the semi-intact cell system and methods for cell resealing...

Advances in Systems Biology VOL.2 NO.1 MARCH 2013 6

Abstract—Omics and bioinformatics approaches provide much

information regarding key factors that cause diseases and their

interactions. Despite such accumulated data, spatial information,

-the localization of proteins and the structure of intracellular

organelles and cytoskeletons- under disease condition remains

insufficiently developed. Cell-based assays provide one solution to

evaluating spatial information, and are increasing in importance

relative to traditional drug screens and investigation of the

mechanisms of drug action. Most current cell-based assays make

use of so-called ‘‘normal’’ cells, however, which do not reflect

intracellular disease conditions. In this review, we introduce the

powerful cell-based assay system of “disease model cells”, which

enables the investigation of perturbed protein networks under

pathogenic conditions while retaining the morphology of

intracellular structures by using semi-intact cells and its resealing

cell technique.

Index Terms—Disease model cells, reconstitution, spatial

information, semi-intact cells, resealing cell technique

I. INTRODUCTION

ECENT advances in analysis of omics data derived from

microarray studies, proteomics, epigenomics, etc. have

allowed us to obtain much information about factors that

are involved in a variety of biological processes in living cells

[1, 2]. Based on the accumulated information, ‘Systems

Biology’ is now making a sizable contribution to the

elucidation of molecular networks among these factors. One

method to validate the relationships between factors in a

network makes use of in vitro reconstitution assays and many

such methods have been developed. For example, functional

networks involved in transcription [3, 4], translation [5, 6],

molecular dynamics and morphological changes in the

This work was supported in part by a grand from Kanagawa Academy of

Science and Technology, and from Technology Development Program for

Advanced Measurement and Analysis, JST.

Fumi Kano is with Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo

153-8902, Japan and with PRESTO, Japan Science and Technology Agency,

4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan (e-mail: fkano@ bio.c.u-tokyo.ac.jp).

Masayuki Murata is with Department of Life Sciences, Graduate School of

Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan (e-mail: [email protected]).

membranes of cellular organelles [7-9] have been reconstituted

using relevant sets of elementary biomolecules that had been

deduced theoretically. More recently, constructive (or synthetic)

approaches using reconstitution assays have gained much

attention as a new analytical concept to elucidate a link between

these factors [10, 11].

In general, in vitro reconstitution assays require biochemical

purification of isolated organelles or cytoskeletal components

from disrupted cells or tissues. This inevitably damages the

integrity/topology of organelles and cytoskeletal components,

and even worse, can disrupt the relative spatio-temporal

relationship between organelles and the cytoskeleton. The

integrity of organelles and the cytoskeleton and their proper

relative spatial localization are important for normal protein

function in cells. As such, the inclusion of spatio-temporal

information in reconstitution assays should allow more precise

determination of molecular networks using systems biology.

The use of semi-intact cell systems coupled with techniques for

resealing of the cells could provide a promising approach to

address this problem.

Semi-intact cells are generated by permeabilization of the

plasma membrane by detergent or bacterial toxins [12] (Fig. 1).

We use streptolysin O (SLO), a streptococcal

cholesterol-binding toxin [13], to permeabilize cells. SLO

binds to cholesterol in the plasma membrane at 4˚C, and forms

a ring-shaped multimer that creates 30-nm diameter pores in the

plasma membrane [14, 15]. These pores allow both depletion of

the cytosol of the permeabilized cells, and delivery of various

molecules, such as proteins, nucleotides, and small chemical

compounds into the cells. One can even replace the cytosol of a

cell in a particular phase of the cell cycle or in a particular state

of differentiation or disease condition with that from a cell with

a different phenotype. In addition, since the integrity of

organelles and the cytoskeleton remain largely intact in

semi-intact cells, semi-intact cells can be considered “cell-type

test tubes” that enable us to examine molecular function at the

level of intracellular substructures. A unique advantage of this

system is that we can obtain information about spatio-temporal

changes in protein localization and the morphology of

intracellular structures under different conditions imparted by

the specific intracellular environment. In particular, the

maintenance of the integrity of the organelles and their

configuration in semi-intact cells makes this system suitable for

analyzing membrane trafficking between organelles in as

The Semi-Intact Cell System and Methods for

Cell Resealing: a Novel Systems Biology Tool to

Elucidate Protein Networks with

Spatio-Temporal Information

Fumi Kano and Masayuki Murata

R


intrinsic an environment as possible. To date, this system has

been used to reconstitute a variety of vesicular transport

pathways [16-21], and to investigate organelle dynamics [7],

[22-25], etc.

