of 22
Article
https://doi.org/10.1038/s41467-024-53078-8
Control of spatio-temporal patterning via
cell growth in a multicellular synthetic gene
circuit
Marco Santorelli
1,10
,PranavS.Bhamidipati
2,3,10
,JosquinCourte
1,10
,
Benjamin Swedlund
1
,NaisargeeJain
1
, Kyle Poon
1
, Dominik Schildknecht
2
,
Andriu Kavanagh
1,4
,VictoriaA.MacKrell
1
, Trusha Sondkar
1
, Mattias Malaguti
5,9
,
Giorgia Quadrato
1
, Sally Lowell
5
, Matt Thomson
2,6,7
&
Leonardo Morsut
1,8
A major goal in synthetic development is
to build gene regulatory circuits that
control patterning. In natural develop
ment, an interplay between mechanical
and chemical communication shapes the dynamics of multicellular gene reg-
ulatory circuits. For synthetic circui
ts, how non-genetic properties of the
growth environment impact circuit behavior remains poorly explored. Here,
we
fi
rst describe an occurrence of mechan
o-chemical coupling in synthetic
Notch (synNotch) patterning circuits:
high cell density decreases synNotch-
gated gene expression in diff
erent cellular systems in
vitro. We then construct,
both in vitro and in silico, a synNotch-based signal propagation circuit whose
outcome can be regulated by cell densi
ty. Spatial and temporal patterning
outcomes of this circuit can be predicte
d and controlled via modulation of cell
proliferation, initial cell density, and/o
r spatial distribution of cell density. Our
work demonstrates that synthetic pa
tterning circuit outcome can be con-
trolled via cellular growth, providing a means for programming multicellular
circuit patterning outcomes.
During embryonic development, morphogenesis emerges through the
interplay between chemical and mechanical processes that occur
simultaneously to generate the architecture of the embryo
1
.Atleastin
part, this is due to the tremendous growth and cell proliferation that
occurs to bring a single cell to generate a multicellular organism. For
example, in the zebra
fi
sh model, the embryo goes from 256 cell stage
to around 22,000 in around 14 h, during which the body plan pat-
terning and morphogenesis is established
2
. It has been shown that,
during natural development chemical and mechanical processes can
proceed in sequence, with patterning providing a template for
mechanical regulation
3
6
.Theinformationcan
fl
ow in the other
direction too, whereby mechanical inputs like substrate stiffness or
cell shape can affect signaling and patterning
7
11
. Finally, the two
aspects (mechanics and chemical) can be intertwined in so-called
mechano-chemical systems; these mechano-chemical systems seem to
abound in developmental transitions
12
22
. Despite emerging examples,
Received: 22 November 2022
Accepted: 1 October 2024
Check for updates
1
Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los
Angeles, CA, USA.
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
3
Keck School of Medicine, University
of Southern California, Los Angeles, CA, USA.
4
Department of Biology, California State University Northridge, Northridge, CA, USA.
5
Centre for Regenerative
Medicine, The University of Edinburgh, Edinburgh, UK.
6
Department of Computing and Mathematical Sciences, California Institute of Technology, Pasadena,
CA, USA.
7
Beckman Center for Single-Cell Pro
fi
ling and Engineering, Pasadena, CA, USA.
8
Department of Biomedical Engineering, Viterbi School of Engi-
neering, University of Southern California, Los Angeles, CA, USA.
9
Present address: Centre for Engineering Biology, Institute of Quantitative Biology,
Biochemistry and Biotechnology, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.
10
These authors contributed equally: Marco
Santorelli, Pranav S. Bhamidipati, Josquin Courte.
e-mail:
mthomson@caltech.edu
;
leonardo.morsut@med.usc.edu
Nature Communications
| (2024) 15:9867
1
1234567890():,;
1234567890():,;
many principles remain obscure regarding how information
fl
ows
between mechanical processes and chemical circuits in general, and
how this contributes to expand, constrain, or regulate patterning and
morphogenetic outcomes. This lack is due at least in part to the
complexity of the embryo which presents inherent challenges to
studying and controlling developmental transitions in general, and
ones where mechanical properties and gene circuit signaling dynamics
are intertwined in particular.
