Liposome-Encapsula ed Esche ichia coli Lysa es o Recons i u e
In acellula Mac omolecula C owding E ec s
Mila a S. Kalache a, Nuno R. da Sil a, and A nold J. Boe sma*
Ci e This: ACS Syn h. Biol. 2025, 14, 901−908
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ABSTRACT: In acellula mac omolecula c owding impac s bio-
mac omolecule beha io , including oligome iza ion, phase sepa a ion,
and di usion. Howe e , unde s anding c owding e ec s in cells is
challenging as cells espond and adap o pe u ba ions. The e o e,
eplica ing in-cell c owding in liposomes would p o ide a good
al e na i e o s udying he consequences o mac omolecula c owding.
He e, we achie e physiological mac omolecula c owding le els using
Esche ichia coli lysa es in liposomes, as e i ied wi h a mac omolecula
c owding senso . We sh ink liposomes wi h a g adien -wise osmo ic
upshi o each he high mac omolecula c owding e ec s. We see
ha lysa e induces highe mac omolecula c owding han BSA a he
same mg/mL, showing he need o use lysa es o eplica e in-cell
beha io . We s udy he consequences o small cosolu es on mac omolecula c owding and see ha suga s and ATP modula e he
lysa e mac omolecula c owding, implying hey would also a ec mac omolecula c owding in cells. These a i icial cells display he
same c owding as E. coli a 220−300 mg/mL lysa e and he same c owding as HEK293T a 50−100 mg/mL lysa e. Hence, hese
a i icial cells a e a pla o m o ob aining in o ma ion on physiologically ele an mac omolecula c owding e ec s in a con olled
en i onmen .
KEYWORDS: mac omolecula c owding, gian unilamella esicles, lysa e, cosolu es, FRET senso , hype osmo ic s ess
■INTRODUCTION
Cells main ain high concen a ions o mac omolecules, whe e a
ange o 50−400 mg/mL is usually assumed o be
physiological.
1−3
These molecules ake up space, and hei
s e ic epulsion excludes olume, which is called mac o-
molecula c owding.
4
C owding excludes olume, which
inc eases he e ec i e concen a ion o a speci ic mac o-
molecule. The eby, associa ion equilib iums shi o sel -
assembled o bound s a es, o example. Mac omolecula
c owding depends on he c owde size, numbe densi y, and
shape. Mac omolecula c owding also depends on he
in e ac ion be ween c owde s o c owde o ganiza ion, as
his would, o example, educe he c owde numbe densi y.
The dependence on bo h c owde p ope ies and o ganiza ion
means ha in-cell c owding depends on an excep ionally la ge
numbe o pa ame e s ha a e challenging o un angle di ec ly
in cells,
5
and unde s anding would bene i om con olled
econs i u ion in a i icial cells.
T adi ionally used c owding agen s such as syn he ic
polyme s (polye hylene glycol and Ficoll) o na u al polyme s
and p o eins (Dex an and bo ine se um albumin) do no
ep esen he in acellula si ua ion well. Mo eo e , a high
concen a ion o a single c owde leads o c owde -speci ic
e ec s ha a e unlikely o be ele an o he cell. Close - o-
physiological c owding would be concen a ing he ac ual
componen s om li ing cells o each in i o mac omolecula
c owding. The e a e a ious ins ances whe e lysa es ha e been
concen a ed o he same concen a ion as li ing cells (in mg/
mL).
6
Because concen a ed lysa es a e highly iscous,
7
concen a ed lysa es a e mo e accessible by inco po a ing
hem in liposomes o simila compa men s and sh inking
hem by osmo ic p essu e. Acco dingly, inc easing lysa e
concen a ions d ama ically inc eases in i o ansc ip ion
ansla ion a es.
8,9
Howe e , concen a ing lysa es does no
au oma ically imply ha c owding beha io o li ing cells has
been achie ed
7,10
because mac omolecula c owding depends
on he c owde o ganiza ion, while he lysa e physical
p ope ies will depend on he lysis and concen a ion me hod.
Thus, while lysa es can be concen a ed, i has emained
unclea i physiological mac omolecula c owding can ac ually
be achie ed.
We p e iously de eloped a me hod o measu e mac o-
molecula c owding, which was based on a gene ically encoded
Recei ed: No embe 27, 2024
Re ised: Janua y 14, 2025
Accep ed: Feb ua y 10, 2025
Published: Feb ua y 20, 2025
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Fo s e esonance ene gy ans e (FRET) senso , c GE2.3
(Figu e 1a).
11,12
This senso con ains a linke wi h wo α-
helices linking a monome ic enhanced g een luo escen
p o ein (mEGFP, dono ) and mSca le -I (accep o ). In
unc owded en i onmen s, he senso emains in a elaxed
con o ma ion; in c owded en i onmen s, i is comp essed,
inc easing he FRET e iciency. An ad an age o a gene ically
encoded senso is ha i can be measu ed pu i ied in bu e as
well as measu ed when exp essed in cells. The e o e, i
p o ides a di ec compa ison be ween di e en c owded
en i onmen s.
He e, we use c GE2.3 o moni o mac omolecula c owding
du ing he sh inkage o gian unilamella esicles (GUVs) illed
wi h bac e ial cell lysa e. A ep oducible and eliable c owding
inc ease could be achie ed h ough a g adien inc ease o he
ex e nal osmo ic p essu e while moni o ing he senso a he
single liposome le el. We he eby gain insigh in o he
c owding in li ing cells, he di e ence wi h pu i ied p o ein
c owde s, and he in luence o small molecules.
