Directional motion of foreign plasmid DNA to nuclear HP1 foci

Directional motion of foreign plasmid DNA to nuclear HP1 foci

Vladan Ondrej1*, Stanislav Kozubek1, Emılie Lukasova1, Martin Falk1, Pavel Matula2, Petr Matula2

& Michal Kozubek2

1Laboratory of Molecular Cytology and Cytometry, Institute of Biophysics AS CR, Kralovopolska 135, Brno,612 65, Czech Republic; Tel: +42-0541-517165; Fax: +42-0541-211293; E-mail: [email protected]; 2Laboratoryof Optical Microscopy, Faculty of Informatics, Masaryk University, Botanicka 68a, Brno, 602 00, Czech Republic* Correspondence

Received 2 March 2006. Received in revised form and accepted for publication by Pat Heslop-Harrison 12 April 2006

Key words: heterochromatin, HP1 protein, nuclear compartments, plasmid, transfection

Abstract

Movement of labelled plasmid DNA relative to heterochromatin foci in nuclei, visualized with HP1-GFP, was

studied using live-cell imaging and object tracking. In addition to Brownian motion of plasmid DNA we found a

pronounced, non-random movement of plasmid DNA towards the nearest HP1 focus, while time-lapse

microscopy showed that HP1 foci are relatively immobile and positionally stable. The movement of plasmid

DNA was much faster than that of the HP1 foci. Contact of transgene DNA with an HP1 focus usually resulted

in cessation of the directional motion. Moreover, the motion of plasmid DNA inside the heterochromatin

compartment was more restricted (limited to 0.25 mm) than when the plasmid DNA was outside heterochromatin

(R = 0.7 mm). Three days after transfection most of the foreign labelled DNA colocalized with centromeric

heterochromatin.

Introduction

Transfection of plasmid DNA into mammalian cells

has become a routine procedure in cellular and

molecular biology experiments, but the mechanism

by which the plasmid DNA reaches its nuclear

destination remains poorly understood. Small circu-

lar DNA molecules invade the nucleus of eukaryotic

cells during viral infection, gene therapy, or exper-

imental transfections (Kashihara et al. 1998). The

most commonly used method, liposome-mediated

gene transfer, involves endocytotic uptake, release

from endosomes, dissociation of DNA from lipid,

diffusion through the cytoplasm, transport across

nuclear pores, and diffusion to nuclear target sites

(Xu & Szoka 1996, Friend et al. 1996). Despite these

common events and experiments, little is known

about the fate and mobility of incorporated DNA

molecules in the nuclei of living cells.

Recent studies (Johnson & Jurcisek 1999, Lukacs

et al. 2000, Verkman 2002, Mearini et al. 2004) have

provided some information about the diffusion of

incoming DNA fragments and small or macromole-

cule-sized solutes in the cytoplasm and nucleus. It

was found that, in the nucleus, the diffusion of DNA

fragments of different sizes was more severely

restricted than in cytoplasm (Lukacs et al. 2000). In

contrast, similar-sized dextrans diffused freely in the

nucleus. At present it is not known which compo-

nents are responsible for the observed immobiliza-

tion of DNA fragments and plasmids inside the

nucleus. It has been suggested that there is an

involvement of the nuclear matrix and scaffold,

binding to nuclear components such as positively

Chromosome Research (2006) 14:505–514

DOI : 10.1007/s10577-006-1058-1

# Springer 2006

charged histones, or formation of DNAYprotein com-

plexes (Lukacs et al. 2000, Mearini et al. 2004).

Therefore, our study focused on the movement and the

destination of foreign DNA in cell nuclei immediately

after transfection. We transfected MCF-7 cells with

Cy3-labelled plasmid DNA bearing a gene coding for

the HP1-GFP fusion protein. Labelling of the con-

structs did not disrupt their function, which could be

seen as light emission from HP1 foci corresponding to

GFP excitation and emission wavelengths. This

allowed simultaneous visualization of the transgene

DNA and the heterochromatin, the latter represented

by HP1 proteins.

Heterochromatin, in contrast to euchromatin, is

very compact and dense. The main component of

heterochromatin is HP1 protein which is recruited to

this compartment by histone H3 methylation on the

lysine 9 (Howe et al. 1995, Berger 2001, Jenuwein &

Allis 2001, Dillon & Festenstein 2002). It is tran-

scriptionally silent and contains tandemly repeated

(satellite) sequences (Gilbert & Allan 2001). HP1 is

a conserved component of heterochromatin, and

plays a key role in heterochromatin formation and

maintenance (Aagaard et al. 1999, Bannister et al.2001, Cheutin et al. 2003, Festenstein et al. 2003,

Verschure et al. 2005). In humans, three subtypes of

HP1 (HP1a, b, g) have been identified, with different

preferences in their heterochromatin location during

the cell cycle. For example, HP1b is closely connected

with centromeric heterochromatin during interphase

as Hayakawa et al. (2003) described. Heterochro-

matin has different functional properties from euchro-

matin, and often causes silencing of active genes in its

proximity (Brown et al. 1997, Francastel et al. 2000,

Bartova et al. 2002).

