600 Research Article

IntroductionLiving tissues are increasingly seen as exceptionally intricate, well-

structured collections of cells embedded in dynamically redefined

extracellular matrices. In order to robustly perform their functions,

cells comprising these tissues have to be in constant communication,

engaging in a complex cooperation in the face of a dynamically

changing environment. Among the various means of cooperative

interaction evolved by living cells, communication by direct

physical intercellular transfer still ranks among the more exotic and

poorly understood.

Intercellular protein transfer (IPT), although relatively recently

identified, has been implicated in a variety of biological processes,

many of which have clear relevance to important pathological states.

For instance, a number of glycosyl phosphatidylinositol (GPI)-

anchored proteins can be directly transferred onto the plasma

membrane of neighboring cells (Ilangumaran et al., 1996; Kooyman

et al., 1995; Liu et al., 2002) including CD55 (19 kDa) and CD59

(70 kDa), two GPI-anchored complement restriction factors. Two

animal models demonstrated in vivo that normal functions of

transferred human CD55 and CD59 were preserved on recipient

cells. In the mouse model, human CD55 and CD59 transferred from

mouse erythrocytes to mouse endothelial cells inhibited the

complement activation in mouse heart vasculature when it was

perfused with human plasma (Ilangumaran et al., 1996). Similar

protection of the heart vasculature from complement activation by

CD55 and CD59 transferred from erythrocytes to endothelial cells

was observed in a transgenic pig model (McCurry et al., 1995). It

was also found that GPI-anchored cellular prion protein PrPC (35

kDa) could be transferred in vitro from donor neuroblastoma cells

onto erythroleukemia cells when they were co-cultured in the

presence of phorbol myristate acetate (PMA), suggesting that

transfer of GPI-anchored proteins might also have implications in

the pathogenesis of prion diseases (Liu et al., 2002).

Intercellular transfer of membrane proteins is not limited to GPI-

anchored proteins, which are only linked to the outer layer of plasma

membrane with a single GPI linkage. It has been reported that

CCR5, a 40 kDa co-receptor for HIV-1 infection, could be

transferred on to CCR5-negative cells through IPT (Mack et al.,

2000). Transferred CCR5 enabled the HIV-1 infection of CCR5-

negative cells, demonstrating the functionality of transferred protein

on recipient cells. Moreover, we have recently shown that P-

glycoprotein (P-gp), a 170 kDa multidrug-resistance-mediating

protein, could be transferred from P-gp+ drug-resistant cells to P-

gp– drug-sensitive cells in vivo within tumors and in vitro between

a number of cell types upon co-culture (Levchenko et al., 2005).

Thus, functional transfer of P-glycoprotein confers the multidrug-

resistance phenotype on recipient cells both in vitro and in vivo.

Recently, it has also been reported that membrane proteins and

cytoplasmic organelles could be transferred through nanotubes

between PC12 cells and several other cell types (Rustom et al.,

2004). The transferred proteins included GFP-labeled Class I MHC

proteins and the farnesylation signal of Ha-Ras. Transfer of GPI-

anchored GFP along the nanotubes, also observed in this study,

suggested that transfer of GPI-anchored membrane protein might

share some similarities with the transfer mediated by nanotubes

(Onfelt et al., 2004).

Recent evidence from different research areas has revealed a

novel mechanism of cell-cell communication by spontaneous

intercellular transfer of cellular components (ICT). Here we

studied this phenomenon by co-culturing different cells that

contain distinct levels of proteins or markers for the plasma

membrane or cytoplasm. We found that a variety of

transmembrane proteins are transferable between multiple cell

types. Membrane lipids also show a high efficiency of

intercellular transfer. Size-dependent cytoplasmic transfer

allows exchange of cytoplasmic macromolecules up to 40 kDa

between somatic cells, and up to 2000 kDa between

uncommitted human precursor cells and human umbilical vein

endothelial cells. Protein transfer, lipid transfer and

cytoplasmic component transfer can occur simultaneously and

all require direct cell-cell contact. Analyses of the properties

of ICT, together with a close examination of cell-cell

interactions, suggest that the spontaneous ICT of different

cellular components might have a common underlying process:

transient local membrane fusions formed when neighboring

cells undergo close cell-cell contact.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/5/600/DC1

Key words: Cell-cell communication, Intercellular transfer,

Membrane fusion, Stem cell

Summary

Physical transfer of membrane and cytoplasmiccomponents as a general mechanism of cell-cellcommunicationXinle Niu1, Kshitiz Gupta1, Joy T. Yang2, Michael J. Shamblott3 and Andre Levchenko1,*1Department of Biomedical Engineering, 2Department of Cell Biology and 3Department of Gynecology and Obstetrics, Johns Hopkins University,School of Medicine, Baltimore, MD 21205, USA*Author for correspondence (e-mail: alev@bme.jhu.edu)

Accepted 24 October 2008Journal of Cell Science 122, 600-610 Published by The Company of Biologists 2009doi:10.1242/jcs.031427

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601Cell-cell communication by intercellular transfer

The mechanism of IPT is still unclear, leading to the suggestion

of several, potentially conflicting hypotheses about how IPT might

occur. For instance, it has been proposed that GPI-anchored proteins

are released from the donor cell membrane or extracted by plasma

lipid-carrier proteins, with some of the GPI-anchored proteins

subsequently being re-inserted into recipient cell membrane

(Ikezawa, 2002). The mechanism of CCR5 transfer has been

proposed to rely solely on secretion of membrane microparticles

by donor cells followed by their re-integration into the membranes

of the acceptor cells. Transfer of P-gp was shown to crucially depend

on cell-cell contact by a mechanism other than one of those

previously proposed. As discussed above, formation of nanotubules

by an as yet unknown mechanism presents a possible explanation

of IPT mediated by cell-cell-contact, although it seems unlikely that

significant amounts of proteins can be transferred through the one

to three nanotubes reported to connect interacting cells. The

mechanism(s) of IPT is thus still, for the most part, a mystery, and

it is not clear whether IPT is a general phenomenon or is restricted

to relatively few transferred proteins in a limited number of cell

types.

In this study we propose that IPT is indeed quite general and can

occur through transient cell-cell fusion following direct cell-cell

contact. We also suggest that this transient fusion might lead not

only to IPT, but also to exchange of other cellular components,

including components of the plasma membrane and the cytosol,

constituting a more general intercellular molecular component

exchange phenomenon we termed intercellular component transfer

(ICT). We provide evidence that ICT might be enhanced when it

occurs between stem and somatic cells.