One particular effector of mechanical input into signaling, pat-
terning and morphogenesis is cell density and its origin through cell
growth and proliferation. Since at least the publication of D
arcy
s
On
Growth and Form
1
, cell growth and proliferation have been recog-
nized as playing an important role in shaping multicellular morpho-
genesis. More recently, cell proliferation is starting to be implicated as
an input for patterning: as a recent example in vivo, a mechanically
induced organizing center was seen to be induced via proliferation-
driven compression in the development of rodent incisors
23
.When
cultivating cells in vitro, it is well known that cell density signi
fi
cantly
impacts growth and differentiation of cells, so much that cell density is
a parameter that needs to be extensively optimized both for cell line
maintenance and directed differentiation protocols
24
43
.
A major emerging theme in the
fi
eld of synthetic development is
the development of gene circuits that enable controlled morphogen-
esis of patterned tissues
19
,
44
52
; building self-organized systems
through biological circuits could become the basis for a new approach
to the engineering of resilient, self-healing and self-forming structures
of multiple scales. Highly simpli
fi
ed engineered systems have been
generated in this fashion that provide controlled and de
fi
ned experi-
mental systems in which to analyze gene circuits within the context of
a multicellular structure
53
60
. Comparatively less is known whether or
how density impacts synthetic gene circuits and synthetic signaling
pathways, and so whether density can be manipulated to control sig-
naling mediated patterning outcomes is not currently known. Engi-
neered systems provide an ideal setting to study mechano-chemical
coupling where signaling and mechanical phenomena can be isolated,
measured and modulated. Additionally, introducing mechanical-
chemical coupling in synthetic gene circuits could uncover novel
strategies for engineering multicellular systems to achieve patterning
and morphogenesis goals.
Synthetic circuits based on Notch signaling, or so-called synNotch
circuits, have emerged as a modular and
fl
exible strategy for engi-
neering multicellular mammalian systems
53
55
. The synNotch system
uses engineered receptors modeled after the endogenous develop-
mental signaling pathway Notch/Delta. This endogenous pathway is
contact-dependent and is used extensively during development to
generate cell-scale patterns
61
63
. In the synthetic version, synNotch,
both the input and output of the pathway have been rendered user-
de
fi
nable and, as such, are orthogonal to the endogenous Notch
pathway. Using this system, developmental circuits have been engi-
neered in 2D culture as well as in 3D
fi
broblast aggregates where a
synthetic signal affects multicellular signaling and mechanics by driv-
ing expression of key adhesion proteins
53
55
. In these synNotch circuits
though, information
fl
ows from engineered signaling proteins to
downstream effects, for example on mechanical properties of the cell
through changes in cell-cell adhesion. To achieve a complete synthetic
mechano-chemical system with reciprocal information
fl
ow between
both modalities, mechanical inputs to signaling must also be char-
acterized. Insights that synNotch could be a good candidate to develop
such a system are emerging. First, although the speci
fi
cmechanisms
may differ based on the cellular context and the endogenous or syn-
thetic nature of the receptors (e.g., ref.
64
), the proposed general
mechanism of activation for Notch and synNotch signaling involve a
mechanical
pulling force
that exposes the protease cleavage site for
further signal transduction
64
69
. Second, cellular mechanical tension,
shear stress, ECM stiffness, and cell density have been shown to play a
role in Notch signaling in certain contexts
70
76
.Third,synNotchhas
been engineered to respond to different degrees of pulling force
77
.
Whether these inputs can be used to create a system where mechanics
or cell proliferation affects not on
ly signaling, but also patterning
outcomes has not been explored.
Mathematical models have been an important tool for under-
standing morphogenesis in natural systems
78
80
and thus provide a
potential strategy for the design and analysis of synthetic systems that
incorporate mechanical-chemical coupling. Cell-based models of
Notch-mediated signaling
81
have uncovered key insights into the self-
organization of regular spatial patterns
82
, the regulation of cell fate
bifurcation by receptor-ligand interactions and cell geometry
63
,
83
,
84
,
and the important roles of ligand expression levels and competition in
robust patterning
61
,
83
,
85
. This tool has also been used to catalyze the
discovery and design of novel circuits for morphogenesis
86
,
87
.Such
models have been used to study natural cases of mechano-chemical
coupling
84
,
88
90
but have not yet been applied to synthetic cell systems.