■RESULTS AND DISCUSSION
Model Sys em o Replica e he C owded In acellula
En i onmen . To ec ea e he c owded in acellula en i on-
men , we p epa ed bac e ial cell lysa e ollowing a p o ocol
adap ed om Fujiwa a e al.
6
(see Ma e ials and Me hods).
The lysa e concen a ions anged om 60 o 80 mg/mL
p o ein in a lysis bu e (30 mM po assium glu ama e, 6 mM
magnesium glu ama e, 10 mM sodium phospha e bu e (NaPi,
pH 7.4), and a cOmple eTM p o ease inhibi o cock ail). We
encapsula ed eshly p epa ed lysa e in GUVs o eplica e he
c owded in acellula con inemen . The GUVs we e c ea ed
using he emulsion ans e me hod,
13
p o iding a ange o
esicle sizes. Each GUV con ained he c owding senso , 50
mg/mL lysa e, and 0.1 M suc ose o s abilize he esicles. The
ex e nal glucose solu ion in 10 mM NaPi, pH 7.4 was
isosmo ic. The esicles we e immobilized in 0.5% w/w low
mel ing aga ose o obse a ion a he single- esicle le el
(Figu e 1b,c).
14
Single- esicle obse a ion p o ided he ela i e
sh inkage o each esicle. The eby, we de e mined he inal
lysa e concen a ion despi e he dis ibu ion in esicle sizes.
To inc ease lysa e concen a ion om 50 mg/mL o
physiological le els, we g adually inc eased he osmola i y by
i a ing NaCl on op o he gel wi h he immobilized GUVs.
The sal was hen allowed o di use in o he gel (Figu e 1b,c).
This sal g adien p e en ed con en leakage om he esicles
and main ained he lysa e soluble. Indeed, sudden s epwise
Figu e 1. Response o single GUVs o inc eases in ex e nal osmolali y. (a) The gene ically encoded c GE2.3 (mEGFP/mSca le -I) c owding senso
used o measu e c owding in he esicles. C owding comp esses he p obe, he eby inc easing FRET e iciency. (b) GUVs immobilized in aga ose
(0.5% w/ ) in a chambe ed co e slip. The osmolali y o he ou e solu ion is slowly inc eased by adding NaCl solu ion in he op solu ion, which
g adually di used in o he aga ose gel. (c) Response o an immobilized GUV con aining lysa e and c GE2.3 o he osmo ic upshi . (d)
Fluo escence o mSca le -I (FRET accep o di ec exci a ion and emission) co ela es linea ly (g ay do ed line) wi h sh inkage up o 5 imes
sh inkage. (e) Sh inkage o a GUV depends on he ex e nal osmola i y, bu no o he same ex en o each c owde . ( ) While GUVs con aining
lysa e a e smalle han ones wi hou c owde , he amoun o sh inkage is independen o he ini ial GUV size. Do s a e anspa en and da ken when
o e laid. (g) The FRET/dono emission a io o c GE2.3 ( ed) inc eases in he p esence o lysa e bu no in he absence o c owde (black).
mEGFP and mSca le -I, as wo sepa a e molecules wi hou a linke (g ay), do no show a FRET/dono inc ease. E o ba s ep esen he s anda d
de ia ion o he a e ages o 3 independen expe imen s. Independen expe imen s a e connec ed wi h dashed lines.
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902
hype osmo ic upshi s led o phase sepa a ion o he lysa e
isible by mic oscopy (Figu e S1b), and a lack o he FRET
inc ease.
Nex , we e i ied he memb ane s abili y by plo ing he
inc ease in mSca le -I luo escence e sus he dec ease in
esicle olume (Figu es 1d and S1c). Bo h lysa e-c owded and
nonc owded esicles main ained linea i y up o a 5- old
dec ease in olume. Beyond his poin , luo escence emission
was lowe han expec ed. The linea i y upon concen a ing
om 50 o 250 mg/mL indica es eliable senso eadou s
wi hin he expec ed physiological c owding ange.
Because c owde s should inc ease osmolali y ia colloidal
osmo ic p essu e, we compa ed he ex e nal osmolali y
equi ed o sh ink he esicles (Figu e 1e). Vesicles con aining
c owde s equi ed highe ex e nal osmolali y o achie e he
same sh inkage as esicles wi hou c owde s. Hence, he
mac omolecules a ec he esicle’s esponse o osmo ic s ess.
In addi ion, esicles con aining cell lysa es equi e a highe
ex e nal osmola i y o sh ink han hose con aining BSA.
Possibly, BSA sel -associa ion lowe s i s colloidal osmo ic
p essu e, making i easie o sh ink he esicles. This aligns
wi h ou p e ious obse a ion whe e sh inkage o Ficoll-loaded
w/o/w emulsions equi ed highe osmola i y han emulsions
con aining BSA.
15
Impo an ly, he ela i e esicle sh inkage
was independen o i s ini ial size wi hin an expe imen (Figu e
1 ).
We moni o ed he c owding senso by lase scanning
con ocal mic oscopy. The FRET emission (ex. 488 nm, em.
600−700 nm) di ided by he dono emission (ex. 488 nm, em.
510−525 nm) inc eased wi h esicle sh inkage in lysa e-loaded
esicles om 0.41 ±0.02 o 0.60 ±0.07 (Figu es 1g and S1a).
In con as , he a io in bu e -only esicles showed a ma ginal
inc ease om 0.40 ±0.03 o 0.45 ±0.02, likely due o
in e molecula FRET, as we no ed be o e.