ResultsModel of membrane-fusion-based IPTIn both cell culture and in vivo, many cell types are actively or

passively motile, with cells forming frequent transient contacts

while undergoing random or directed movement. Our previous

analysis of P-gp IPT strongly suggested that cell-cell contact is

of crucial importance for efficient protein transfer. Moreover, we

indeed observe that cells in culture can move with respect to each

other, forming and breaking contacts, which might lead to transient

fusion of cellular membranes of two neighboring, cells

(supplementary material Movies 1 and 2). We therefore

hypothesized that IPT can occur during transient cell-cell contacts

made by cells while they move with respect to each other, and

that the IPT is mediated by transient membrane fusion events,

allowing a limited time for protein molecules to migrate by lateral

diffusion to an adjacent cell. This hypothesis can be expressed in

the form of a simplified model relating the level of expression of

the transferred protein on the surface of acceptor cells to the

expression of the same protein on the surface of donor cells. This

model (see Materials and Methods) yields the following formula:

where φA and φD are the surface densities of the transferred protein

on the acceptor and donor cells respectively, β is a normalization

factor reflecting the extent of the transient cell-cell fusion, LC is

the average size (diameter) of a cell, μ is the lateral membrane

mobility of the transferred protein, cD is the relative density of donor

cells in the cell population, and γ is the rate of loss of the protein

from the acceptor cells (through dilution due to cell division or

φA = ,β LC μ cD

γ + β LC μ cD

φ D (1)

protein degradation). If γ is much greater than the product: β LC μcD, formula (Eqn 1) can be reduced to the following expression:

where E=φA/φD is defined as the efficiency of IPT. An indirect way

of establishing the validity of this approximation is to evaluate how

close φA and φD are to each other. If γ is relatively small, than φA<φD,

whereas if γ is relatively large, φA can be much smaller than φD,

which, as shown below, is frequently the case. Therefore the

approximate formula (Eqn 2) might be applicable in most cases.

From the above formulas, it is clear, that the efficiency of IPT is

expected to increase with increased lateral membrane mobility of

the protein of interest, which can be enhanced for smaller proteins

or with increased membrane fluidity. In addition, IPT is expected

to be augmented if the relative density of donor cells or the

expression of the protein on donor cells increases. These predictions

can be directly tested experimentally. Additionally, the proposed

hypothesis directly implies further properties of IPT: it should be

a general phenomenon, not confined to a small number of proteins

or cell types; the transfer should be bidirectional, with cells capable

of being simultaneously both donors and acceptors; in addition to

IPT one also expects transfer of membrane lipids and cytosolic

molecules: the intercellular component transfer (ICT). Finally,

continuous IPT and ICT are expected to critically depend on the

constant presence of donor cells, with transferred intracellular

components otherwise being quickly lost from the acceptor cells.

Protein transfer is bidirectionalTo test whether surface proteins on co-cultured cells of distinct types

can be mutually exchanged – or IPT is bidirectional – we explored

potential transfer of three proteins not previously shown to undergo

IPT. Specifically, we studied Chinese hamster ovary (CHO) cells

overexpressing human membrane proteins CD36, ICAM-1 and a

GFP-conjugated integrin subunit α4-GFP. Although all these

proteins can mediate cell-cell adhesion, their ligands are absent in

CHO cells. We then used flow cytometry to investigate protein

transfer, because this technique allows the identification of the levels

of fluorescence-labeled cellular components of both donor and

recipient cell populations in one experiment. The results suggested

that these proteins could indeed be transferred to the wild-type

parental CHO cells. To investigate whether cell-cell contact was

indeed required for protein transfer, we cultured untransfected CHO

cells separated from transfected CHO cells with a 0.45 μm filter.

Flow cytometry analysis demonstrated that, as previously observed

for P-gp transfer, the transfer required cell-cell contact and the

continuous presence of the protein donor cells (Fig. 1A-C). A time-

course observation of protein transfer in a co-culture of donor CHO-

CD36+ cells and recipient wild-type CHO cells revealed that

transfer of membrane protein can be observed within 1 day of co-

culture and continues for more than 4-5 days.

Although the appearance of GFP-conjugated α4 on

untransfected CHO cells demonstrated that it was physically

transferred onto the recipient cells, there existed a very remote

possibility that the CD36 and ICAM-1 detected on untransfected

CHO cells using mouse antibodies for human CD36 and ICAM-

1 might be endogenous CD36 or ICAM-1 induced in untransfected

CHO cells during co-culture. To provide an additional test of

transfer of these proteins, we co-cultured CHO-CD36+ cells and

CHO-ICAM-1 cells with mouse NIH 3T3 fibroblasts, followed

φA = ,βLC μ cD

γϕD ≡ Eφ D

(2)

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by immunostaining with mouse anti-human CD36 or ICAM-1

antibody that could not recognize endogenous mouse CD36 or

ICAM-1 on the recipient NIH 3T3 cells. A pronounced increase

of CD36 and ICAM-1 in the mouse recipient cells (supplementary

material Fig. S1L,N) indicated that CD36 and ICAM-1 could

indeed be transferred. We provided further evidence for transfer

by directly immunostaining the co-culture of CHO-

CD36+ and CHO-α4-GFP cells and quantifying the

protein levels by fluorescent microscopy (Fig. 1F). These

two cell types were differentiated in fluorescent images

by their distinct levels of phycoerythrin (PE)-labeled

CD36 and α4-GFP. The results were consistent with flow

cytometry analysis.

We next co-cultured CHO-CD36+, CHO-α4-GFP and

CHO-ICAM-1 cell lines in a pair-wise fashion. We

observed that the corresponding proteins could be mutually

exchanged between the respective protein donor and

acceptor cells leading to a stable increase in transferred

protein concentrations in the acceptor cells (Fig. 2A-C),

suggesting that IPT is indeed bidirectional. The efficiency

of the transfer was found to be different for different

proteins, with CD36 being transferred with approximately

double the efficiency (E ~4%) of ICAM-1 and α4-GFP (E~2% for both). The differential transfer efficiency was

possibly due to differences in the lateral membrane mobility

of these proteins, which have considerably different

molecular masses: 88 kDa for CDF36; 180 kDa for ICAM-

1 homodimer and 280 kDa for α4β1 integrin heterodimer

(Pinco et al., 2002), in agreement with the mathematical

model.