In this study we investigated the mobility of the

plasmid DNA, which is transiently active in tran-

scription without requiring prior integration into the

genome, in two physically and functionally different

compartments of nucleus. We found that, as well as

random motion of plasmid DNA, there was a distinct

directional motion towards a single heterochromatin

focus, usually the nearest one. This was followed by

restriction of movement of the plasmid DNA. Three

days after transfection, most of the foreign labelled

DNA colocalized with centromeric heterochromatin.

Figure 1. The monitoring of labelled plasmid DNA (red) and heterochromatin foci represented by expressed HP1-GFP (green) in the cell

nucleus after transfection. Maximum image computed from 3D images in XY, XZ and YZ planes through the nucleus is shown with DNA

signals in an MCF7 cell. (A) Much of the labelled DNA was observed in the cytoplasm, as well as accumulated against the nuclear

membrane. Most nuclear signals are localized close to or in the centromeric heterochromatin, defined by expressed HP1b-GFP fusion protein,

and a minority of signals are remote from heterochromatin in cells with transgene expression. In (B), the single cut, 2D images in the XY, XZ

and YZ planes demonstrate the position of a single foreign DNA signal inside the cell nucleus localized on the surface of a heterochromatic

region.

506 V. Ondrej et al.

Materials and methods

Cell culture, cell transfection and DNA constructlabelling

A human MCF7 mammary carcinoma cell line was

grown in DMEM medium supplemented with 10%

fetal calf serum (FCS), penicillin (100 U/ml) and

streptomycin (100 mg/ml) in humidified air with 5%

CO2, at 37-C.

One day before transfection, 0.8Y1 � 106 cells

were plated in 2 ml of growth medium without

antibiotics in a 35 mm diameter Petri dish so as to be

90Y95% confluent at the time of the transfection. For

measurements of heterochromatin motion the cells

were transfected with expression vector containing

Figure 2. The dynamics of plasmid DNA in the living cell nucleus within a 1-min period. (A) Changes in the distances between two DNA

signals are shown in relation to time up to 60 s. DNA signals close to an HP1 focus (closed circles) and outside heterochromatin (open circles)

are shown. (B) The Dd2 vs time plot for plasmid DNA remote from heterochromatin (open circles) and that associated with HP1 foci (closed

circles).

Figure 3. Short-term movement of the HP1b foci in the nuclei of MCF7 cells: (A) scatter plot of distances between two foci (d) in relation to

time in a living cell. The intervals between individual measurements were 400 ms; the total observation time was 60 s. The deviations of the

distances between subsequent measurements were about 0.04 mm; the mean distance between the two foci increased from 2.85 to 2.90 mm.

(B) The relationship of Dd2 to time for living (green) and fixed cells (yellow) averaged from 10 cell nuclei. In each cell nucleus approximately

five to-10 foci were analysed using the object tracking algorithm, and distances between all pairs were used for calculations.

Directional motion of foreign plasmid DNA to nuclear HP1 foci 507

GFP-tagged HP1b. The transfection was performed

using Lipofectamine 2000 (Invitrogen) in OPTIMEM

medium according to the manufacturer’s protocol.

After 5 h of cell transfection in the presence of Lipo-

fectamine 2000, the medium was replaced with

DMEM containing 10% FCS without antibiotics,

and the cells were incubated for 48 h before imaging.

The vector bearing the HP1b-GFP gene was

labelled before transfection with Label IT Tracker

reagent Cy3 (Mirus Co.), at the ratio of Cy3:DNA

Figure 4. Medium-term observation of transgene loci in relation to the HP1 foci. Schematic illustration of the movement of the signals of

foreign DNA (red circles) in 3D space of the cell nucleus during medium-term observation. The plasmid DNA moved directionally (see

arrows) in most cases to the nearest HP1 focus (green circles). The nuclear spatiofunctional compartments corresponding to the HP1 foci

(transgene loci) are shown by blue lines. XY, YZ and XZ projections are shown (A). Distances between individual DNA signals (each colour

indicates a different signal) and the nearest HP1 foci in the cell nucleus decreased with time in most cases during observation (B).


recommended by the manufacturer, at 37-C for 1 h.