We have been working on a mammalian, semi-intact cell

system to reconstitute cell cycle-dependent changes in

organelle morphology and membrane trafficking between

organelles [21-25]. We recently observed that SLO-induced

pore formation can be reversed by calcium ions under the

appropriate conditions, and that such “resealed cells” restored

various intracellular functions that were difficult to reconstitute

in the original semi-intact cell system due to the

permeablization of the plasma membranes. In this review, we

describe reconstitution assays for the study of membrane

trafficking and signal transduction in cultured cells under

various conditions using semi-intact and “resealed” cells, and

show the usefulness of these systems for validating and

elucidating protein networks in living cells.

II. SEMI-INTACT CELL SYSTEM

Cell cycle-dependent changes in the morphology of both the

Golgi and ER occur transiently and simultaneously in a

concerted fashion in mammalian cells during mitosis, rendering

these processes difficult to dissect both morphologically and

biochemically. In addition, the analysis is complicated further

by the asynchronous progression of the cell cycle in individual

cells of a population.

Since the cytoplasmic environment of most semi-intact cells

can be synchronized by addition of interphase or mitotic

cytosol, analysis of cell cycle-dependent events can be

monitored at a high signal-to-noise ratio. In addition, since the

intracellular location and relative position of the organelles is

largely conserved in semi-intact cells, we can manipulate

intracellular conditions and then observe the resulting

morphological changes in GFP-tagged, pre-existing organelles

using fluorescence microcopy. Consequently, we can dissect

complex morphological processes in cells and investigate the

biochemical requirements and kinetics of the latter.

A. Reconstitution of Cell Cycle-dependent Changes in the

Morphology of the Golgi and Endoplasmic Reticulum (ER) in

Semi-intact Cells

The Golgi apparatus forms a ribbon-like structure near the

nucleus during interphase, but is dispersed throughout the

cytoplasm prior to mitosis so as to ensure equal partitioning to

the two daughter cells [26-28]. We reconstituted the Golgi

disassembly process induced by mitotic cytosol in semi-intact

cells [22]. We first established a cell line (MDCK-GT) that

expresses constitutively a fluorescent Golgi marker protein,

GT-GFP. MDCK-GT cells were permeabilized with SLO, and

incubated with Xenopus mitotic extracts. As shown in Fig. 2,

the GT-GFP-containing Golgi apparatus was disrupted into

large vesicles followed by further dispersion into the cytoplasm

in response to exposure to mitotic extract. The Golgi

disassembly was thus dissected into two elementary processes:

early disruption of the intact Golgi into large vesicles, followed

by further dispersion throughout the cytoplasm. We next

identified the kinase responsible for each of the two steps of the

process. Use of kinase inhibitors and immunodepletion

experiments revealed that MEK and cdc2 kinase were primarily

responsible for the first and subsequent steps of the dispersion

process, respectively (Fig. 2). It was striking that MEK was

required for Golgi disassembly prior to the action of the mitotic

master kinase Cdc2. In vivo experiments by other researchers

provide support for this processive model of Golgi disruption

and provide evidence that MEK induces the unlinking of the

Golgi stacks. The Golgi comprises several stacks, which are

connected by Golgi matrix proteins such as GRASP65 and 55

[29]. Feinstein and Linstedt demonstrated that MEK

phosphorylates GRASP55, thereby inhibiting binding between

adjacent stacks and leading to their unlinking [30, 31].

+SLO

Leakage of

cytosol

Semi-intact

cells

Preparation of cytosol

from organs of

disease model mouse

Exchange

of cytosol

Ca2+

cytosol

Proliferation of

resealed cells

Resealed

cells

disease

model mouse

Intact cells

Figure 1Fig. 1. A schematic of semi-intact and resealed cells.

Fig. 2. Reconstitution of Golgi disassembly in semi-intact MDCK-GT cells:

MEK and cdc2 kinase are required sequentially in Golgi disassembly.