IPT depends on the culture conditions membranefluidityWe next tested whether other properties of IPT were

consistent with our model. We have shown in a previous

study that the efficiency of P-gp transfer increased as the

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ratio of donor cells to recipient cells increased (Levchenko et al.,

2005). Similar results were observed for ICT of CD36 (Fig. 3A),

ICAM-1 (Fig. 3B), α4-GFP (supplementary material Fig. S1H) and

EGFR (170 kDa) (supplementary material Fig. S1H,T), in agreement

with the model predicting an increase in transfer efficiency in

proportion to CD/CA. We also found an increase in the efficiency of

Fig. 1. Physical transfer of membrane proteins inCHO cells. (A-C) Untransfected CHO cells (solidblack lines) were co-cultured respectively with labeledcells (dashed pink lines): CHO-CD36+ cells (A),CHO-ICAM1 cells (B) and CHO-α4-GFP cells (C).Cell populations were separated by a 0.45 μm filter(dotted black lines), or were grown in co-culture(green lines). (D) Time course of CD36 transfer fromCHO-CD36 cells to untransfected CHO cells beforeco-culture (blue), and after 1 day (green), 2 days (red),3 days (cyan), 4 days (purple) and 5 days (light green).(E) Average fluorescence intensity per unit celldetermined by epifluorescence in co-culture of CHO-CD36+ cells and CHO-α4-GFP cells. Each barrepresents the average epifluorescence intensity of atleast 50 cells.

Fig. 2. Bidirectional transfer of membrane proteins between CHO-CD36+, CHO-ICAM1 and CHO-α4-GFP cells co-incubated a pair-wise fashion. (A) CD36 and α4-GFP transfer between CHO-CD36+ cells and CHO-α4-GFP cells. (B) ICAM-1 and α4-GFP transfer between CHO-ICAM1 cells and CHO-α4-GFP cells. (C) CD36 andICAM-1 transfer between CHO-CD36+ cells and CHO-ICAM1 cells.

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CD36 and ICAM-1 transfer under conditions of higher initial cell

density (Fig. 3C; supplementary material Fig. S3), which was also

consistent with our model.

Consistent with the model predictions and our previous studies

(Levchenko et al., 2005), the transfer was transient and reversible.

The CHO-CD36 cells distributed by ATCC were derived from CHO

cells by transfection with linearized cDNA encoding human CD36

followed by stable selection with G418. We found that the cells

were actually a mixture of two distinct subpopulations: CHO-CD36+

with a higher level of CD36, and CHO-CD36– with a much lower

level of CD36. After these two subpopulations were separated by

fluorescence-activated cell sorting, the CD36 level on CHO-CD36–

cells rapidly decreased to that of untransfected CHO cells. When

the sorted CHO-CD36– cells were co-cultured again with the sorted

CHO-CD36+ cells, the CD36 level on CHO-CD36– cells increased

to a level close to that of the low-CD36 subpopulation in the original

heterogeneous CHO-CD36 cell culture (Fig. 3D). These results

indicate that constant contact between co-cultured cells is required

to maintain a steady protein transfer.

Our model also predicts that an increase in the lateral membrane

mobility might enhance the efficiency of transfer. Linoleic acid,

an unsaturated omega-6 fatty acid known to increase membrane

fluidity (Nano et al., 2003) was found to promote protein transfer.

In its presence, the ICAM-1 levels on the recipient cells and the

donor cells did not change, but the transfer of ICAM-1 was

enhanced approximately twofold (Fig. 3E). Cholesterol is a rigid

amphipathic molecule that has an important role in plasma

membrane integrity and function (Simons and Ikonen, 2000).

Cyclodextrin has been demonstrated to be an effective method to

manipulate cellular content of cholesterol (Christian et al., 1997).

In the presence of methyl-β-cyclodextrin, levels of CD36 in

acceptor CHO cells increased, compared with that in the control

(Fig. 3F). The cases above indicate that physical properties of the

plasma membrane might contribute to the regulation of intercellular

protein transfer.

IPT can involve different proteins and occurs in and amongdifferent cell linesThe results presented so far have strongly suggested that IPT can

involve different proteins within the same cell line. However, our

model suggests that IPT can occur between different cell types and

involve almost any surface protein, as long as transient membrane

fusion occurs and the proteins have sufficient lateral membrane

mobility. To explore the generality of IPT, we extended the co-

culture experiments from the exclusive use of CHO cells to other

cell types including human epidermal A431 carcinoma cells, which

naturally express high levels of EGFR (170 kDa), human

neuroblastoma cells BE(2)C/CHC(0.2) selected for high levels of

P-glycoprotein (170 kDa), human ovary carcinoma cells SKOV3

naturally expressing high levels of ErBb2, and other cells including

Fig. 3. Properties of membrane protein transfer. Donor cells (CHO-CD36+ and CHO-ICAM-1) are represented by dashed pink lines, and acceptor CHO cellsrepresented by thin black lines. (A) CHO-CD36+ cells co-cultured with CHO cells at the seeding ratio of 1:3.6 (thick green line) or 1.2:1 (thick blue line).(B) CHO-ICAM1 cells co-cultured with CHO cells at the ratio of 1:1 (thick blue line) or 1:2 (thick green line). (C) CHO-CD36+ cells and CHO cells co-cultured ina 75 cm2 flask at the ratio of 1:1 with initial cell numbers of 1�106 (thick green line) and 2�106 (thick blue line). (D) Transfer of membrane protein is transient. 5days after separation from CHO-CD36 cells (thick blue line) by FACS, CHO-CD36+ cells (dashed pink line) and CHO-CD36– cells (thin black line) were co-cultured again (thick green line) Black dotted line, untransfected CHO cells. (E) CHO-ICAM1 cells and CHO cells co-cultured in the presence of linoleic acid(25 μM) (thick blue line) and without linoleic acid (dotted pink line). (F) CHO-CD36 and CHO cells co-cultured without (thick red line) and in the presence ofmethyl-β-cyclodextrin (1 mM) (thick green line) for 2 days.

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human umbilical vein endothelial cells (HUVECs), rat

pheochromacytoma cells (PC12), and, more interestingly, goat

mesenchymal stem cells (GMSCs) and an embryoid-body-derived

(EBD) cell line (LVEC) (Shamblott et al., 2001). EBD cells are

uncommitted human precursor cells established from embryonic

germ cells and have been used as models of human stem cells in

vitro and in vivo (Frimberger et al., 2005; Kim et al., 2005; Mueller

et al., 2005). In this study, LVECs were used as an accepted surrogate

for human stem cells for co-culture with other somatic cells, in part

because they could be cultured in a monolayer without feeder cells.

As summarized in Fig. 4, all the co-culture experiments that were

tested clearly showed protein transfer, except the co-culture of A431

cells with NIH 3T3 cells, where no EGFR transfer was detected. In

addition, no transfer of EGFR was observed in the presence of linoleic

acid (supplementary material Fig. S4). All the antibodies we used

recognized only the extracellular domains of the transmembrane

proteins of interest. This indicated that the normal topology of

transferred proteins was preserved on the plasma membrane of

recipient cells. These results suggest that IPT is indeed a general

phenomenon, occurring within and between different cell types and

involving various proteins with different topology and molecular sizes.