15
To es i he
inc ease in a io in he p esence o lysa e was due o FRET, we
compa ed ou da a wi h unlinked mEGFP and mSca le -I ha
should no FRET. Indeed, he a io was lowe , wi h a small
inc ease om 0.30 ±0.01 o 0.32 ±0.02 upon sh inkage, likely
due o in e molecula FRET as p oposed abo e. Hence, he
inc ease in in amolecula FRET o c GE2.3 ollows he
p oposed comp ession due o mac omolecula c owding. Thus,
we c ea ed s able GUVs wi h lysa e-induced mac omolecula
c owding ha can be inc eased by applying a sal g adien .
C owding Depends on he C owde and I s
Solubili y. BSA is a model c owde because i is a globula
p o ein and eadily a ailable in la ge quan i ies. Since we
obse ed a di e en p opensi y o esicles o sh ink when
loaded wi h BSA, we compa ed BSA-induced c owding wi h
lysa e-induced c owding. We encapsula ed 50 mg/mL BSA
wi hin ou GUVs and measu ed he c owding wi h he
c GE2.3 senso . We saw ha a he same p o ein weigh %, he
FRET e iciency was lowe in BSA-c owded GUVs compa ed
o lysa e-c owded GUVs, indica ing lowe c owding (Figu e
2a). Speci ically, he inc ease in he FRET/mEGFP a io was
1.17 ±0.07 o BSA-c owded GUVs and 1.4 ±0.1 o lysa e-
c owded GUVs a 4.5- old sh inkage. A possible explana ion is
ha BSA-induced mac omolecula c owding is lowe due o i s
endency o sel -associa e, simila o ou easoning why hese
esicles sh ink mo e (see abo e). While lysa es will also
con ain a signi ican mass o RNA, hei numbe densi y will
be ∼100- old lowe ,
16
and we expec di ec c owding e ec s o
o igina e p ima ily om he lysa e p o eins. The e o e, BSA is
a less e icien c owde han cell lysa es.
The c owding o cell lysa es is po en ially a ec ed by he
olding o i s cons i uen p o eins. Un olded p o eins ake up
mo e olume han olded p o eins and may inc ease c owding
unless hey agg ega e. The e o e, we aimed o de e mine he
ole o c owde dena u a ion on mac omolecula c owding. To
his end, we mixed na i e BSA wi h hea -dena u ed BSA in
bulk in he p esence o c GE2.3. BSA dena u a ion was
con i med wi h DLS measu emen s (Figu e S5b). Inc easing
na i e BSA om 0 o 200 mg/mL inc eases he FRET a io
om 0.076 ±0.001 o 0.092 ±0.001 (Figu e 2b), while
exchanging 10% o he na i e BSA wi h dena u ed BSA
p o ided sligh ly lowe a ios. Hence, un olding BSA somewha
lowe s he c owding. The solu ion u bidi y also inc eased
(Figu e S5a), sugges ing agg ega ion (o p ecipi a ion), which
could lowe he mac omolecula c owding. We conclude ha
p o ein dena u a ion does no inc ease c owding pe se, likely
due o inc eased agg ega ion.
Small Cosolu es Modula e Mac omolecula C owd-
ing. Nex , we in es iga ed he impac o suga s on ou lysa e-
c owded sys em. We ini ially inco po a ed suc ose o imp o e
memb ane s abili y.
17,18
Howe e , suga s could a ec mac o-
molecula c owding by (i) changing c owde con o ma ion o
hyd a ion s a e o (ii) changing mac omolecula c owde
numbe densi y by modula ing c owde −c owde in e ac ions:
suc ose and ehalose ha e been desc ibed o s abilize p o ein
olding and inducing p o ein sel -assembly.
19
Thus, we added
0.1 M suc ose o ehalose o 50 mg/mL lysa e-con aining
GUVs and compa ed his o he absence o added suga s.
These GUVs we e osmo ically sh unk as be o e. We saw ha
GUVs wi hou suga s had he highes a ios, ollowed by
suc ose and ehalose (Figu e 3a). A a ound 70 mg/mL
(a ound 1.4- old sh inkage), he FRET/mEGFP a ios we e
0.53 ±0.02 in he absence o suga s, 0.44 ±0.01 o suc ose,
Figu e 2. Mac omolecula c owding depends on he c owding agen . (a) FRET/mEGFP (no malized o isosmo ic condi ions) o lysa e-c owded
( ed), BSA-c owded (b own), o unc owded GUVs (black) plo ed agains he deg ee o sh inkage o hese GUVs showing c owde -dependence.
(b) FRET/mEGFP dependence on BSA concen a ion. BSA solu ions con aining 0%, 10%, 50%, o 100% hea -dena u ed BSA. E o ba s ep esen
he s anda d de ia ion o he a e ages o 3 independen expe imen s. Independen expe imen s a e connec ed wi h dashed lines.
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903
and 0.42 ±0.05 o ehalose. The di e ences became mo e
p onounced a 170 mg/mL lysa e (3.4- old sh inkage) wi h
0.58 ±0.02 in he absence o suga s, 0.52 ±0.04 o suc ose,
and 0.44 ±0.04 o ehalose. A 5- old sh inkage, 250 mg/mL,
he di e ences we e less as he c owding inc ease le eled o in
he absence o suc ose. Hence, suga s educe mac omolecula
c owding.
We hypo hesized ha i suga s educe c owding h ough
inc easing c owde −c owde in e ac ions, he di usion o a
es pa icle would inc ease wi h suga s. This con as s wi h he
inc eased iscosi y when dissol ing suga s a high concen-
a ions. We hus measu ed he senso di usi i y by
luo escence eco e y a e pho obleaching (FRAP). We saw
ha he lysa e-c owded senso in esicles con aining suc ose
indeed displayed as e eco e y imes ( 0.5 = 0.7 ±0.3 s a 158
±11 mg/mL) han hose wi hou suc ose ( 0.5 = 1.4 ±0.5 s a
147 ±17 mg/mL) (Figu e 3b), implying highe di usi i y.