Incubating semi-intact MDCK-GT cells with Xenopus egg mitotic extracts induced the disassembly of the Golgi apparatus. The intact Golgi apparatus

(stage I) was first disrupted to form large vesicles (stage II) in a

MEK-dependent manner, and was then further disassembled into cytoplasm

(stage III) in a cdc2 kinase-dependent manner.


Furthermore, both Golgi disassembly and mitotic entry were

delayed in cells expressing a mutant GRASP55 containing a

substitution of alanine at its mitotic MEK1 phosphorylation

sites [30]. It is interesting that changes in the morphology of the

Golgi are deeply connected to other physiological processes.

Next, to investigate cell cycle-dependent changes in the

morphology of the ER network in mammalian cells, we created

a clonal cell line derived from Chinese Hamster Ovary (CHO)

cells that constitutively expressed GFP-HSP47 (CHO-HSP).

Using confocal microscopy, we found that, during interphase,

GFP-HSP47 is associated with polygonal structures with

three-way junctions that are located at the periphery of

CHO-HSP cells and in the cisternae in the perinuclear region.

Interestingly, at the onset of mitosis, the ER appears to retain its

network structure rather than being disrupted into vesicles, in

contrast to the Golgi apparatus, that is consistent with the

reports by others [32, 33]. Further observation by fluorescence

microscopy revealed that the ER is partially severed at the onset

of mitosis.

Using the reconstitution system in semi-intact cells, we

reconstituted mitotic disruption of the ER [23]. We first

preincubated CHO-HSP cells with nocodazole to disrupt

microtubules and then permeabilized the cells with SLO. Then,

we incubated the semi-intact CHO-HSP cells with mitotic

cytosol prepared from synchronized mitotic L5178Y cells, and

found that the continuous network of the ER was partially

severed, as was seen in intact mitotic CHO-HSP cells, in a cdc2

kinase-dependent manner (Fig. 3). Fluorescence microscopy

also revealed that partial disruption of the ER network by

mitotic cytosol results from inhibition of the fusion process of

ER tubules, rather than inhibition of tubulation/bifurcation

(unpublished data). With regard to the fusion process, some

cytosolic proteins or their regulators that are downstream of

cdc2 kinase are thought to be inactive in mitotic cytosol [22,

34]. Extrapolating from these findings, the disruption of the ER

network by mitotic cytosol in vitro could also result from the

blocking of fusion events by cdc2 kinase-mediated

phosphorylation. One of the candidates for this inhibition is p47,

a cofactor of p97, which mediates the fusion of Golgi

membranes [35]. Uchiyama et al. [36] found that Ser140 of p47

was selectively phosphorylated by cdc2 kinase and that this

phosphorylation was involved in Golgi disassembly during

mitosis. Therefore, we investigated the effect of a

non-phosphorylatable form of p47, p47 (S140A), which we

refer to as p47NP, which we show inhibits mitotic Golgi

disassembly. Phosphorylation of p47 by cdc2 kinase

dissociates the p97/p47 fusion complex from membranes,

which might inhibit membrane fusion between ER tubules. To

test this, we investigated the effect of p47NP on partial

disruption of the ER network that is induced by mitotic cytosol

in semi-intact cells. In the presence of p97/p47NP, mitotic

cytosol failed to induce partial disruption of the ER network.

Therefore, we conclude that disruption of the ER during mitosis

requires phosphorylation of p47 by cdc2.

Interestingly, using other semi-intact cell assays we also

found that the cdc2-dependent phosphorylation of p47 in

mitotic cytosol induces disassembly of ER exit sites (ERES),

which are specialized membrane domains within the ER from

which bud vesicles that contain cargo proteins destined for the

Golgi [24]. Conversely, the mitotic disassembly of ERES in

semi-intact cells was completely blocked in the presence of

mitotic cytosol containing p47NP and p97. Collectively,

mitotic disassembly of the Golgi, the disassembly of ERES and

partial disruption of ER networks appear to be controlled, at

least partly, by cdc2-dependent phosphorylation of p47.

B. Reconstitution of Anterograde or Retrograde Vesicular

Transport between the Golgi and the ER in Semi-intact cells

The Golgi apparatus and the ER are connected through

dynamic membranous flow by membrane trafficking

(hereinafter, referred to as vesicular transport), while the

organelles sustain their unique structures during interphase.