One advantage of the generality of IPT is that it allows the

investigation of potentially important heterotypic cell-cell

interactions. To illustrate this, we analyzed CD36 transfer between

human platelets and endothelial cells. Platelets express high amounts

of CD36 and normally circulate in the bloodstream, coming in

frequent contact with endothelial cells (Lou et al., 1997; Philippeaux

et al., 1996; Philippeaux et al., 2000). Therefore, platelets might be

natural donors of CD36, transferring the protein to endothelial cells

lacking CD36 expression. We did indeed find that the CD36 levels

Journal of Cell Science 122 (5)

on iHUVECs following co-culture were considerably higher than

those in the control (supplementary material Fig. S5). We next tested

whether enhancement of a specific molecular interaction controlling

cell-cell adhesion could increase the IPT efficiency. We took

advantage of the expression of LFA-1 on the surface of platelets,

and the expression of ICAM-1 (the LFA-1 binding partner) on the

surface of iHUVECs. It is well established that the expression of

ICAM-1 can be further induced by pre-treatment of TNFα (Lou et

al., 1997). We found that pretreatment of iHUVECs with TNFαconsiderably increased CD36 transfer from platelets to endothelial

cells (supplementary material Fig. S5). This increase in IPT was

reversed by adding an anti-LFA-1 antibody capable of blocking the

interaction between LFA-1 and ICAM-1 (Bechard et al., 2001).

These results suggest that the enhancement of cell-cell adhesion

can increase the efficiency of IPT, potentially by facilitating the

close juxtaposition of the plasma membranes of the two interacting

cells, leading, in turn, to a higher probability of transient membrane

fusion.

Transfer of plasma membrane lipidsTransient cell-cell fusion is expected to result not only in IPT, but

also in intercellular transfer of membrane lipids. We therefore tested

whether this was the case, using a lipid fluorescent dye PKH67.

PKH67 is a green fluorochrome with long aliphatic carbon tails that

stably incorporate into the lipid region of plasma membrane, allowing

the dye to remain in the membrane for over 2 weeks or longer with

little leakage and cytotoxic effects (Horan and Slezak, 1989; Waters

et al., 2002). CHO-CD36+ cells were labeled with PKH67, and, after

extensive washing, co-cultured with unlabeled CHO cells. A

substantial transfer of PKH67 onto the unlabeled CHO cells was

detected (Fig. 5A), suggesting that membrane lipids could indeed be

transferred. The lipid transfer could be also directly visualized in the

fluorescent images of PKH67-labeled SKOV3 cells (Fig. 5B,C),

which showed high levels of PKH67 staining. As observed in the

case of protein transfer, lipid transfer is also dependent on cell-cell

contact. When PKH67-labeled CHO-CD36+ cells were separated

from unlabeled parental CHO cells using a 0.45-μm-pore filter, lipid

transfer was dramatically inhibited (Fig. 5A).

We further found that membrane lipids and membrane proteins

can be co-transferred from donor cells to recipient cells. PKH67-

labeled CHO-CD36+ cells were co-cultured with non-labeled CHO

cells followed by immunostaining with a PE-conjugated antibody

against CD36. Dual fluorescence plots for PE and PKH67 showed

that lipids were transferred onto the recipient CHO cells as the donor

CHO-CD36+ cells transferred CD36 onto the recipient CHO cells

(Fig. 5C).

Exhibiting bidirectionality of lipid transfer, protein donor cells

could also receive lipids from recipient cells as they transferred

proteins onto recipient cells. When A431 cells were used as

membrane protein donors, they acquired membrane lipids from the

PKH67-labled CHO-CD36+ cells while transferring EFGR onto

them (Fig. 5D). Again, when CHO-CD36+ cells were used as

membrane protein donors, they acquired lipids from PKH67-labled

A431 cells while transferring CD36 onto them (Fig. 5E). These

results suggest an exchange of plasma membrane lipids between

co-cultured cells as they exchange membrane proteins.

The lateral mobility of a membrane dye is expected to be much

higher than that of a membrane protein, suggesting that the

efficiency of the dye transfer (closely approximating membrane lipid

transfer) should be much higher than the efficiency of IPT. The

experiments reported in Fig. 5C-E allowed us to test this hypothesis.

Fig. 4. Generality of membrane protein transfer. Human cells expressing highlevels of certain membrane proteins [P-gp on BE(2)C/CHC(0.2) cells, ErbB2on SKOV3 cells and EGFR A431 cells] and stably transfected CHO cellsCHO-CD36+, CHO-ICAM1 and CHO-α4-GFP cells were co-cultured witheach other or with other recipient cells (PC12 cells, NIH 3T3 cells,untransfected CHO cells, HUVECs, iHUVECs, LVECs and GMSCs). Aftereach co-culture, cells were labeled with fluorescence-conjugated monoclonalantibodies for specific membrane proteins, followed by flow cytometrymeasurement. This diagram summarizes the results of the co-cultureexperiments in this study (see supplementary material Fig. S1 and Figs 1, 3and 5 for the corresponding data). A solid line represents transfer of a specificprotein in a co-culture experiment with its arrow pointing to the recipient cells,whereas a dashed line represents no transfer detected in a co-cultureexperiment. All primary antibodies were raised from mouse and onlyrecognized the extracellular domain of the transmembrane proteins.

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Indeed, we consistently found that the efficiency

of PKH67 transfer was approximately ten times

greater than that for CD36 between CHO cells,

seven times more than that observed for EGFR

between A431 and CHO cells and ten times

greater than the efficiency of CD36 transfer

between CHO and A431 cells.

Transfer of cytoplasmic componentsIn addition to IPT and membrane lipid transfer,

transient cell-cell fusion is expected to result in

transfer of diffusible cytoplasmic components,

including proteins and small metabolites. To test

this, we used cytosolic markers of different

molecular sizes, as well as a marker for

endosomes.

Calcein-AM (~1 kDa) is a membrane-

permeable molecule with low intrinsic

fluorescence. Within a cell it is hydrolyzed by an

endogenous esterase, yielding a highly negatively

charged and thus membrane-impermeable

metabolic product, calcein. Calcein can emit

fluorescence in the green spectrum and is retained

in the cytoplasm, making it a widely used

cytosolic marker. We found that calcein was

efficiently transferred from the calcein-AM-

labeled CHO-CD36+ cells to the unlabeled CHO

cells, with the transfer blocked if the labeled cells

were separated from unlabeled cells by a filter

with 0.45 μm pores during the co-culture (Fig.

6A). When the culture medium from the calcein-

labeled CHO-CD36+ cells was collected, filtered

(pore size of 0.2 μm) and used to culture the

unlabeled cells, no calcein transfer was observed

(Fig. 6B). These results demonstrated the

dependence of ICT of small cytosolic molecules

on direct cell-cell contact, which is similar to our

findings for membrane protein transfer and lipid

transfer.