Hence, he lowe c owding wi h suc ose aligns wi h highe
di usi i y despi e ha ing he same mg/mL lysa e, om which
we in e ha suc ose likely al e s c owde o ganiza ion.
To exclude ha suc ose educes c owding by inducing lysa e
agg ega ion (o p ecipi a ion), we assessed he u bidi y o
dense lysa es in bulk wi h and wi hou suc ose using Pu -A-
Lyze Midi Dialysis ubes. We ind ha he u bidi ies a e
simila : suc ose induced sligh ly lowe u bidi y (277 ±17
mAu a 91 ±6 mg/mL) compa ed o lysa es wi hou suc ose
(310 ±16 mAu a 87 ±6 mg/mL) (Figu e S4a). The e o e,
lysa e agg ega ion does no explain he di e ence in c owding
e ec s. To de e mine i suc ose a ec ed he senso di ec ly, we
i a ed suc ose o c GE2.3 in bu e . The FRET a ios display
a much smalle dec ease han in he liposomes upon adding 0.4
M suc ose (Figu e S3), and we can exclude ha di ec suc ose
in e ac ion wi h he senso plays a signi ican ole. Hence,
suc ose modula es mac omolecula c owding induced by cell
lysa es, likely by changing i s o ganiza ion.
Nex , we es ed adenosine iphospha e (ATP) as i was
sugges ed o al e p o ein s abili y and dispe se p o ein
assemblies,
20,21
which could hus change c owding as well.
We s a ed a 2.5 mM ATP in lysa es o each physiological
concen a ions o ∼10 mM ATP
22
upon esicle sh inking. We
saw ha ATP does no a ec he FRET a ios signi ican ly
(0.55 ±0.02 a 275 ±3 mg/mL lysa e; Figu e 4a). When we
ins ead s a ed wi h 10 mM ATP, we saw a highe c owding
indeed (0.61 ±0.02 a 273 ±7 mg/mL lysa e). These ATP
and suc ose e ec s do no compensa e: ATP in he absence o
suc ose gi es a lowe a io (0.46 ±0.07 a 272 ±3 mg/mL)
(Figu e 4a), implying a complex mechanism.
To ob ain insigh in o he e ec o ATP on mac omolecula
c owding, we measu ed FRAP. We saw ha eco e y wi h 10
mM ATP is slowe han wi hou ATP and as e han he
eco e y wi hou ATP and suc ose ( 0.5 = 1.1 ±0.3 s a 198 ±
10 mg/mL) (Figu es 4b and 3b). This obse a ion aligns wi h
ou hypo hesis ha when c owding inc eases, eco e y is
slowe . He e, we assume ha lysa e e ec s domina e o e
iscosi y changes om ATP i sel , simila ly as we no ed o
suc ose. The u bidi y o lysa es concen a ed in he p esence
o 10 mM ATP and 0.1 M suc ose emained a he simila
compa ed o he absence o ATP, sugges ing no ex ensi e
c owde solubili y change equi ed o change c owding (Figu e
S4b and dena u ed BSA expe imen s abo e). Mo eo e , ATP
does no a ec he senso di ec ly (Figu e S3b). Hence,
inco po a ing high ATP concen a ions beyond physiological
concen a ions, in combina ion wi h suc ose, enhances mac o-
molecula c owding.
We es ed he e ec o a ew o he addi i es. The small
zwi e ionic p o ein-p o ec i e osmoly es be aine and ime-
hylamine N-oxide (TMAO) did no signi ican ly in luence
mac omolecula c owding (Figu e S2c,d). The FRET a io is
somewha educed when using 40 mM ins ead o 6 mM
magnesium glu ama e (Figu e S2b). When we used lysa es
om cells adap ed o g ow wi h a 300 mM NaCl osmo ic
upshi , we obse ed he same c owding as lysa es om cells
g own in a egula LB medium (Figu e S2a). In conclusion,
small molecules can change mac omolecula c owding, and in
he case o suc ose and ATP, his eadou co esponds o a
concomi an change in p obe di usion.
Di ec Mac omolecula C owding Compa ison be-
ween A i icial Cells and Li ing Cells. Nex , we es ed i
mac omolecula c owding in he a i icial cells compa es o
li ing cells o (i) indica e i we achie ed physiological c owding
le els and (ii) assess how c owded li ing cells a e, based on
hei p o ein con en . To measu e he FRET a ios in li ing
cells, we exp essed c GE2.3 in E. coli BL21(DE3).
11
In con as
o in i o expe imen s, he ma u a ion o he luo escen
p o eins in E. coli is incomple e.
23
The e o e, we ea ed he
cells wi h chlo amphenicol o s op ansla ion, allowing he
Figu e 3. Suga s modula e mac omolecula c owding induced by
lysa es. (a) Lysa e-c owded GUVs con aining ei he suc ose (s a ing
concen a ion 0.1 M) ( ed) o ehalose (s a ing concen a ion 0.1
M) (blue) ha e lowe FRETs/mEGFP han esicles con aining no
suga s (g een) a he same o al p o ein lysa e concen a ion; (b)
FRAP expe imen ollows he measu ed c owding, showing he
a e age ime needed o lysa e-c owded GUVs con aining suc ose is
sho e han in he absence o suga s. E o ba s ep esen he
s anda d de ia ion o he a e ages o 3 independen epea s.
Independen expe imen s a e connec ed wi h dashed lines.