Disturbance of vesicular transport during mitosis would affect

both the level of protein that flows in and out of the organelles

and overall organelle morphology [37, 38]. So, quantitative

analysis of cell cycle-dependent vesicular transport between the

Golgi and the ER should provide insight into the regulatory

mechanisms governing the morphology of mitotic organelles.

However, perturbation of the biochemical composition of the

Golgi and ER during mitosis complicates analysis of the

efficiency of vesicular transport between these organelles using

quantitative microscopic methods, such as fluorescence

recovery after photobleaching (FRAP).

To solve this issue, we reconstituted cell cycle-dependent

vesicular transport between the Golgi and the ER in semi-intact

cells in the presence of interphase and mitotic cytosol [21]. We

first established CHO-GT cells, which stably express GT-GFP.

Although GT-GFP traffics between the ER and Golgi by

vesicular transport, most of the protein localizes to the Golgi

under steady-state conditions. We then used FRAP to bleach

the fluorescence of GT-GFP in the Golgi region by repetitive

laser illumination. After bleaching, fluorescence in the Golgi

recovers owing to anterograde transport of GT-GFP from the

Fig. 3. Partial severing of the endoplasmic reticulum induced by mitotic

cytosol in semi-intact CHO-HSP cells. Semi-intact CHO-HSP cells, in which HSP47-GFP was constitutively expressed to visualize the

endoplasmic reticulum (ER), were incubated with interphase (I cytosol) or

mitotic cytosol (M cytosol). Incubation with M cytosol caused partial severance of the ER network (Lower left figure). Right panel shows a

schematic illustration of ER morphology in semi-intact CHO-HSP cells

incubated with I or M cytosol. Morphological changes of the ER were evaluated by counting the number of three-way junctions of the ER

network in a defined area.


ER to the Golgi. By measuring the fluorescence recovery in the

Golgi area, we can estimate the extent of transport of GT-GFP

from the ER to the Golgi. To examine retrograde transport from

the Golgi to the ER, fluorescence in the ER region was

bleached and the fluorescence recovery of the ER (whole area

of the cell except for the nucleus) was determined. Figure 4

shows representative kinetic curves for the anterograde and

retrograde transport of GT-GFP that were obtained from this

assay. In the presence of mitotic cytosol, anterograde transport

was selectively inhibited, whereas retrograde transport

remained intact. In addition, we found that cdc2-depleted

mitotic cytosol induced anterograde transport normally, which

indicates that mitotic inhibition of anterograde transport also

requires cdc2 kinase. This result is consistent with the finding

that in the semi-intact cell assay, the ERES was disrupted by

mitotic cytosol in a cdc2 kinase-dependent manner.

Remarkably, the retrograde transport assay using semi-intact

cells revealed that mitotic cytosol can induce various aspects of

vesicular transport, including vesicle budding, transport, and

fusion, and results in the translocation of Golgi components to

the ER.

C. Schematic model for the coupling of the disruption of the

mitotic ER network and Golgi disassembly to vesicular

transport

On the basis of our results and those of others, we have

developed a model for the relationship between cell

cycle-dependent morphological changes in the ER and Golgi

and the regulation of vesicular transport between them in

mammalian cells (Fig. 5). During the earlier steps of mitosis,

activation of cdc2 kinase induces the phosphorylation of p47

and other proteins. The phosphorylated p47 causes disassembly

or vesiculation of the Golgi, and simultaneously causes partial

severance of the ER network and disassembly of ERES. The

disassembly of ERES inhibits anterograde transport from the

ER to the Golgi, whereas retrograde transport remains intact.

As a result, some components of the Golgi translocate to the ER

network during the early phase of mitosis. The severed ER

network and vesiculated Golgi membranes are easily

distributed into two daughter cells in a stochastic fashion.

In mitotic cells, a single event, e.g. phosphorylation of p47

by cdc2 kinase, seems to induce orchestrated and simultaneous

changes in the morphology of the Golgi, ERES and ER. Thus it

is likely that the precise roles of certain proteins in particular

events in mitotic cells might be difficult to discern. Our

semi-intact cell assay is suitable for investigating the

biochemical requirements of specific processes. For example,

the ER disassembly assay revealed that a p97/p47-mediated

fusion process plays a crucial role in the maintenance of the ER

network when microtubules are disrupted by nocodazole.

