix and the steady-state probability distribution (i.e., the best uninformed guess).

ChTxB+

ShTxB1a+/2e+

ShTxB1a+ShTxB2e+ Triple

Other

ShTxB2e+ Triple

ShTxB1a+/2e+ Other

ChTxB+

00:00

42:00

21:00

Time (h)

Cell Lineage

Ch+ Ch+ Ch+ Ch+

Gb3 (42:1 )Gb4 (42:1 )SM(42:1)

closeup Toxins

MALDI

MALDI-

MSI

Toxins

MALDI

ShTxB1 a ShTxB2 e Ch TxB A

H I

J K

L

ChTxB+ ShTxB1a+ ShTxB1a+/2e+ ShTxB2e+ Triple Other

*** *** ** *** * *** * *** ** *** ** *** *** ** *** *** ***

Sister pair frequency deviation

−3σ

−2σ

−σ

0

σ

2σ

3σ

Z-scor

e

*** p < 0.00005 ** p < 0.0005 * p < 0.005

0.500.751.001.251.501.75 (Normalized Intensity)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

(Normalized Intensity)

ChTxB Level (R=0.62)

0.500.751.001.251.501.75 (Normalized Intensity)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

ShTxB1a Level (R=0.65)

0.5 1.0 1.5 (Normalized Intensity)

0.25

0.50

0.75

1.00

1.25

1.50

1.75

ShTxB2e Level (R=0.69)

5

10

15

20

Time since last division (hours)

ChTxB+49%

3.4% 0.097% 2.1% 13% 32%

5.5%

ShTxB1a+

48% 5% 9%

13% 20%

5.6%

ShTxB1a2.8% +/2e+

63%

3.7%

15% 10%

6%

31% 1.1% 20%ShTxB2e+

13% 29%

6%

20% 0.7% 8.5% Triple 32% 33%

14%

10% 0.32% 5.3% 24% Other 46%

Probability of state transition from one division to the next (21h)

0 24 48 72 96 120144168 Time (hours)

0.0

0.2

0.4

0.6

0.8

1.0

Population Abundanc

e

(fraction of the total

)

Simulated evolution from a pure ChTxB+ population

ChTxBShTxB1a++ ShTxB1aShTxB2e++/2e+ TripleOther median time divisionsteady state reached

0 24 48 72 96 120144168 Time (hours)

0.0

0.2

0.4

0.6

0.8

1.0

Population Abundance(fraction of the total

)

Simulated evolution from a pure ShTxB1a+/2e+ population

0 12243648 Time (hours)

0.0

0.2

0.4

0.6

0.8

1.0

KL-divergence (t^0

-normalized)

Predictability

E Time Lapse 42 h Toxin Staining ShTxB1a ShTxB2e ChTxB

F Endpoint Toxin Staining Determinaton of type G

ChTxB+

Triple

S

C D

Legend

ChTxB

+

ShTxB1

+a

ShTxB1a

+/2e

+

ShTxB2

+e TripleOther

ChTxB+

EEA1 staining 0 Max Beta-COP staining 0 Max

ChTxB in Celli ShTxB1a in Celli ShTxB1a in Celli

ChTxB in Cell

Sisteri

ShTxB1a in Cell

Sisteri

ShTxB2e in Cell

Sisteri

0 6E3

Area

*

*** ***

***

0 1.0 COPI Area

**

***

******

0 1.0

EE Area

**

***

ChTxB+ ShTxB1a+ ShTxB2e+ ShTxB1a+/2e+ Triple Others

ShTxB1a ShTxB2e Ch TxB

Toxin staining

B

RESEARCH | RESEARCH ARTICLE

Science - USA (2022-04-15)

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