Figu e 4. ATP modula es he mac omolecula c owding o
concen a ed lysa es. (a) FRET/mEGFP was plo ed e sus he
o al p o ein concen a ion in lysa e-c owded GUVs con aining
s a ing concen a ions o suc ose (0.1 M) and ATP (2.5 mM o 10
mM, g een and da k blue espec i ely), ATP (10 mM) wi hou
suc ose (g ay), o suc ose (0.1 M) wi hou ATP ( ed); (b) FRAP
measu emen o lysa e c owded GUVs con aining ATP (10 mM) and
suc ose (0.1 M). E o ba s ep esen he s anda d de ia ion o 3
biological epea s.
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904
luo escen p o eins o ma u e and di ec ly compa e he a ios
wi h pu i ied p o ein in in i o expe imen s. We used he same
mic oscope se ings as he a i icial cells o di ec a io
compa ison. Chlo amphenicol ea men inc eased he FRET
a ios as he accep o ma u ed maximally (Figu e S6).
Applying an osmo ic upshi by adding NaCl o he medium
showed he expec ed FRET a io inc ease om 0.62 ±0.04 o
0.77 ±0.08 upon osmo ic upshi (Figu e 5, igh g een line).
We p e iously no ed ha adding suc ose ins ead o NaCl
p o ided simila esul s,
11,24
excluding NaCl-speci ic e ec s.
Nex , we compa ed he in-cell mac omolecula c owding
wi h ha o a i icial cells. Besides using he same mic oscopy
se ings and maximal luo escen p o ein ma u a ion, he
senso ’s insensi i i y o ele an cellula molecules acili a es
he compa ison.
11,24
In addi ion, lysa es ha e a simila
composi ion as he cy osol. We saw ha a i icial cells had
eached he same a ios as E. coli wi hin he biological a ia ion
o he expe imen s. This indica es ha he a i icial cells each
physiologically ele an mac omolecula c owding. The mac o-
molecula c owding in a i icial cells wi h a high ATP
concen a ion co esponds o 225−300 mg/mL cell lysa e
based on p o ein con en , which compa es wi h li e a u e
alues o E. coli (∼280−300 mg/mL).
25
Lysa es wi hou ATP
each he window o in-cell c owding a ∼300 mg/mL lysa e
p o ein. We canno inc ease he lysa e c owding u he , and
his aligns wi h ou obse a ions ha he luo escence inc ease
o mSca le -I is no linea wi h he dec ease in cell olume a e
250−300 mg/mL lysa e (Figu e 1 ). This has been seen be o e
o GFP luo escence in lysa es,
6
and co ela es wi h a d ama ic
inc ease in lysa e iscosi y,
7
which could be due o an al e ed
physical s a e o he lysa es. Appa en ly, addi ional lysa e o
memb ane componen s a e needed o highe c owding le els.
None heless, concen a ing cell lysa es p o ide mac omolecu-
la c owding le els o E. coli and a simila p o ein concen a ion
in mg/mL.
Since ou a i icial cells p o ide ele an mac omolecula
c owding le els, we assessed how c owding in mammalian cells
compa ed o E. coli. The mac omolecula c owding in
mammalian cells is epo ed o be lowe han in bac e ial
cells.
1
We measu ed he c owding in HEK293T cells s ably
exp essing he c GE2.3 c owding senso o ou model sys em.
Since HEK293T cells a e di iding much slowe han E. coli, we
assumed he luo escen p o eins ha e ully ma u ed. These
cells exhibi ed a FRET a io o 0.45 ±0.01. I was p e iously
shown ha lysa es om E. coli and mammalian cell lines ha e
simila physical p ope ies,
7
and we hus compa ed HEK293T
FRET a ios wi h ou bac e ia lysa e-c owded GUVs and ound
ha HEK293T c owding co esponded o 50−75 mg/mL in
GUVs wi h 10 mM ATP, o 70−122 mg/mL in GUVs wi hou
ATP (Figu e 5). We hus con i m wi h c GE2.3 ha c owding
is lowe in a human cell line and ha i is s aigh o wa d o
each HEK293T mac omolecula c owding wi h E. coli lysa es.
■CONCLUSION
In his s udy, we encapsula ed E. coli lysa es in liposomes o
achie e he o iginal in-cell mac omolecula c owding le els.
Ou app oach demons a ed ha concen a ed bac e ial lysa e
can mimic he in acellula en i onmen , p o iding a mo e
accu a e ep esen a ion han adi ional p o ein o polyme
c owde s. This sys em b idges he gap be ween in i o and in
i o condi ions by p o iding a complex he e ogeneous cell-like
en i onmen ha includes di e se su ace chemis ies c i ical
o cellula unc ion. The FRET-based c owding senso ,
c GE2.3, enabled us o quan i a i ely compa e c owding le els.
The senso showed ha E. coli lysa es eached c owding
concen a ions equi alen o hose in E. coli and HEK293T
cells. The obse a ion ha small molecules such as suc ose and
ATP modula e c owding in a i icial cells implies ha hey ha e
consequences o mac omolecula c owding in li ing cells: his
would be challenging o in es iga e in li ing cells because
changing he concen a ion o a molecule in a cell leads o a
physiological esponse. Thus, hese a i icial cells wi h
physiological c owding o e mo e con ol han in acellula
s udies, making hem an excellen subs i u e o he na i e
cy osol.
■MATERIALS AND METHODS
Gene Exp ession and P o ein Pu i ica ion. Chemically
compe en E. coli BL21(DE3) cells we e ans o med wi h
plasmid pRSET A con aining he c owding senso c GE2.3
gene.