When microtubules are intact, the contribution of the fusion

process to the maintenance of the ER network appears to be

masked. In another case, our transport assay also revealed that,

even in the presence of mitotic cytosol, the retrograde transport

of GT-GFP occurs normally when microtubules remain intact,

but ERES are disassembled easily under these conditions.

These findings suggest that mitotic cytosol can facilitate

retrograde transport as long as microtubule integrity is

maintained, but that anterograde transport ceases rapidly in the

presence of mitotic cytosol. Thus, the ability to manipulate the

cytoskeleton easily in semi-intact cell systems will be useful in

elucidating the role of the cytoskeleton in morphological

changes affecting organelles or membrane trafficking during

mitosis.

III. RESEALED CELL SYSTEM

The partial disruption of plasma membrane integrity makes

semi-intact cells unsuitable for reconstituting endocytic

processes such as the internalization of receptor/ligand

complexes or the recycling and degradation of receptors and/or

ligands in lysosomes, and consequently, for analyzing signal

transduction from the cell surface to the nucleus via the

cytoplasm. However, recent advances in cell resealing

techniques by calcium ions [39-42] have solved some of the

problems that are caused by the partial disruption of the plasma

membrane by SLO-mediated pores. Resealing renders

semi-intact cells intact again and some of the resealed cells

proliferated for several days [39]. Hence, the cell resealing

Fig. 4. Anterograde and retrograde transport between the ER and the Golgi

in semi-intact CHO-GT cells incubated with interphase or mitotic cytosol. Semi-intact CHO-GT cells were incubated with interphase (I) or mitotic

(M) cytosol, and the anterograde or retrograde transport of GT-GFP

between the ER and the Golgi was measured by fluorescence recovery after bleaching (FRAP). Anterograde transport from the ER to the Golgi

was specifically inhibited by mitotic cytosol.

Fig. 5. Schematic model of cell-cycle dependent morphological changes of

the ER and the Golgi coupled with the balance of vesicular transport

between the organelles.


technique is a unique method that enables molecules or a

variety of cytosolic factors delivered into living cells to exert

their effects on the cells for a substantial period of time. In fact,

the Collas group have used this technique successfully to

perform epigenetic reprogramming of DNA methylation and

histone modification within the regulatory region of Oct4 and

Nanog by introducing embryonal carcinoma extracts into 293 T

cells [43, 44], and have also induced 293 T cells to express T

cell-like functions by exposing the cells to extracts from T cells

[45]. A separate group have generated iPS cells by delivering

ES cell extracts to fibroblasts [46].

We first determined the optimal conditions for preparing

resealed HeLa cells by incubating semi-intact HeLa cells with

cytosol prepared from L5178Y cells. The Ca2+

-mediated

resealing process was reported to require membrane trafficking

functions, such as endocytosis [40], exocytosis [41], and

ectocytosis [42], which are largely dependent on the integrity of

the cytoskeleton and its associated motor proteins. In addition,

cytoskeletal integrity affects the structure and function of

various organelles, and the relative spatial localization between

organelles and the cytoskeleton. Thus, integrity of the

cytoskeleton is a key factor ensuring a high efficiency of

resealing.

To limit damage to the morphology of the cytoskeleton and

organelles, we optimized the resealing conditions for HeLa

cells. Resealing efficiency was estimated by calculating the

percentage of cells in which exogenously added

fluorescein-dextran (Mw>40kDa) remained 30 min after

resealing. This allowed us to determine that 0.13 mg/ml SLO

was sufficient for permeabilization of HeLa cells when other

conditions (e.g. the time of incubation of cells with SLO at 37

degree for resealing) were kept constant. We also found that

incubation of HeLa cells with 1 mM CaCl2 for 5 min was

sufficient for resealing and that cytosol (> 1.5 mg/ml) enhanced

this process [47]. In addition, immunofluorescence microscopy

using antibodies against marker proteins for several organelles

(e.g. early endosomes, the Golgi apparatus, the ER networks,

nuclear membranes, actin filaments and microtubules) revealed

that the resealing protocol had little effect on organelle

morphology, using intact HeLa cells as a control. Electron

microscopy revealed that the Golgi was swollen slightly,

possibly owing to perturbation of the ion balance in resealed

cells. Indeed, just after resealing, the mitochondria appeared

fractionated and slightly swollen, probably also owing to

perturbation of the ion balance. The perturbation of

mitochondrial morphology was restored ~90 min after

resealing at 37 degree.