To test whether larger cytoplasmic components

could be transferred, we used quantum dots, now

widely used for live cell labeling. These

fluorescent nanocrystals become stably localized

in endosomes upon cellular uptake (Jaiswal et al.,

2003). We found no observable transfer of

quantum dots in the co-culture of quantum-dot-labeled CHO-CD36+

cells with unlabeled CHO cells (Fig. 6C). This result suggested that,

over the course of the experiment, no detectable transfer of

endosomes occurs, which also rules out the possibility of extensive

complete cell-cell fusion.

The contrast between the prominent transfer of smaller calcein

and the relatively lesser transfer of quantum-dot-labeled organelles

implies that the cytoplasmic component transfer may be size

dependent. To further explore this dependence, we used Fluorescein-

conjugated dextrans with molecular masses ranging from 3 kDa to

2000 kDa. These polysaccharides with low cell toxicity enter cells

through phagocytotic and endocytotic pathways followed by gradual

dispersion into the cytosol. Supplementary material Fig. S2 shows

an example of cytosolic distribution of a dextran molecule. After

incubation with high concentrations of Fluorescein-conjugated

dextran (10 kDa) for 2 hours at 37°C followed by extensive washing,

about 30-40% of HUVECs showed a uniform distribution of

dextran in the cytosol.

CHO-CD36+ cells were labeled by dextrans of different molecular

sizes followed by co-culture with unlabeled CHO cells (Fig. 6C-

J). We found that smaller dextrans (3 kDa, 10 kDa, 40 kDa)

displayed highly efficient ICT (Fig. 6C,E,G), whereas the larger

ones (500 kDa, 2000 kDa) did not. The transfer of larger dextrans

was not enhanced even at higher ratios of labeled cells to unlabeled

cells (Fig. 6I,J), which otherwise promoted cytoplasmic component

transfer (Fig. 6L). Similarly to calcein transfer, dextran transfer was

dependent on cell-cell contact. Separation of labeled cells from

unlabeled cells with a 0.45 μm filter in the co-culture, or growing

unlabeled cells using 0.2-μm-filtered culture medium from labeled

cells showed little transfer. It is important to note that the transfer

of dextrans was not likely to occur through gap junctions, because

they have been estimated to selectively permit the intercellular

Fig. 5. Intercellular transfer of membrane lipids. (A) PKH67-labeled CHO-CD36+ cells (dashedpink line) were co-cultured with unlabeled CHO cells (solid black line) in a monolayer (thickgreen line) or separated by a filter with 0.45 μm pores (thick black line). (B) PKH67-labeledSKOV3 cells were co-cultured with unlabeled SKOV3 cells (white arrows). (C) PKH67-labeledCHO-CD36+ cells were co-cultured with CHO cells. (D) PKH67-labeled CHO-CD36+ cells wereco-cultured with A431 cells. (E) PKH67-labeled A431 cells were co-cultured with CHO-CD36+

cells. CD36 and EGFR levels in C-E were determined by flow cytometry.

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transport of cytoplasmic molecules with molecular mass not

exceeding 1 kDa (Kumar and Gilula, 1996).

In another experiment, after the cells attached on the growth

surface, the flasks of the co-cultured cells, the labeled cells and the

unlabeled cells were gently shaken together on an orbital shaker so

that the culture medium in each flask was always well mixed and

homogeneous. The co-culture under shaking conditions showed

similar transfer to that under normal static culture conditions (Fig.

6D). This ruled out the possibility that dextran might be released

individually or within micro-membrane vesicles shed into the cell

medium followed by integration into unlabeled cells.

We verified that CD36 and cytoplasmic dextran (10 kDa) could

be co-transferred from dextran-labeled CHO-CD36+ cells onto

recipient CHO cells (Fig. 6M). Conversely, after co-culture of

dextran-labeled CHO cells with protein donor CHO-CD36+ cells,

Journal of Cell Science 122 (5)

we found that the CHO-CD36+ cells received dextran from the

dextran-labeled CHO cells when they transferred CD36 onto the

CHO cells (Fig. 6N). This suggested a close relationship between

protein transfer and cytoplasmic component transfer.

Stem cells can receive large cytoplasmic macromoleculesIt was recently reported that embryonic-stem-cell-derived

microvesicles reprogrammed hematopoietic progenitors by transfer

of mRNA and proteins (Ratajczak et al., 2006). This raises the

possibility of enhanced transfer of large cytoplasmic macromolecules,

such as mRNA, between stem cells and surrounding somatic cells

compared with the relatively inefficient transfer between somatic cells.

We examined the properties of the transfer of cytoplasmic components

in the context of uncommitted human precursor cells LVECs and

HUVECs, the latter representing a potentially interesting human

Fig. 6. Analysis of intercellular transfer of cytoplasmic components by flow cytometry. (A-L) Unlabeled CHO cells (solid black line) and CHO-CD36+ cells(dashed pink line) labeled with calcein-AM, Fluorescein-conjugated dextrans of different molecular sizes and quantum dots, were co-cultured (thick green line),separated with a 0.45 μm filter (dotted blue line), and with higher ratios of labeled to unlabeled cells (thick blue lines). (M-N) Cytoplasmic macromolecules andmembrane proteins were co-transferred onto recipient cells (M) or mutually transferred between co-cultured cells (N).

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somatic cell type. Surprisingly, both 10 kDa and 2000 kDa dextrans

were significantly transferred (Fig. 7C,D) from dextran-labeled

HUVECs to LVECs after co-culture. A parallel experiment showed

a considerable transfer of 10 kDa dextran from labeled HUVECs to

NIH 3T3 cells but little transfer of 2000 kDa dextran (Fig. 7A,B),

which was consistent with the findings in CHO cells.

Stem cells show a propensity of fusion with somatic cells (Cowan

et al., 2005; Ogle et al., 2004; Ogle et al., 2005). To verify that

cytoplasmic transfer between HUVECs and LVECs was not

mediated by permanent cell fusion, we co-cultured LVECs with

iHUVECs. The GFP gene in iHUVECs is transcriptionally coupled

to the telomerase hTERT gene by an IRES element in the construct

(Freedman and Folkman, 2004). The GFP protein is localized both

in the nucleus and in the cytoplasm. The co-culture of iHUVECs

with LVECs showed no detectable transfer of GFP (Fig. 7E),

indicating that there was little or no whole cell fusion during the

co-culture for 1-3 days. The mechanism by which LVECs receive

large dextrans (2000 kDa), but not much smaller GFP (about 30

kDa), is currently not clear. There was no noticeable transfer of

GFP from iHUVECs to CHO-CD36+ cells either (Fig. 7F), even

though CD36 was readily transferred from CHO-CD36+ to

iHUVECs during the same co-culture (supplementary material Fig.