Here, we
fi
rst identify cell density as a non-genetic parameter of
cell culture that affects synNotch signaling, through a screening of
mechanical inputs in a murine
fi
broblast cell line (L929) and in mouse
embryonic stem cells (mES). Cell density above a critical threshold
robustly dampens synNotch signaling, not only in 2D, but also in 3D
and with multiple synNotch receptor/ligand pairs. This is due, at least
in part, to a transcriptional repression at high density, which particu-
larly affects membrane-bound signaling partners (ligands and recep-
tors). We then build, both in vitro and in silico, a synNotch-based
patterning circuit to study the effe
cts of cell density on patterning
outcomes. We construct a spatial-propagation multicellular synNotch
circuit, which contains a local relay circuit with a sender (signal-origi-
nating) cell type and a transceiver (signal-propagating) cell type that
both receives and propagates a
fl
uorescent signal. With this simple
genetic circuit, we show that cell density and proliferation can affect
patterning outcomes, such that the same genetically identical cells can
generate spatial and temporally distinct patterns depending on how
cell density is regulated in time and/or in space. We
fi
nally discuss how
our work could provide insight in mechano-chemical patterning cir-
cuits, and how cell density can be used as a control point for pro-
gramming multicellular circuit patterning outcomes.
Results
Cell density impacts SynNotch signal transduction
With the goal of integrating mechano-chemical control in synNotch-
based multicellular synthetic gene circuits, we
fi
rst decided to evaluate
the impact of non-genetic factors such as tissue mechanics and cell
density on synNotch signal transduction. To quantify the impact of
individual perturbations to the physical environment on synNotch
signaling, we employed a previously reported in vitro assay for syn-
Notch activation based on a sender-receiver cell signaling paradigm in
the mouse
fi
broblast cell line L929 (Figs.
1
A, B and S1). Brie
fl
y, two L929
mouse
fi
broblast cell lines, a sender cell line and a receiver cell line, are
engineered such that signaling between a sender cell and receiver cell
can be assessed by the presence of a red
fl
uorescent reporter in
receiver cells. Sender cells constitutively express membrane-bound
green
fl
uorescent protein (GFP), which acts as the ligand for an anti-
GFP synNotch receptor on receiver cells (anti-GFP synNotch, Fig.
1
A).
The intracellular portion of the anti-GFP synNotch receptor contains a
tetracycline-controlled transactivator (tTA) which is freed from the
membrane upon contact-dependent activation and translocates to the
nucleus where it activates expression of cytosolic mCherry. To assay
synNotch activity, sender and receiver
fi
broblasts are co-cultured in a
1:1 ratio for 24 h, by which time mCherry
fl
uorescence in alive activated
receiver cells can be assayed via a
fl
uorescence activated cell sorter
(FACS) machine (see Fig. S1 for FACS gating scheme).
In this experimental setup, we varied extracellular matrix (ECM)
composition, substrate stiffness, cytoskeletal tension, and cell density,
Article
https://doi.org/10.1038/s41467-024-53078-8
Nature Communications
| (2024) 15:9867
2
then evaluated via FACS their impact on synNotch activation of the
reporter gene mCherry. Cells grown on different substrates or at
varying stiffnesses exhibited similar mCherry activation as the refer-
ence condition (Figs.
1
C and S2A). Cytoskeletal tension modulation,
modulated by the addition of three drugs known to affect cytoskeletal
contractility and actin polymerization (Y-27632 (ROCK-inhibitor)
91
,
blebbistatin
92
, and latrunculin-A
93
,
94
), affected cellular morphology
(Fig. S2B, C), but did not affect signaling activity in the presence of
sender cells (Fig.
1
D and Supplementary Fig. S2D). The absence of a
difference of signaling with these treatments was con
fi
rmed with a
statistical test (Fig.
1
I).
In contrast, when cells were grown across a range of cell densities
from 0.008x
8x con
fl
uency (1x = 1250 cells/mm
2
), signal outcome
followed a bell-shaped curve going from lower to medium to higher
densities. Densities outside a central optimal window between 0.125X
and 2X exhibited signi
fi
cant and reproducible signal inhibition in
receivers (Fig.
1
E, F and Supplementary Fig. 3). Signaling was sig-
ni
fi
cantly compromised above the critical threshold density of 4x
(Fig.
1
J). Although not signi
fi
cant, signaling was reduced at lower
densities, presumably because of less frequent cell-cell contacts.