Permeabilization by SLO activated several stress-response

kinases, such as p38 MAPK [48], JNK [49], and NFkB [50].

We investigated the level of phosphorylation of p38 MAPK,

JNK, and p42/44 MAPK by Western blot analysis using

phospho-specific antibodies. We found that phosphorylation of

p38 MAPK and p42/44 MAPK was greater in resealed cells

than in intact cells, but gradually decreased—but was not

completely eliminated—with ongoing incubation. The extent

of JNK phosphorylation was similar in resealed and intact cells,

although this might be related to the cell type. Thus, at this time,

we cannot determine the experimental conditions to fully

eliminate cell stress caused by permeabilization and resealing.

Therefore, we must consider the effect of permeabilization and

resealing on the activation of signaling pathways when

reconstituting biological processes.

A. Reconstitution and Analysis of Intracellular Events under

Pathogenic Conditions Using Disease Model Cells

Next, we established a basic protocol for preparing diseased

or healthy model cells from semi-intact HeLa cells using

pathogenic or healthy cytosol. For this purpose, we established

diabetes model cells (Db cells) by introducing cytosol prepared

from the liver of db/db diabetes model mice (Fig. 6). As a

control, wild type cells (WT cells) containing the liver cytosol

from WT mice were used. The results and experimental

conditions established for the permeabilization/resealing of

HeLa cells should be useful for other researchers who adopt the

resealing cell technique, because HeLa cells are commonly

used by many researchers.

Immunofluorescence microscopy of the morphology of

organelles and the cytoskeleton in resealed cells revealed that

the early endosome marker protein EEA1 was less associated

with endosomal membranes in Db than in WT cells (Fig. 7).

Since EEA1 is reported to be involved in the early endocytic

pathway [51], we hypothesized that endocytosis might be

disturbed in Db cells. Therefore, we investigated three

endocytic pathways that act via early endosomes in WT and Db

cells: the transferrin recycling pathway, degradation of the EGF

receptor, and retrograde transport of cholera toxin (Ctx) from

endosomes to the Golgi. Interestingly, retrograde transport of

Ctx from early endosomes to the Golgi was delayed in Db cells

in a p38 MAPK-dependent manner (Fig. 8). In addition, upon

EGF stimulation, the degradation of EGFR was greater in Db

than in WT cells (Fig. 8), and occurred in a manner independent

of p38 MAPK.

Furthermore, we found that

phosphatidylinositol-3-phosphate (PI3P), a lipid that

accumulates specifically in the early endosomes [52], was

+SLO

WT

cytosol

Db

cytosol

WT mouse

Diabetes model mouse

liver

Semi-intact cells

WT model cells

Db model cells

liver

Figure 6Fig. 6. Establishment of disease model cells by the cell resealing technique

To change the intracellular cytoplasmic condition to the disease state,

semi-intact cells were incubated with disease cytosol, which was prepared from the organ of the disease model mouse. The cells were further treated

with calcium chloride to reseal the permeabilized plasma membrane.


depleted in Db cells in a p38 MAPK-dependent manner. The

depletion of PI3P in Db cells was detected using the semi-intact

cell assay [53]. Briefly, semi-intact HeLa cells were incubated

with WT or Db cytosol at 32˚C for 30 min, then further with

recombinant GST-2xFYVE protein, a probe that binds to PI3P

through its PI3P-binding domain (FYVE domain) for 15 min.

The cells were fixed and stained with fluorescently labeled

antibody against GST. As shown in Fig. 7, GST-2xFYVE

signals became faint in Db cells. This result was also confirmed

by lipid blot analysis. Furthermore, restoration of PI3P in Db

cells treated with a p38 MAPK inhibitor (SB203580) indicated

that the depletion of PI3P was dependent on p38 MAPK. The

physiological role of PI3P in endosomes has received much

attention with respect to endosomal processing and the

transduction of extracellular signals such as EGF and TGFß to

the nucleus via endosomes [54]. This is because PI3P in the

endosomal membrane appears to act as a microdomain for the

recruitment of various proteins that contain a PI3P-binding

(FYVE) domain. The functions of FYVE-domain proteins

range from endosomal fusion and processing to signal

transduction [55, 56]. In addition, depletion of PI3P from early

endosomes can modulate signaling by growth factor receptors

such as EGFR by extending or shortening the sojourn time of

the receptors in endosomes [54]. Therefore, depletion of PI3P

from endosomes under diabetic conditions could have

pleiotropic effects on the cell, especially in terms of endosomal

processing and signal transduction. It is noteworthy that a

stress-responsive kinase, p38 MAPK, is activated in Db cells.