S1C). In a report of cytosolic and membrane transfer between PC12

cells through nanotubes, no transfer of cytosolic GFP was observed

(Rustom et al., 2004). Among the possible explanations of this

phenomenon is potential GFP multimerization or its association with

cytosolic components that are not readily transferred.

DiscussionIntegration of cell function in the context of a tissue has long been

the focus of analysis of various biological organisms. As a

mechanism, direct exchange of cellular components between

neighboring eukaryotic cells remains poorly characterized and

understood. Our analysis here suggests that this phenomenon is quite

general, capable of occurring in multiple cell types and involving

multiple proteins and other cellular components. Moreover, the

totality of our results is consistent with the hypothesis that ICT

involves transient fusion of the plasma membranes of two

neighboring cells as a result of cell-cell contact. Among the various

pieces of evidence for this mechanism presented in this study,

perhaps the stronger ones are the experimental data suggesting that

an increase in membrane fluidity and cell-cell adhesion can enhance

the transfer efficiency. Indeed, an increase of the membrane fluidity

either through cholesterol depletion or addition of an omega-6 fatty

acid increased the efficiency of ICT. The efficiency of ICT from

LFA-1-expressing platelets was also increased following

upregulation of the LFA-1 adhesion partner, ICAM, on acceptor

cells.

Our results also argue that transfer by membrane microparticles,

although possible, might not be the chief mechanism of ICT. We

could not detect ICT following incubation of cells with filtered

medium conditioned by donor cells. Moreover, if membrane

microparticles shed by donor cells are indeed a major route of ICT,

one would expect that the transfer of proteins and lipids and

cytosolic markers would occur in proportion to their relative

content in the donor cells. However, we found that the efficiency

of the transfer of the lipid dyes and cytosolic dextrans was

considerably higher than that of the proteins in the same experiment.

The transient cell fusion hypothesis does not exclude the

possibility of ICT occurring through nanotubules. However, direct

observations of cells through time-lapse microscopy suggest that

the usual depictions of protein transfer through a few long

nanotubules connecting a few neighboring cells might be a transient

point in a more dynamic cell interaction process. In particular, we

generally observed that cells actively moving within relatively dense

layers, both in cell culture and tissues, can have extensive

opportunities for the formation of tight transient contacts and

possibly membrane fusions. As cells move away, they can

transiently form multiple short tethers transiently connecting them,

which ultimately either merge into a few residual long nanotubules

or break off. The residual nanotubules are also very transient, rapidly

disintegrating if cells continue separation (supplementary material

Movies 1 and 2).

During membrane adhesion, small portions of the plasma

membranes of two contacting cells might be brought into sufficiently

close contact, making the polar and hydrophilic head groups of

phospholipids of the outer membrane layer face each other. This

energetically unfavorable state might lead to transient fusion of the

outer layers of the membranes, which could then disintegrate into

separated membranes or evolve into a full local membrane fusion,

forming a small pore, which are both lower energy states. The

intermediate state during which only the outer membranes layers

fused has been demonstrated in hemagglutinin (HA)-induced

membrane fusion (Melikyan et al., 1997; Melikyan et al., 1995), and

Fig. 7. Stem cells can accept large cytoplasmic macromoleculesfrom somatic cells. (A-D) Primary HUVECs labeled with 10 kDaand 2000 kDa Fluorescein-conjugated dextrans (dashed line) wereco-cultured with NIH 3T3 cells or uncommitted LVECs (thin blacklines), and co-cultures (gray lines) studied by flow cytometry.(E,F) iHUVECs transfected stably with GFP were co-cultured withLVECs (E) and CHO-CD36+ cells (F).

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608 Journal of Cell Science 122 (5)

has been proposed in spontaneous whole cell fusion between stem

cells and somatic cells. Low efficiency of transfer of large

cytoplasmic macromolecules or big organelles between somatic cells

indicates that the fusion occurs within very small areas and there is

very limited whole cell fusion. Rather, there might be several such

small fusion events at the interface of two juxtaposed membranes,

because a few small fusions would not enable considerable transfer

of membrane and cytoplasmic components. These sieve-like multi-

fusions have been revealed during HA-induced membrane fusion

between erythrocyte and fibroblasts (Melikyan et al., 1997; Sarkar

et al., 1989) and in the co-culture of neutrophils with parasites

Schistosoma mansoni (Caulfield et al., 1980). Through these small

continuities of the membrane and the cytosol, cellular components

can be transferred passively by diffusion.

Recent studies of cell fusion have focused on fusogenic proteins

that irreversibly bring two membranes close enough to induce a

stable membrane fusion, which might be followed by complete cell

fusion (Chen and Olson, 2005; Oren-Suissa and Podbilewicz, 2007).

The apparent lack of cell-cell fusogens that resemble class I viral

fusion proteins and SNARE proteins in some observations of cell

fusions indicates the possible involvement of other mechanisms (Tse

et al., 1993). Consistent with this prediction, our results suggest a

complementary, but not exclusive mechanism: transient membrane

fusion initiated by reversible membrane juxtaposition followed by

local membrane destabilization. This transient membrane

juxtaposition might be induced by weaker but ubiquitous adhesion

molecules on many cells types, such as the LFA-1–ICAM interaction

(supplementary material Fig. S5), or by mechanical impinging of

neighboring cells during locomotion and local deformation, and

might not be maintained long enough to lead to complete cell-cell

fusion, in contrast to some of the known fusogenic proteins.

The sizes of individual fusions might depend on the cell type. The

transfer of cytoplasmic macromolecules as large as 2000 kDa

between some stem cells and somatic cells might allow the stem cells

to exchange many types of cellular macromolecules with somatic

cells, including mRNAs and proteins. This could further induce

subsequent changes in neighboring cells. As the duration of cell-cell

co-incubation increases, small fusions might occasionally combine

into larger ones, potentially resulting in a complete whole-cell fusion

for certain cell types. This might explain the observations that stem

cells can fuse with somatic cells after more than 10 days of co-culture

in vitro (Cowan et al., 2005) or in vivo (Ogle et al., 2004; Ogle et

al., 2005). Our data suggest that stem cells might start exchanging

large cytosolic molecules or membrane proteins with somatic cells

much earlier. Because complete whole-cell fusion is much less

frequent, as suggested in Figs 6 and 7, the earlier and more frequent

instances of ICT through transient and local membrane fusions might

be an important factor for stem cell differentiation induced by

surrounding somatic cells, as well as for somatic cell reprogramming

by adjacent, not fully differentiated cells.