26
The cells we e g own in 200 mL Lu ia−Be ani (LB)
medium (NaCl 10 g/L, yp one 10 g/L, yeas ex ac 5 g/L)
con aining 50 μg/mL ca benicillin a 37 °C un il he OD600
eached 0.6. P o ein exp ession was induced o e nigh wi h 1
mM isop opyl β-D-1- hiogalac opy anoside (IPTG) a 28 °C
and 180 pm. The cells we e ha es ed by cen i uga ion a
4000 g o 40 min and washed once wi h isosmo ic sodium
phospha e bu e (NaPi, pH 7.4). The cell pelle was hen
esuspended in lysis bu e (500 mM NaCl, 50 mM NaPi, pH
7.4, 6 mM MgCl2, 1 mg/mL lysozyme, cOmple e EDTA- ee
p o ease inhibi o cock ail), incuba ed a 4 °C o 30 min, and
lysed wi h a high-p essu e homogenize (Cons an Sys ems
L d.) a 20 kpsi. The cell ex ac was ea ed wi h
deoxy ibonuclease I (DNase I) o 30 min, clea ed by
cen i uga ion a 4 °C o 30 min a 16000 g, and
supplemen ed wi h 10 mM imidazole. The ecombinan
p o ein was pu i ied using he NGC Disco e 10 Ch oma og-
aphy Sys em (BioRad) wi h a HisT ap FF column (Cy i a).
The wash and elu ion bu e s con ained imidazole (20/250
mM), NaPi, pH 7.4 (50 mM), and NaCl (500 mM). The
pu i ied p o ein was analyzed using 10% SDS-PAGE. The
ac ions ha showed pu e p o ein we e combined, and he
bu e was exchanged o 10 mM NaPi, pH 7.4, aliquo ed, and
s o ed a −80 °C.
Figu e 5. Compa ison o lysa e-c owded GUVs wi h he c owding o
cells. Le : FRET/mEGFP o osmo ically sh unken GUVs con aining
no c owde (black), lysa e ( ed), o lysa e wi h 10 mM ATP s a ing
concen a ion (da k blue), plo ed agains he o al p o ein
concen a ion in he GUVs. Da a is om Figu e 4a. Righ : FRET/
mEGFP o osmo ically s essed E. coli cells (g een) ea ed wi h 0.2
mg/mL chlo amphenicol, and HEK293T cells (ligh blue do ).
Do ed lines and g ay shading ep esen he a e age a io and
s anda d de ia ion o uns essed cells. E o ba s ep esen he
s anda d de ia ion o 3 biological epea s. Independen expe imen s
a e connec ed wi h dashed lines.
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h ps://doi.o g/10.1021/acssynbio.4c00824
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905
Gene a ion o Cell Lysa es. A s a e cul u e o E. coli
BL21(DE3) was g own a 37 °C in 50 mL LB wi hou a
selec ion ma ke . Then, he cul u e was dilu ed in 1 l esh LB
media o OD600 0.05 and g own a 37 °C and 180 pm un il
OD600 o 0.9−1.0. The bac e ia we e ha es ed by
cen i uga ion a 4000 g, washed once wi h isosmo ic NaPi,
pH 7.4, and he pelle was esuspended wi h lysis bu e . The
cells we e lysed by unning he cell suspension wice h ough a
high-p essu e homogenize (Cons an Sys ems L d.) a 20
kpsi. DNase I was added, and he lysa e was incuba ed o 30
min a oom empe a u e. The lysa e was cen i uged o 90
min a 21300 g a 4 °C. The inal p o ein concen a ion o he
soluble ac ion was measu ed wi h he Pie ce BCA P o ein
Assay. The lysa es we e used immedia ely o aliquo ed and
s o ed a −80 °C.
Depending on he expe imen , he lysis bu e con ained a
combina ion o he ollowing componen s: 10 mM NaPi, pH
7.4, 6 mM o 40 mM magnesium glu ama e, 30 mM po assium
glu ama e, comple e EDTA- ee p o ease inhibi o cock ail, 0.1
o 0.25 M TMAO, 0.1 o 0.25 M be aine, 2.5 mM o 10 mM
ATP.
Gene a ion and Immobiliza ion o GUVs. Gian
unilamella esicles (GUVs) we e gene a ed wi h he in e ed
emulsion me hod, also known as he wa e -in-oil emulsion-
ans e me hod.
13,27
In sho , he oil phase consis s o a
mix u e o 5 mg/mL 1-palmi oyl-2-oleoyl-sn-glyce o-3-phos-
phocholine (POPC):1-palmi oyl-2-oleoyl-sn-glyce o-3-phos-
pho-(1′- ac-glyce ol) (POPG):choles e ol (8.5:1:0.5 weigh
a io) in liquid pa a in oil (FUJIFILM Wako). Depending on
he expe imen , he inne solu ion con ained a combina ion o
c owde (cell lysa e, BSA, o none), lysis bu e , c GE2.3, and
suga s (0.1 M suc ose, 0.1 M ehalose, o none). The c GE2.3
concen a ion should be a leas 4 imes he backg ound
luo escence in all mic oscopy channels (see below). We ha e
seen no dependence o he eadou s in he senso
concen a ion ange o ∼25−200 μg/mL. The ou e solu ion
was an isosmo ic solu ion o glucose in 10 mM NaPi, pH 7.4.
Osmolali ies we e measu ed wi h Gono ec Osmoma 3000.