This was confirmed by Western blot using antibodies against

phosphorylated p38 MAPK. It is possible that several cellular

stresses cells could be increased under diabetic conditions

possibly owing to the high glucose content in blood or the

associated inflammation. This would lead to activation of

stress-responsible kinases including p38 MAPK, which could

contribute to the various defects found in Db cells. Our results

showed that activation of p38 MAPK plays a role in the

depletion of PI3P and the inhibition of Ctx transport in Db cells.

(Mechanistic model of p38 MAPK-dependent depletion of

PI3P in endosomes is shown in Fig. 7C).

In the above-noted study, we observed cytosol-dependent

depletion of PI3P from endosomes, inhibition of Ctx transport

to the Golgi, and enhanced EGFR degradation in Db cells. The

detection of these dysfunctions in diabetic liver or in isolated

diabetic hepatocytes would be required to validate the model

system. In fact, the enhanced degradation of EGFR that we

observed in Db cells seems to be one example of detecting

defects in disease model cells that mirror those found in the

diseased organs or primary cell cultures from the disease organs.

Several studies have also shown that the number of EGFR

molecules is decreased on the surface of hepatocytes isolated

from streptozotocin-induced diabetic rats [57, 58]. If enhanced

degradation of EGFR occurs under diabetic conditions in

hepatocytes, this could explain the decrease in EGFR receptors

at the cell surface in such cells. The next step is to investigate

the physiological relevance of the enhanced degradation using

the resealed cell system or intact hepatoma cells. In addition,

validation of the decreased level of PI3P and the inhibition of

Ctx transport in diabetic hepatocytes should be performed in

future studies.

IV. APPLICATION OF DISEASE MODEL CELLS TO CELL-BASED

ASSAYS FOR DRUG DISCOVERY AND ANALYSES OF

PATHOGENESIS

WT DbA

B GST-2xFYVE

Figure 7

C

Golgi

Early endosome

Late endosome

lysosome

Recycling

endosome

Retrograde transport

: inhibited

Degradation pathway

: enhanced

Recycling pathway

: unchanged

Degradation of EGF receptor

Ctx transport

Figure 8

in Db cells

Fig. 7. Depletion of phosphatidylinositol-3-phosphate (PI3P) in diabetic model cells

A. Semi-intact HeLa cells were incubated with liver cytosol prepared from

wild type (WT) or diabetic model (Db) mice, and then were resealed. After incubation with medium for 30 min, the cells were subjected to

immunofluorescence microscopy using antibodies against EEA1, a marker

of early endosomes. B. Semi-intact HeLa cells were incubated with WT or Db cytosol in the

presence or absence of SB203580, an inhibitor of p38 MAPK. By further

incubating the cells with recombinant GST-2xFYVE proteins, intracellular PI3P was labeled and stained with fluorescently-labeled antibodies against

GST. The GST-2xFYVE signal was reduced in Db cells, but was restored

following SB203580 treatment. C. Schematic model of p38 MAPK-dependent PI3P depletion in Db model

cells. The increase in activation of p38 MAPK in Db cells due to stress

inhibits the targeting of hVps34, an enzyme that synthesizes PI3P, to the endosomes through its binding of the small GTPase, Rab5. This leads to

depletion of PI3P in endosomes, and subsequent dissociation of

PI3P-binding proteins including EEA1 from the latter, and perturbation of

various endocytic pathways and signal transduction.

Fig. 8 Perturbation of several endocytic pathways in diabetic model cells.

We investigated several endocytic pathways in WT and Db model cells: transferrin/ transferrin receptor recycling, degradation of the EGF receptor,

and retrograde transport of cholera toxin to the Golgi apparatus.

Retrograde transport of cholera toxin was inhibited in Db cells, whereas

degradation of the EGF receptor was enhanced.