It was observed that human embryonic stem cells incorporate

nonhuman sialic acids from the mouse feeder layer cells (Martin

et al., 2005). In this study we demonstrated that both LVECs and

GMSCs could receive transmembrane proteins from neighboring

differentiated cells during co-culture (Fig. 4). Of particular

significance is the transfer of EGFR from A431 cells to LVECs, as

well as its increasing efficiency as the ratio of A431 cells to LVECs

increased (supplementary material Fig. S1T). Stem cells in adults

are usually surrounded by a larger number of fully differentiated

somatic cells. These naturally occurring high cell ratios might

remarkably augment ICT between stem cells and somatic cells. In

addition, because of the powerful amplification along the pathways

of signal transduction, even a temporary acquisition of growth factor

receptors could initiate sufficient downstream signaling events for

stem cells to make irreversible decisions.

Not all cellular components might be available for transfer. It

was reported that activation of EGFR could increase its association

with the cytoskeleton. This would impair the diffusive movement

of EGFR on the membrane. A possible reason for little EGFR

transfer from A431 cells to NIH 3T3 cells could be that some

signaling molecules secreted by NIH 3T3 cells could partially

activate EGFR, which would inhibit the lateral membrane mobility

of EGFR and thus ICT of EGFR.

In addition to cellular locomotion and local deformation, ICT

might occur under many other conditions in vivo and in vitro. ICT

of surface proteins, such as antigens and receptors, was also

observed at immune synapses (Carlin et al., 2001; Hudrisiers and

Bongrand, 2002; Poupot and Fournie, 2003) and has been suggested

as a commonplace and important phenomenon in the functioning

of the immune system (Davis, 2007). Although some of the ICT

of surface proteins at immune synapses are mediated by direct

ligand-receptor recognition followed by internalization, spontaneous

ICT of other membrane components and cytoplasmic components

might also exist where immune cells undergo transient cell-cell

contact.

Although mammalian cells have been thought to be largely

discrete functional units, increasing evidence suggests that they

frequently undergo transient exchange of cellular components with

their neighbors. Our results point to ICT as a potentially powerful

general mechanism of cell-cell communication and provide insight

into the underlying mechanisms. For instance, ICT might quickly

modify the behavior of large groups of neighboring cells, not

requiring all of them to change their genetic makeup. This could

blur the boundaries between distinct cell types within complex

tissues and thus the associated beneficial or potentially harmful

phenotypes. Further analysis of the mechanisms might enable

control or utilization of this phenomenon.

Materials and MethodsCell culture and co-cultureCHO-CD36 cells, CHO-ICAM1 cells, SKOV3 cells and A431 cells were obtainedfrom the ATCC (Manassas, VA) and cultured as recommended. Two subpopulationsof CHO-CD36 cells: CHO-CD36+ and CHO-CD36– cells were separated by clonalselection or two rounds of fluorescence-activated cell sorting (FACS) and maintainedin the same medium with CHO-CD36 cells. SKOV3 cells were selected from originalSKOV3 cells from ATCC for high ErbB2 expression by FACS except for resultsshown in supplementary material Fig. S1, where original SKOV3 cells were used.Untransfected CHO cells were maintained in DMEM with F12 and 10% FBS and100 U/ml penicillin-streptomycin. CHO-α4-GFP cells were maintained in the samemedium with 0.8 mg/ml G418. BE(2)C/CHC(0.2) cells were cultured as described(Levchenko et al., 2005). PC12 cells were maintained in DMEM, supplemented with10% heated-inactivated FBS, 5% horse serum and 50 U/ml penicillin-streptomycin.HUVECs and their medium EGM bullet kit were purchased from Clonetics (SanDiego, CA). iHUVECs were a gift from Deborah Freedman (Harvard University,Boston, MA) and were maintained as described (Freedman and Folkman, 2004). NIH3T3 cells were maintained in low glucose (1 mg/ml) DMEM with 10% calf serumand 100 U/ml penicillin-streptomycin. LVECs were cultured as described (Shamblottet al., 2001). GMSCs were a gift from Jennifer Elisseeff (Johns Hopkins University,Baltimore, MD) and used at passage 4. GMSCs were cultured in DMEM with 10%FBS, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine.

Platelets prepared by two methods from peripheral blood of healthy donors wereused and no noticeable difference between the two isolation methods was found. Inthe first method, fresh platelets were prepared by density gradient centrifugation andused on the same day as the blood was drawn. Heparin-anticoagulated peripheral bloodfrom healthy donors was centrifuged through Ficoll-Hypague gradients (30 ml of bloodon 15 ml of Ficoll at 300 g for 25 minutes). The buffy layer with platelets was collectedand washed in PBS with 2% FBS. After centrifuging at 300 g for 15 minutes,supernatant was collected as a fresh platelet suspension. In the second method, Interstate

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Blood Bank (IBB, Memphis, TN) platelet-rich plasma (PRP) was prepared bycentrifugation of fresh ACD-anticoagulated blood at 800 g for 4 minutes and thencollection of plasma. After receipt from IBB on the following day, PRP were centrifugedat 1000 g for 8 minutes and resuspended in PBS with 0.025 M EDTA. Platelets werefurther purified by two rounds of centrifugation (700 g for 2 minutes), collection ofsupernatant, and collection of IBB platelet suspension. For both fresh platelet and IBBplatelet isolation methods, the platelet suspensions were centrifuged (1000 g for 8minutes), resuspended in culture medium and counted for co-culture with other cells.

For co-culture, two types of cells were seeded together and cultured for about 2days until they were close to a confluent monolayer. The initial numbers of the twocell types were adjusted according to their growth rates so that after co-culture theirnumbers would be comparable. The same medium was used for each flask in a co-culture experiment, usually the medium for recipient cells, or combination of bothmedia for recipient and donor cells to ensure both cell types grew well.

Chemicals and antibodiesLinoleic acid (L5900) was purchased from Sigma (Carlsbad, CA) and was added tothe co-culture medium at a concentration of 25 μM at the time of cell seeding, andmedium was changed each day. Methyl-β-cyclodextrin (4555) purchased from Sigma,was added at a concentration of 1 mM at the time of cell seeding, and medium waschanged each day. Recombinant human TNFα and IL-1βwas from PeproTech (RockyHill, NJ). Fluorescence-conjugated antibody solutions for human CD36 (clone NL07),ICAM-1 (clone HA58), and EGFR (clone EGFR.1), and unconjugated antibodysolutions for human CD36 (clone NL07), and EGFR (clone EGFR1) were purchasedfrom BD Biosciences (San Jose, CA) for flow cytometry and fluorescent imaging.Other monoclonal antibodies for P-glycoprotein (clone MRK 16, Kamiya Biomedical,Seattle, WA), ErbB2 (clone 2G11, Chemicon, Temecula, CA) and a PE-conjugatedgoat anti-mouse IgG2a antibody (SouthernBiotech, Birmingham, AL) were also used.FITC-conjugated goat anti-mouse secondary antibody was purchased from Invitrogen(Carlsbad, CA).