Importantly, we exclude that at high densities the cells are not sig-
naling because they are dead, since in our gating structure we only
ACD
E
F
B
ECM material and stiffness
Cytoskeletal tension
modulators
mCherry signal in Receivers
(log
10
Arb. Units)
mCherry signal in Receivers
(log
1
0
Arb. Units)
Senders
Matrix
-+
+
+
+
+
++
None
None
Fibronectin
8.0 kPa
0.2 kPa
Matrigel
Gelatin
64 kPa
Senders
Treatment
-+
+
+
None
None
Y-27632 100 μM
blebbistatin 25 μg/mL
+
latrunculin-A 200 nM
Seeding density (in L929 mouse fibroblasts)
Seeding density (in L929 mouse fibroblasts)
Seeding density (in mouse embryonic stem cells)
Seeding density (in mouse embryonic stem cells)
Senders
Seeding density
Seeding density:
Seeding density:
-+
+
+
+
+
+
+
+
+
+
+
mCherry signal in Receivers
(log
10
Arb. Units)
1 x
8 x
4 x
2 x
1 x
0.5 x
0.25 x
0.125 x
0.064 x
0.032 x
0.016 x
Senders
Seeding density
-+
+
+
+
+
+
+
+
+
+
+
mCherry signal in Receivers
(log
10
Arb. Units)
1 x
16 x
8 x
4 x
2 x
1 x
0.5 x
0.25 x
0.125 x
0.064 x
0.032 x
0.016 x
+
+
0.008 x
0.004 x
0.125 x
0.5 x
4 x
Brightfield
0.125 x
0.5 x
4 x
Brightfield
GFP-lig
tagBFP
mCherry
GFP-lig
tagBFP
mCherry
G
H
0.008 x
IJ
K
Log-Likelihood Ratio (LLR)
-1500
-1000
-500
0
500
1000
1500
-15000
-10000
-5000
0
5000
10000
15000
Log-Likelihood Ratio (LLR)
Log-Likelihood Ratio (LLR)
Seeding density (L929)
Seeding density (mESCs)
Perturbations
-4000
-2000
0
2000
4000
Ref. OFF
Ref. ON
Fibronectin
Gelatin
Matrigel
0.2 kPa
8 kPa
64 kPa
Y-27632
blebbistatin
latrunculin-A
Ref. OFF
Ref. ON (0.5 x)
0.008 x
0.016 x
0.032 x
0.064 x
0.125 x
0.25 x
1 x
2 x
4 x
8 x
Ref. OFF
Ref. ON (0.5 x)
0.008 x
0.016 x
0.032 x
0.064 x
0.125 x
0.25 x
1 x
2 x
4 x
8 x
0.004 x
16 x
**
**
***
*
*
*
*
*
*
*
GFP-lig
anti-GFP synNotch
receiver cell
sender cell
mCherry
tagBFP
Article
https://doi.org/10.1038/s41467-024-53078-8
Nature Communications
| (2024) 15:9867
3
evaluate signaling from the non-dead cells (see Fig. S1). Cells at high
densities do seem to become
stressed
(see more below, in the
Mechanistic insights
section). We then tested if the reduction of
synNotch signaling at high density is restricted to a GFP/anti-GFP
synNotch pathway or extends to other pairs of ligand-receptor. To do
so, we co-cultured an alternative pair of L929 Senders and Receivers
which express mCherry-ligand (a fusion of mCherry with a PDGFR
transmembrane domain) as the ligand and anti-mCherry/GAL4-VP64
synNotch
55
as the receptor that induces the expression of tagBFP in
activated receiver cells. We found that a similar bell-shaped curve of
synNotch activation is obtained in this system; we noted a shift in the
optimal culture density, which ranges from 0.5x to 4x in this system
(Supplementary Fig. S3C), meaning that although qualitatively the
phenomenon is replicated, the exact quantitative impact of cell den-
sity on synNotch activation may be different for different pairs and/or
ligand-receptor af
fi
nities.
We then asked if density-dependency of synNotch signaling is a
feature of the speci
fi
c cell line, or a phenomenon that would apply to
other cellular contexts; to do so, we tested cell density effects on
synNotch signaling in mouse embryonic stem cells. We co-cultured
mouse embryonic stem cells (mESCs) Senders and Receivers expres-
sing the ligand-receptor system of GFP-lig (PDGFR-GFP) and anti-GFP
synNotch
95
. synNotch signaling in mESCs is similarly affected by initial
seeding density, with a bell-shaped curve of activation of alive receiver
cells at increasing cell densities; the optimal signaling occurs here in a
central window between 0.064x and 1x (where 1x is 6000 c/mm
2
;see
Fig.
1
G, H and Supplementary Figs. S3D and S4). Alive receiver cells
coming from densities below 0.008x and above 4x showed statistically
signi
fi
cant compromised signaling (Fig.
1
K).