An emulsion o inne solu ion and oil was c ea ed by mixing
30 μL inne solu ion in 300 μL oil phase. The emulsion was
incuba ed o 10 min on ice, ca e ully laye ed on op o he
ou e solu ion, and incuba ed o an addi ional 10 min. Then,
he GUVs we e c ea ed by cen i uga ion o 5 min a 5000 g
and 4 °C. The clea oil phase and mos o he ou e solu ion
we e ca e ully pipe ed ou so as no o dis u b he pelle . A
esh ou e solu ion was added, and he pelle was ca e ully
esuspended. The gene a ed GUVs we e immobilized in
aga ose, as desc ibed in.
14
Top Vision Low Mel ing Poin
Aga ose (The mo Scien i ic) was dissol ed in NaPi, pH 7.4 o
4% (w/ ). The mol en aga ose was mixed wi h he GUVs
solu ion o c ea e a 0.5% (w/ ) inal concen a ion o aga ose.
Then 200 μL o he GUV-aga ose solu ion was placed in a
chambe ed co e slip wi h 8 wells (Ibidi) and le o
polyme ize. Once he aga ose was ully polyme ized, a 200
μL ou e solu ion was laye ed on op. Single GUVs we e
measu ed and hen osmo ically s essed by ca e ully adding
200 μL hype osmo ic NaCl solu ion on op. A e 10 min o
incuba ion, new pic u es we e aken o he same GUVs. The
osmo ic s ess s ep was epea ed un il he GUVs showed
signi ican sh inkage. The olume o he GUVs a each s age
was de e mined by hei diame e .
Fluo escen Mic oscopy and FRET Measu emen s.
Scanning con ocal luo escen mic oscopy was pe o med on
Leica TCS SP8 wi h 63×(1.2 NA) wa e imme sion objec i e
and 3.5 Ai y. The monome ic enhanced g een luo escen
p o ein (mEGFP) was exci ed a 488 nm and de ec ed a 510−
525 nm emission (dono channel), whe eas mSca le -I was
exci ed a 561 nm and de ec ed wi hin he 600−700 nm band
(accep o channel). The FRET channel was de ec ed by
exci a ion a 488 nm and de ec ion in he 600−700 nm
emission band. The de ec ion o all channels was done wi h
pho omul iplie ube (PMT) de ec o s wi h 1000 gain. The
backg ound signal o he cell lysa e was sub ac ed as indica ed
by GUV/cells wi hou c GE2.3. Image quan i ica ion was done
wi h ei he Leica Applica ion Sui e o wi h ImageJ Fiji.
FRAP Measu emen s. FRAP expe imen s we e conduc ed
using a con ocal lase scanning mic oscope, Ca l Zeiss LSM880
Fas Ai yScan, wi h a 63×glyce ol imme sion objec i e (1.2
NA). Images we e cap u ed a 128 ×128 pixel esolu ion, wi h
a ime se ies o 40 p ebleach ames, 3 bleach cycles, and 100
eco e y ames, o aling 340 ames a 51.61 ms pe ame. A
sphe ical egion o in e es (ROI) was bleached using a 561
nm lase a 100% powe o 15 i e a ions. Reco e y images
we e aken a 1% powe , and de ec ion was ia a PMT
de ec o .
Fo each condi ion, h ee esicles we e used o acquisi ion
pho obleaching measu emen and h ee o lysa e backg ound
measu emen . A leas se en esicles pe condi ion we e
pho obleached. The lysa e backg ound was sub ac ed, and
acquisi ion pho obleaching was co ec ed. F ames 30−40 o
he p ebleach phase we e used o no maliza ion. Reco e y
cu es we e i ed using G aphPad P ism wi h a one-phase
exponen ial associa ion equa ion.
Concen a ing Cell Lysa es in Bulk. Bac e ial cell lysa es
we e p epa ed as desc ibed abo e. These lysa es we e hen
concen a ed ia e apo a ion in Pu -A-Lyze Midi Dialysis
ubes. In sho , 800 μL o lysa e a a concen a ion o 50 mg/
mL was placed in each dialysis ube and incuba ed a 4 °C in a
en ila ed oom. A each ime poin (0, 18, 24, 30, and 43 h),
aliquo s we e aken o measu e p o ein concen a ion (Pie ce
BCA p o ein assay), e ac i e index, and u bidi y.
P epa a ion o Dena u ed BSA. BSA was dissol ed in 10
mM NaPi, pH 7.4, and incuba ed o e nigh a 4 °C o c ea e a
s ock BSA solu ion wi h a concen a ion o 300 mg/mL.
Subsequen ly, u he dilu ions o he BSA s ock wi h 10 mM
NaPi, pH 7.4 we e made o achie e inal concen a ions o 0,
50, 100, 150, and 200 mg/mL BSA. A 50 mg/mL BSA dilu ion
was dena u ed by incuba ing i a 95 °C o 1 h a 1000 pm,
ollowed by cooling o oom empe a u e. The dena u a ion
was con i med by dynamic ligh sca e ing (DLS) (Figu e
S5b). By mixing he dena u ed 50 mg/mL BSA solu ion wi h
he nondena u ed 300 mg/mL s ock BSA, we p epa ed he
BSA solu ions used in Figu e 2b. These solu ions had inal
concen a ions o 50, 100, 150, and 200 mg/mL BSA, wi h
10% o he BSA being dena u ed. Fo he solu ion wi h a inal
concen a ion o 50 mg/mL, addi ional solu ions we e
p epa ed in which 50% o 100% o he BSA was dena u ed.