Flow cytometry and fluorescence-activated cell sortingCells were incubated in the dark with monoclonal antibodies at concentrationsrecommended by their manufacturers at 4°C for 30 minutes, during which time cellswere gently vortexed every 10 minutes. In the case of unconjugated antibodies, cellswere washed twice and incubated in fluorescent-conjugated secondary antibodies for20 minutes. After washing three times, cells were resuspended in cold PBS for flowcytometry measurement. Cells labeled with membrane dye or cytoplasmic markerswere resuspended in cold PBS or basal culture medium, followed by immediateexamination. Cells were filtered through 40 μm cell strainers (BD Bioscience, SanJose, CA) before fluorescence measurement with a FACSCalibur system or sortingby a FACSVantage SE system (BD Bioscience, San Jose, CA). At least 10,000 cellsin each sample for histograms or 2500 cells for dot plots were analyzed. All curvesin flow cytometry histograms were smoothed by Cellquest software.

Cell imagingCells were cultured in customized glass-bottom dishes (MatTek, Ashland, MA) withFisher growth glass (Fisher Scientific, Hampton, NH), washed with warm PBS, fixedwith 2% paraformaldehyde in PBS for 15 minutes at room temperature, washed twicefollowed by blocking with 1% BSA in PBS at room temperature for 1 hour. Afterremoval of blocking solution, 200 μl labeling solution was added in the 15 mm wellin a glass-bottom microwell dish and incubated for 45 minutes at room temperature.Labeling solution was made by adding 50 μl PE-conjugated anti-human CD36antibody to 150 μl PBS. After incubation, cells were washed three times in PBS, andwere kept in 0.5% paraformaldehyde before taking images using a Nikon EclipseTE2000-U microscope with a Spot RT monochrome camera (Diagnostic Instruments,Sterling Heights, MI) at room temperature. The objective was �40 or �10 and thecamera software was Spot v3.5. Fluorescence per unit cell area in the original imageswas measured using MetaMorph version 6 (Universal Imaging, Downingtown, PA).For live-cell imaging, cells were cultured and imaged under the same conditions exceptthe control of temperature and humidity as well as supply of 5% CO2. Videos weregenerated by Spot software from sequential images.

Live-cell labelingQtracker 585 cell-labeling kit (Invitrogen) and membrane dye PKH67 (Sigma) wereused following the manufacturers’ step-by-step protocols. Low PKH67 concentrationswere used in dual fluorescence experiments for better fluorescence compensation.Lysine-fixable dextrans conjugated with Fluorescein was dissolved in culture mediumwithout serum at a concentration of 5-10 mg/ml. Cells were harvested and resuspendedin dextran solution at a concentration of about 107 cells/ml, incubated at 37°C for 2hours in a cell culture incubator, washed at least three times before co-culture withother cells. During the incubation, cells were mixed every 20 minutes to avoid cell-cell adhesion. Calcein-AM (Invitrogen) stock solution (4 mM in DMSO) was dilutedin basal culture medium without serum. A final concentration of Calcein-AM of 1-5μM was used. The final concentration of DMSO in labeling solution was about 0.1%or lower and no innocuous effects on the cell types used in this study were observed.Cells were resuspended in this labeling solution for 15-20 minutes at room temperatureand washed three times in complete medium before co-culture with other cells.

Mathematical analysis of intracellular protein transferIn setting up the model we made the following assumptions. First, we assumed thatcells can be represented as essentially two-dimensional objects. Second, we assumedthat IPT can only take place during formation of a transient cell-cell contact, whichcan be seen in a simplified fashion as a ‘collision’ between two moving cells. Third,we assumed that prior to such collisions, two interacting cells move in relativelystraight trajectories with the same speed V, the characteristic speed of migration fora given cell type. These assumptions make it possible to follow the model developmentusually used in molecular interaction theory, deriving the rate of chemical reactions.Instead of molecular concentrations, one can use here cell densities; the molecularsizes would correspond to the cell diameter. We can therefore assume that within afinite time period Δt a donor cell sweeps the total area equal to LcVΔt, with theprobability of encountering an acceptor cell equal to this area multiplied by the densityof the acceptor cells: LcVΔtcA. Since this logic can be extended to all the donor cellsin the limited experimental space, the total number of donor-acceptor cell interactionswill be also proportional to the density of the donor cells, cD: LcVΔtcAcD. The amountof protein transferred in each such cell interaction will depend on the efficiency oftransient cell fusion µ, the difference in the protein expression levels in donor versusacceptor cells (φD-φA), and the duration of the cell-cell interaction, which is, in turn,inversely proportional to the cell speed V. The amount of the transferred protein overthe same finite amount of time Δt is then proportional to the number of donor-acceptorcell interactions multiplied by the amount of the transferred protein per cellinteraction:ΔT=βLcVΔtcAcDµ(φD-φA)(1/V), where β is a proportionality coefficient.Taking the limit of Δt r �, which justifies the assumption of approximately straightcell movement at a constant speed, we arrive at the following expression ofacquisition of the transferred protein by acceptor cells:

Note that the cell speed is no longer present in the equation, having been canceled.Since we also assume that the transferred protein is not produced within acceptorcells, but can be lost either through degradation or dilution in the process of celldivision, equation 3 needs to be expanded to describe the loss of the transferred proteinas follows:

At the steady state, equation 4 is equal to zero, yielding the final formula:

We note that this formula leads to reasonable predictions for several extreme cases.For instance, if cD = 0, then φA = 0; if cD is extremely large, then φA < φD; if γ = 0,then φA < φD and if γ is very large, φA < 0. We also note that although for simplicitywe have postulated the parameter to describe the efficiency or extent of the transientcell fusion, this parameter will also depend on the lateral mobility of the protein withthe membrane, because, if the lateral mobility was zero, no IPT would take place.Finally, we note that the implicit assumption made here is that the level of the proteinof interest in the donor cells does not appreciably change because of the transferprocess, a simplifying assumption that is nevertheless supported by data from all theexperiments presented.

We thank Jennifer Elisseeff, David Sullivan, Monique Stins, LeslieLoew, Paul Roepe, Charles Flexner, Laurence Schramm and membersof the Levchenko laboratory for helpful discussions and sharing ofmaterials. The research was supported in part by the Johns HopkinsMalaria Research Institute Pilot Grant.

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