Based on the density-dependent changes in synNotch signaling in
2D cultures, we wondered if similar effects would arise in 3D cultures.
To test this, we seeded L929 Senders:Receivers 3D spheroids of dif-
ferent sizes, and found that, similar to the 2D setting, increasing cell
numbers (hence potentially cellular crowding) dampens synNotch
signaling in 3D systems, with a critical threshold of around 8000 cells,
which corresponds to approximately 700,000c/mm
3
(Supplementary
Fig. S5A
D). Interestingly, when we repeated the same experiment in
3D structures that are elongated and not spherical, activation seems to
be restricted in localized domains at the tip of the structures (Sup-
plementary Fig. S5E).
In sum, these results expose a previously unreported effect of cell
density on synNotch signaling, such that signaling is supported in a
system-speci
fi
ccelldensitywindow.
Mechanistic insights on the sensitivity of synNotch signaling to
density
After discovering that synNotch signaling was decreased at high cell
culture densities, we sought to understand how different factors were
contributing to this phenomenon.
We
fi
rst asked if the nature of the mechanism is through a
secreted molecule in the media, by performing conditioned-media
experiments: media conditioned by L929 cells cultured at high den-
sities applied on an L929 Senders:Receivers co-culture did not reca-
pitulate the impact of high culture densities on signaling, making a
soluble-molecule mediated mechanism less likely (Supplementary
Fig.S6A).WethenaskedifthemechanismwasthroughaclassicalYAP-
mediated mechanotransduction
11
, by visualizing YAP nuclear localiza-
tion at different densities; Supplementary Fig. S6B shows that YAP
localization is mainly cytoplasmic both at the 1x density and at the 4x
density used here in L929 cells, making it less likely that synNotch
inhibition at high cellular densities is due to a YAP-dependent
mechanism.
It has been suggested that cellular crowding causes reduced
proliferation, cell movement and an overall reduction in transcription,
a phenomenon sometimes referred to as
contact-inhibition
96
.Totest
if this was at play in our system, we measured total RNA content per
cell at different densities 24 h after seeding. We found that total mRNA
levels decreased substantially at higher seeding densities in L929 cul-
tures (Fig.
2
A). We con
fi
rmed this phenomenon by showing that, at
high densities, the induction of reporter gene expression from the
dox-inducible tTA-VP64 transcription factor is strongly reduced
(Supplementary Fig. S6C). Additionally, L929 cell motility was
decreased at higher densities (Supplementary Fig. S6D), and both L929
and mESCs have reduced cell sizes at high densities (Supplementary
Fig. S6E, F). We also measured cell death percentage in the cultures at
high densities and found that: L929 cells display a low baseline cell
death that increases to around 10% at 8X densities (equal to 10,000 c/
mm
2
, Fig. S6G); mES cells display a basal cell death percentage of
around 10
20% at low con
fl
uency, which increases with increasing
con
fl
uenceupto80%at16X(equalto96,000c/mm
2
, Fig. S6H), again
indicating a progressive increase in cell stress at higher con
fl
uency.
(WeremindherethatforthesynNotchsignalingexperimentsofFig.
1
,
we only measure synNotch reporter induction in non-dead cells, both
for L929 and mES cells). These data suggest that cells at the high
densities used here are in a general state of reduced activity.
We then asked whether high cell density speci
fi
cally affects syn-
Notch signaling components. To address this question, we evaluated
expression levels of synNotch ligand GFP and anti-GFP synNotch
receptors via FACS; we found that both in L929 (Figs.
2
B, C and S8) and
mES cells (Figs. S7A, B and S8), synNotch ligand GFP and anti-GFP
receptor protein abundance decreased after 24 h of culture at high
densities, whereas cytoplasmic GFP protein expression was not affec-
ted. We con
fi
rmed that other overexpressed cytoplasmic or nuclear
proteins, both in L929 and mESCs, are not affected by 24 h culture at
high cell densities (Supplementary Figs. S7C
EandS8).
For some signaling receptors, receptor-ligand complexes clus-
tering is relevant for signaling. We set out to assess if ligands of the
synNotch family are affected in their abundance and/or localization by
Fig. 1 | Screening of mechanical perturbations reveals cell density-dependence
of synNotch receptor activation. A
Schematic of Sender-Receiver synNotch sig-
naling. Membrane-bound GFP-ligand in Senders binds synNotch in Receivers
cleaving synNotch, freeing the intracellular domain (tTA-VP64) to translocate to the
nucleus and activate mCherry reporter.