An important application of this assay system is for

cell-based assays. Cell-based assays are increasing in

importance relative to traditional drug screens and investigation

of the mechanisms of drug action. Most current cell-based

assays make use of so-called ‘‘normal’’ cells, which do not

reflect intracellular disease conditions. The culture and use of

primary cells from a patient’s diseased tissues for cell-based

assays is difficult owing to their lack of uniformity. Disease

model cells enable us to perform various cell-based assays in

cells under disease conditions, and should be helpful for

elucidating which intracellular events are perturbed during

pathogenic conditions.

More recently, a new approach using differentiated cell lines

derived from induced pluripotent stem (iPS) cells that have

been generated from patients is receiving attention from

researchers for drug discovery [59]. This system will be useful

for the study of genetic disorders, but not for chronic diseases

such as lifestyle-related diseases. In this regard, our assay

system should allow us to produce model cells representative of

chronic disease such as diabetes and hyperglycemia. The

intracellular conditions of chronic diseases cannot be

reproduced by iPS cell technology since it takes several years to

display symptoms. In contract, our disease model cells and the

resealing cell technique could replicate the intracellular

conditions existing in pathogenic organs immediately by

switching the cytosol with that from diseased cells.

The other important goal of assays using “disease model

cells” is to detect the various phenotypic differences between

healthy and pathogenic (diseased) cytosol. By focusing upon

phenotypic differences between healthy and disease model

cells, we can control for the effects of stress or other artifacts

induced during permeabilization and resealing. Given that

phenotypic differences can be attributed to functional

differences between the two types of cytosol, it might be

possible to identify the cytosolic factors that perturb the

reconstituted reactions in disease model cells by biochemical

analyses. For example, immunodepletion or addition of

function-blocking antibodies to the cytosol should enable key

pathological factors to be identified. Thus, established disease

model cells can be used for both drug screening and

identification and evaluation of the etiologic factors

contributing to disease progression at the cellular level.

Finally, to validate and elucidate protein networks deduced

by systems biology that are involved in intracellular

physiological events, assays using resealed cells (or disease

model cells) should provide spatio-temporal information,

which could not be obtained using standard in vitro

reconstitution methods. In particular, information about the

morphological changes of organelles, changes to the relative

spatio-termporal positioning between organelles and the

cytoskeleton or perturbation of protein localization under

pathogenic conditions should be observable by fluorescence

microscopic observations comparing resealed cells containing

either healthy or pathogenic cytosol. Therefore, using

morphological or spatio-temporal information on organelles or

proteins under pathogenic conditions might provide novel

criteria for disease diagnosis. Furthermore, we will be able to

screen drug candidates that can restore the perturbation of

morphology or protein localization in disease model cells.

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Fumi Kano received the B.S. degree in

biology from Faculty of Science, Kyoto

University in 1997, and the M.S., and the

Ph.D. degrees in biology from Graduate

School of Science, Kyoto University,

Japan, in 1999 and 2002. From 1999 to

2003, she was a Research Fellow of the

Japan Society for the Promotion of

Science (DC1 and PD). Since 2003, she

has been an Assistant Professor with Department of Life

Sciences, Graduate School of Arts and Sciences, The

University of Tokyo, Japan. From 2007 to 2011, she was a

Research Scientist, PRESTO, Japan Science and Technology

Agency (Life Phenomena and Measurement Analysis). She is

concurrently serving as a Research Scientist, PRESTO, Japan

Science and Technology Agency (Design and Control of

Cellular Functions) from 2012.

Masayuki Murata received the B.S.

degree in biophysics from Department of

Chemistry, Kyoto University of

Education, Japan, in 1982, and the M.S.

and the Ph.D. degrees in biophysics from

Graduate School of Science, Kyoto

University, Japan, in 1985 and 1988.

From 1989 to 1996, he was an Assistant Professor with

Graduate School of Science, Kyoto University. He was

concurrently a Visiting Scientist of European Molecular

Biology Laboratory (1994-1995, Humboldt Foundation

Scholarship), and University of California, Berkely (1995-1996,

HIMM Scholarship). From 1996 to 2003, he was an Associate

Professor with Department of Molecular Physiology, National

Institute for Physiological Sciences. From 2003, he has been a

Professor with Department of Life Sciences, Graduate School

of Arts and Sciences, The University of Tokyo, Japan.

the semi-intact cell system and methods for cell resealing...

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