The unbound Cy3 reagent was separated by DNA

precipitation. The Cy3-labelled DNA of the trans-

gene enabled us to observe simultaneously the local-

ization of the transgene (red) and the expressed

protein tagged with GFP. Green fluorescence of HP1

protein in the cell nucleus indicates expression of the

transgene.

Cell fixation and immunolabelling

The transfected cells were grown on glass coverslips

for 72 h. The cells were washed for 2 � 3 min with

PBS (37-C), fixed for 10 min with 4% paraformal-

dehyde/PBS, washed for 3 � 5 min with PBS,

permeabilized in 0.2% Triton X-100 in PBS, washed

for 3� 5 min in PBS, and sequentially incubated with

the primary and secondary antibodies. The primary

antibody against CENP-A was from UPSTATE (Lake

Placid, NY), diluted 1000-fold. The secondary anti-

rabbit FITC-conjugated antibody (dilution 50�) was

from Jackson ImmunoResearch (West Grove, PA,

USA). The chromatin was counterstained with a

freshly prepared 1 mM solution of TOPRO-3 (Molec-

ular Probes) in 2� SSC for 5 min. After brief washing

in 2 � SSC, the antifade mounting medium Vecta-

shield (Vector Laboratories) was placed on the

labelled area and covered with a coverslip.

Living cell observation and time-lapse microscopy

Twenty-four hours before image acquisition the cells

were trypsinized and resuspended in fresh DMEM

medium without phenol red, supplemented with 10%

FCS and 200 mM Trolox (Sigma), and put into a

special chamber for microscope imaging. The images

were obtained by means of a high-resolution confo-

cal cytometer (Kozubek et al. 2004). The cytometer

is based on a completely automated Leica DM RXA

fluorescence microscope equipped with a confocal

unit CSU-10a (Yokogawa, Japan), a CoolSnap HQ

CCD camera (Photometrix) or alternatively an iXon

DV 887ECS-BV (Andor), and an Ar/Kr laser Inova

70C (Coherent). Three types of in-vivo observations

were performed: short-, medium- and long-term. For

the short-term observations 2D transgene images

were acquired every 2 s for a period of 1 min. For

medium-term observations 3D images were acquired

from 15 optical sections taken at 0.6 mm z-steps.

Intervals of 2 min were allowed between each series

of 15 sections, and observations were continued for a

total of 18 min. For the long-term observations 3D

images were acquired as for the medium-term

observations, but with longer intervals (40 min)

between each series of 15 sections. Except for mea-

surement of DNA mobility, we also measured move-

ment of HP1 foci for comparison. The light exposure

was kept as low as possible to avoid phototoxic

effects.

Analysis of motion of loci and calculationof diffusion constants

The changes in the positions of the fluorescence

signals (object tracking) were determined using the

FISH 2.0 software and a 3D viewer (Kozubek et al.2004), which allowed us to assign 2D or 3D coor-

dinates to the fluorescence signals. The coordinates

were taken at the centre of gravity of the visualized

objects, and corrected for the rotation of the cell

nucleus and drift of the images acquired during

the longer time-lapse observations. The objects were

traced in the time-lapse series on the basis of

matching algorithms. In 2D the distances between

two transgene loci or HP1 foci, and between the

DNA signal and an HP1 focus, were calculated

using the equation: d = ¾[(x1jxn)2 + (y1jyn)2]; or

in 3D: d = ¾[(x1jxn)2 + (y1jyn)2 + (z1jzn)2], where

x1, y1 and z1 (xn, yn and zn) were coordinates for

the first measurement and the nth measurement of the

same object. The mean square of differences in the

distance (Dd2) between two objects at each time

point (t) was calculated as Dd2 = (dtjdt+Dt)2, where

Dt was the time interval between measurements.

The diffusion coefficient (Dc) in 2D was calculated

as Dc = Dd2/(4 � $ t).

Results

To monitor movements of foreign DNA in living

cells we transfected MCF-7 cells with Cy3-labelled

plasmid DNA bearing the gene for the fusion protein

HP1b-GFP. Three-dimensional observations of fixed

cells showed two to 12 transgene DNA signals per

nucleus (n = 50). A large amount of labelled DNA


Figure 5. Four-dimensional tracking of the plasmid DNA (red) and the HP1 foci (green) in the cell nucleus. A 3D time-lapse image series

(nine frames in 8 min) displays the movement of the plasmid DNA, marked as TL1 and TL2, towards the nearest HP1 foci (HF1, HF2) in a

dynamic and directional manner.