B
Schematic of synNotch signaling assay.
Senders and Receivers are co-cultured at a 1:1 ratio and mCherry activation in
receivers is measured at 24 h.
C
E
Violin plots depict the distributions of mCherry
fl
uorescence (log
10
scale) measured via FACS in L929 Receiver cells (
n
=4660
6733
cells) cultured in the indicated conditions for 24 h, seeded at an overall density of
1x
(1250 cells/mm
2
, counting Senders and Receivers), except where indicated
otherwise. In (
C
), SynNotch signaling assay is performed with cells on different
growth substrate materials and stiffnesses, in (
D
) with chemical modulators of
cytoskeletal tension, in (
E
) with different initial cell densities at the same Sen-
ders:Receivers ratio 1:1. mCherry signal is speci
fi
cally measured in Receiver cells
(Fig. S1 shows gating scheme). Gray violin plots are reference samples for OFF and
ON Receiver states. Black dots indicate medians. Dashed gray lines indicate the
separation between ON and OFF populations. * indicates the sample is more likely
OFF than ON, as determined by the log-likelihood ratio (LLR). Representative bright
fi
eld and
fl
uorescent micrographs of L929 (
F
)andmES(
H
) sender/receiver co-
culture after 24 h of culture at the indicated densities. GFP-lig is expressed in sen-
ders, tagBFP in the receivers. Scale bars 500
μ
m.
G
Violin plots of mCherry
fl
uor-
escence in mESCs Receivers after 24 h of coculture with mESCs Senders at the
indicated densities, 1x is 6000 c/mm
2
. Dashed gray lines and * as in (
E
).
I
K
Plots of
the LLR calculated for each sample. The circle represents the LLR of the measured
data and the error bars denote 95% CI with
n
= 1e6, calculated by bootstrapping.
Points above zero indicate the sample resembles the ON state more than the OFF
state. Error bars represent 95% con
fi
dence intervals (see
Methods
). All experi-
ments were repeated at least 3 times with similar results. Source data are provided
as Source Data
fi
le.
Article
https://doi.org/10.1038/s41467-024-53078-8
Nature Communications
| (2024) 15:9867
4
the presence of their cognate receptors and at different densities. We
fi
rst assessed the levels of GFP-ligand in presence of receiver cells at
different densities (Fig. S9A), and we con
fi
rmed that GFP-ligands in
sender cells decrease at the increase of cell density. We noted that the
decrease of the levels of GFP ligand seem to be more pronounced
when the sender cells are cultivated with receiver cells; this prompted
us to assess the microscope localization of GFP ligand in presence or
absence of neighbor cells with an anti-GFP synNotch receptor. To do
so, we cultivated a mixed culture of sender cells and parental cells on
one case, and a mixture of sender cells with receiver cells on a different
well; in both cases we evaluated GFP localization via confocal micro-
scopy. As shown in Fig. S9B, C, localization of GFP ligand changes
A
Seeding
density
RNA per cell (ng)
0.25 x
0.5 x
2 x
4 x
anti-GFP
SynNotch receptor
(L929)
RNA amount/cell
(L929)
GFP-ligand in L929
GFP-lig
(synNotch ligand)
(L929)
cytoplasmic GFP
(L929)
Cells
synNotch
staining
parental
parental
receiver
sender
-
+
-
++
+
+
+
+
++
C
0.25 x
1 x
4 x
GFP-lig
Brightfield
B
Seeding
density
1 x
1 x
0.064 x
0.125 x
8 x
4 x
2 x
1 x
0.5 x
0.25 x
1 x
1 x
0.064 x
0.125 x
8 x
4 x
2 x
1 x
0.5 x
0.25 x
Seeding
density:
D
GFP signal
(normalized between 0 and 1)
GFP:
28 h half-life
GFP-lig:
19 h half-life
time (h)
0 h: dox addition
0 h: dox removal
GFP signal
(normalized between 0 and 1)
GFP:
16.5 h half-life
GFP-lig:
7.5 h half-life
time (h)
Degradation kinetics
of GFP versus GFP-lig
(mES)
Degradation kinetics
of GFP versus GFP-lig
(L929)
E
parental
cytoplasmic GFP
1 x
0.064 x
0.125 x
8 x
4 x
2 x
1 x
0.5 x
0.25 x
Normalized fluorescence
(log
10
Arb. Units)
Article
https://doi.org/10.1038/s41467-024-53078-8
Nature Communications
| (2024) 15:9867
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