Spec o luo ome e Measu emen s. The luo escence
spec um o c GE2.3 was measu ed wi h an exci a ion
wa eleng h o 465 nm and an emission ange o 490−700
nm using a Va ian Ca y Eclipse Spec opho ome e and Qua z
Glass High Pe o mance cu e e (1 mL). Fi s , he spec um o
he solu ion wi hou c GE2.3 was measu ed o de e mine he
backg ound signal. Subsequen ly, c GE2.3 (25 μg/mL) was
added, and he spec um was measu ed again. The FRET/
dono was calcula ed a e sub ac ing he backg ound and
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h ps://doi.o g/10.1021/acssynbio.4c00824
ACS Syn h. Biol. 2025, 14, 901−908
906
di iding he a e age emission in he 583−597 nm band (FRET
emission) by he emission in he 505−515 nm band (dono
emission). Addi ionally, he accep o was di ec ly exci ed a
569 nm, wi h he emission de ec ed in he 580−700 nm band
(accep o emission).
Tu bidi y and DLS Du ing The mal Un olding.
Tu bidi y and DLS measu emen s we e pe o med using he
P ome heus Pan a (NanoTempe ) wi h P ome heus high-
sensi i i y capilla ies. Tu bidi y measu emen s we e conduc ed
a 50% lase powe ac oss a empe a u e ange om 15 o 95
°C (un olding s age) and om 95 o 15 °C ( e olding s age).
Fo he DLS measu emen s he samples we e dilu ed o 2 mg/
mL and measu ed a 20 °C and 50% lase powe .
E. coli Fluo escence Con ocal Mic oscopy. E. coli
BL21(DE3) was ans o med wi h c GE2.3, pla ed on LB
aga pla es wi h ca benicillin (50 μg/mL), and hen incuba ed
o e nigh a 37 °C. Non ans o med E. coli BL21(DE3) cells
we e also pla ed on LB aga wi hou a selec ion ma ke o
con ol o au o luo escence. Single clones om bo h pla es
we e g own in MOPS minimal medium
28
o e nigh a 37 °C
wi h shaking a 200 pm. The o e nigh cul u es we e dilu ed
in esh medium o an OD600 o 0.015−0.025 and g own a 37
°C, 200 pm, un il an OD600 o 0.14−0.15. Cells we e hen
ans e ed o wo new lasks and spli in o con ol and
chlo amphenicol- ea ed g oups (0.2 mg/mL), wi h non-
ans o med cells added o bo h. Bo h g oups we e incuba ed
a 37 °C, 200 pm, o 1.5 h. A e incuba ion, aliquo s om
bo h g oups we e collec ed and washed wice in esh MOPS
medium (wi hou po assium and glucose) by cen i uga ion a
3000 g o 1 min a 37 °C. NaCl was added o induce osmo ic
s ess o cells in he absence o po assium, and he cells we e
placed on co e slips. Images we e aken as desc ibed abo e
wi hin 5 min o NaCl addi ion.
Mammalian Cell Cul u e. Human emb yonic kidney
293T (HEK293T) cells and HEK293T cells exp essing
c GE2.3 we e cul u ed in Dulbecco’s Modi ied Eagle Medium
(DMEM) supplemen ed wi h 10% e al bo ine se um (FBS)
and 1% penicillin-s ep omycin a 37 °C wi h 5% CO2. Fo
mic oscopy analysis, he cells we e mixed and pla ed a a
densi y o 190,000 cells pe well in an 8-well chambe ed
co e slip (Ibidi). The cells we e incuba ed o e nigh a 37 °C
wi h 5% CO2. The nex day, he old medium was eplaced wi h
esh media (DMEM + 10% FBS + 1% penicillin−
s ep omycin, no phenol ed). Fluo escen images we e
ob ained as desc ibed abo e, wi h he cells main ained a 37
°C du ing imaging.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acssynbio.4c00824.
Cha ac e iza ion o liposome sh inkage and FRET,
e ec s o cosolu es on mac omolecula c owding, di ec
in e ac ion o cosolu es wi h he FRET senso , u bidi y
measu emen s o lysa es, un olding and DLS o BSA, he
ole o chlo amphenicol on c owding in E. coli, and he
lis o DNA sequences o he genes encoding he
c GE2.3 o E. coli and HEK293T (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
A nold J. Boe sma −Cellula P o ein Chemis y, Bij oe
Cen e o Biomolecula Resea ch, Facul y o Science, U ech
Uni e si y, U ech 3584 CH, The Ne he lands; DWI-
Leibniz Ins i u e o In e ac i e Ma e ials, Aachen 52074,
Ge many; o cid.o g/0000-0002-3714-5938;
Email: [email p o ec ed]
Au ho s
Mila a S. Kalache a −Cellula P o ein Chemis y, Bij oe
Cen e o Biomolecula Resea ch, Facul y o Science, U ech
Uni e si y, U ech 3584 CH, The Ne he lands; DWI-
Leibniz Ins i u e o In e ac i e Ma e ials, Aachen 52074,
Ge many
Nuno R. da Sil a −Cellula P o ein Chemis y, Bij oe Cen e
o Biomolecula Resea ch, Facul y o Science, U ech
Uni e si y, U ech 3584 CH, The Ne he lands; CEB -
Cen e o Biological Enginee ing, Uni e sidade do Minho,
4710-057 B aga, Po ugal; LABBELS - Associa e Labo a o y
in Bio echnology, Bioenginee ing, and Mic oelec omechanical
Sys ems, B aga 4710-057, Po ugal
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acssynbio.4c00824
Au ho Con ibu ions
A.J.B. concei ed he p ojec . M.S.K. and A.J.B. designed and
concep ualized expe imen s. M.S.K. p epa ed and pe o med
expe imen s and acqui ed da a. M.S.K., N.R.S., and A.J.B.
analyzed and in e p e ed da a. M.S.K. and A.J.B. w o e he
manusc ip .
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
The wo k was unded by he ERC Consolida o G an
(PA Cell; No. 864528).
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