Figure 6. In-vivo images from long-term observations of plasmid DNA. Changes in the labelled DNA (red) in the cell nucleus and the HP1-

GFP foci (green) are shown for a period of observation of 120 min. The labelled plasmid DNA (TL) became associated with the nearest HP1

focus (HF) within the first 40 min; after that the plasmid DNA movement was restricted only for compartment of relevant HP1 focus.


was observed in the cytoplasm, as well as accumu-

lated against the nuclear membrane. The majority of

signals in the nucleus were localized in or close to

the centromeric heterochromatin, defined by

expressed HP1b-GFP fusion protein; a minority of

signals were distant from the heterochromatin in cells

with transgene expression (Figure 1). The cells were

observed in vivo, 48 h after transfection, when tran-

sient expression usually peaks (around 60% trans-

fected cells showed GFP signals) similarly as

described by Johnson & Jurcisek (1999); after that

transient expression decreases.

The movement of individual plasmid signals in

human cells is restricted, but there are differences in

the level of restriction. The shortest time interval

for plasmid DNA image acquisition was 2 s and

therefore we were able to collect 30 2D frames in

1 min. The occurrence of several plasmid DNA

structures in the cell nucleus allowed calculation of

two-dimensional distances between them. As shown

in Figure 2, the movement of plasmid DNA outside

the heterochromatin (up to 1 mm/min, with an

average Dc = 7 � 10j3 mm2/s) was significantly

faster than the movement of plasmid DNA inside the

heterochromatin compartments (Dc = 1.5 � 10j3

mm2/s); the speed of movement was very variable. As

expected, the plot of Dd2 values against time showed

that transgenes had a significantly higher rate of

diffusion than the HP1b foci, which were also

measured at 1 min intervals using a 400 ms time-

lapse series (Figures 2 and 3). The average value of

Dd2 for 60 s was estimated to be 0.35 mm2, i.e. 15

times higher than the value for the HP1b foci. For

plasmid DNA inside the heterochromatin compart-

ment the average value of Dd2 was around 0.06 mm2,

which is just three times higher than the value for the

HP1b foci.

To check the correctness of the distance measure-

ments in living cells, the distances between HP1bfoci were measured in paraformaldehyde-fixed cells

under identical conditions. The distances measured in

fixed cells thus quantify the apparent motion attrib-

Figure 7. The image shows a cell nucleus fixed 3 days after transfection with the plasmid DNA (red) localized near centromeric regions

(green). Green signals correspond to the centromeric protein CENP-A visualized using antibodies. Chromatin was stained with TO-PRO. XY,

XZ and YZ sections through the transgene; because of the sectioning, only a relatively small number of centromeres can be seen.


utable to measurement errors resulting from external

influences. A comparison of the dependence of Dd2

on time for HP1 foci in living and fixed cells is

shown in Figure 3b. Displacement of HP1 foci in

living cells is relatively small, but significantly

different from that observed in fixed nuclei.

Simultaneous observation of HP1 foci and trans-

gene loci revealed several important aspects of

transgene DNA mobility and HP1 focus function.

For any one transgene and its closest HP1 focus, the

distance between them usually decreased with time

(Figures 4 and 5). Although the movement was pre-

dominantly in one direction, the paths of the trans-

genes were neither straight nor the shortest possible.

These results led us to the hypothesis that the cell

nucleus can be divided into several compartments, as

shown in the simple sketch in Figure 4.

During long-term observation we found that trans-

gene DNA movement is still located and restricted

within space of relevant HP1 focus. A typical example

is shown in Figure 6, where the transgene locus

moved to the heterochromatin and surrounded it for

a period of 120 min. Expression of transgenes in the

cell population was dramatically reduced by 3 days

after cell transfection (nearly 10% of cells showed

GFP signals at different levels), although the presence

of plasmid DNA in the cell population could be

detected. At this time, colocalization of the plasmid

DNA with centromeres (Figure 7) could be detected

using antibodies in cell nuclei without expression of

the fusion protein.

Discussion

Plasmid DNA bearing a transgene for an HP1-GFP

fusion protein was labelled to visualize the process of

silencing. Labelling of the constructs did not disrupt

their function, which could be seen as light emission

from HP1 foci. Thus transgene expression was moni-

tored by the light emission in parallel with spatial

positioning of the transgenes and the HP1 foci. The

transgenes were expressed shortly after cell transfec-

tion and penetration into the cell nucleus (see Figure 1).

However, after some time (3 days after transfection)

the transient expression of fusion protein was

silenced. At that time point the plasmid DNA signals

colocalized with centromeric protein CENP-A, which

was visualized using antibodies.

Before associating with heterochromatin the plas-

mid DNA in the cell nucleus moved along complex

pathways. Fast diffusive motion, observed in the

course of the first minute for free transgenes (Dc = 7

� 10j3 mm2/s), was superimposed on a systematic

directional movement towards the nearest HP1 focus

during medium-term observations (Figures 2 and 4).

The motion of plasmid DNA outside the heterochro-

matic region corresponds to the dynamic parameters

for small circular DNA in the mammalian nucleus

(Mearini et al. 2004). After the capture of the

plasmid DNA by an HP1 focus, its motion became

restricted (R = 0.25 mm) with parameters similar to

the values published by Chubb et al. (2002) for lacO

array loci integrated in the nuclear periphery or

nucleolar neighbourhood (R = 0.3 mm).

In this study we compared the motion of plasmid

DNA bearing an HP1-GFP transgene with that of

large heterochromatin foci visualized by the HP1-

GFP protein. Analysis of the movement of HP1 foci

in the cell nucleus during short-term observation

(Figure 3) demonstrates a very limited diffusion of

these large heterochromatin structures. Our time-

lapse measurements revealed that the rate of diffusion

of HP1 foci as measured by their diffusion coefficient

(Dc = 1.1 � 10j4 mm2/s) was similar to that for Cajal

bodiesYDc = 1.1 � 10j4 mm2/s, PML bodiesYDc =

1.2 � 10j4 mm2/s (Gorisch et al. 2004), or nucleo-

plasmic chromatin with integrated lacO arraysYDc =

1.3 � 10j4 mm2/s (Chubb et al. 2002). Our results

concerning HP1 foci correspond to experiments of

Cheutin et al. (2003), in which the authors estimated

similar relative displacement of heterochromatin

domains (0.14 mm/min), but calculated over a longer

time interval. The random motion of HP1 foci was

limited to approximately R = 0.13 mm. In agreement

with the findings of these authors, our results show

that HP1 foci are among the structures with the most

restricted motion in the cell nucleus. This suggests a

structural role of HP1 foci as anchoring elements in

the chromatin.

HP1 protein is known to contribute to the formation

of heterochromatin, which plays an important func-

tional role in silencing of genes or transgenes (Howe

et al. 1995, Jenuwein & Allis 2001, Verschure et al.2005). In this study heterochromatin not only

affected the mobility of plasmid DNA because of

its physically dense structure, but probably changed

the expression level of transgenes because of its


functional character. Many authors (Dobie et al.1996, Francastel et al. 1999, Cryderman et al. 1999,

Porteus et al. 2003) have observed the colocalization

of silent integrated transgene loci with centromeres

or centromeric heterochromatin and their neighbour-

hood and, moreover, that transgenes integrated into

such sites are usually strongly silenced (Dobie et al.1996, Francastel et al. 1999, Mutskov & Felsenfeld

2004). As we have demonstrated, transgene DNA

finished its journey in the centromeric regions. It is

not known if this foreign DNA integrated into these

nuclear sites or not.

The foreign DNA was subject to directional

movement to the nearest HP1 focus. These move-

ments were restricted to some spatiotemporal com-

partments of the cell nucleus (Figure 4). The driving

force behind this directional movement to the nearest

heterochromatin and to the transcriptionally silent

regions is not yet known. It is likely that an extended

network exists in the nucleus which provides a

platform for the organization of nuclear subdomains

and for supporting their functionality. This leads us

to hypothesize that plasmid DNA could bind to the

network of nuclear matrix and scaffold which is

dynamic and maintained nuclear architecture (Ma

et al. 1999). The studies of nuclear actin (reviewed in

Pederson 2000) remind us that filament-forming

protein families are present in cells. We can also

hypothesize that plasmid DNA might tether to short

nuclear actin filaments where actin is partly a

component of nuclear matrix, plays a direct part in

the nuclear export of retroviral RNA and cellular

proteins and is necessary for transcription by RNA

polymerase II (Clubb & Locke 1998, Kimura et al.2000, Hofmann et al. 2004). Moreover, we can

speculate that the plasmid DNA movement to the

transcriptionally silent regions represents some kind

of cell defence mechanism against alien genetic

information. To our best knowledge this kind of

movement in the cell nucleus has not been described

before.

Acknowledgments

We thank T. Misteli, who kindly provided the

backbone vector for HP1b-GFP expression. This

work was supported by the Grant Agency of the

Czech Republic GA202/02/0804 and the Academy of

Sciences of the Czech Republic A1065203.

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Directional motion of foreign plasmid DNA to nuclear HP1 foci

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