Dynamic imaging of neutrophil migration in three ... - CiteSeerX

Dynamic imaging of neutrophil migration in three dimensions: mechanical interactions between cells and matrix John

T. H. Mandeville,

Department

Abstract: to

Pathology,

of

Fluorescence

obtain

and

a spatial

through a temporal

Neutrophils tant gradient

migrating within

of

Univeisity

microscopy (3-D)

resolution

A. Lawson,

Columbia

confocal

three-dimensional

neutrophils migrating motic membrane with 5

Moira

College

was

images

of

used

in response a 3-D matrix

in the were

z-dimension.

to a chemoattracapparently able

to generate traction by use of lateral pseudopods serted into footholds in the matrix as evidenced matrix distortion. Similar anchored pseudopods seen

in

branes increased

cells with

with cells ofpseudopods trix

and

migrating

on porous

filters

Contact fibrils

matrix show

that

neutrophils

Leukoc.

Key

Words:

observed

ofneutrophils observed. These in 3-D

migrating

structures to generate 61:

Riot.

constriction

appeared

to

crawling along observations are

in the matrix, traction for

188-200;

ring

.

able

to the

polymerization,

motors,

[2,

12],

and

the

ECM.

2-D

their net migration number of cells

and

there

through crossing

studies of how cells actually Migrating cells undergo

have

been

3-D substrates a barrier. There

many

studies

of

by counting have been

the few

crawl through 3-D matrices. cycles of adhesion, generation

.

Journal

of

Leukocyte

Biology

must

it

[8-li] signaling. by a combination

traction

to differences

lead

ECM.

Third, that the

cell

that on by the

and

surface

architecture may on the substrate

allow with

receptor-mediated adhesion. Secare not homogeneous, and they dimensions, orientations, compo-

and mechanical properties. These to contact guidance of migration,

can

the

differ from is enveloped

in cytoskeletal of a 3-D purchase

pnes-

between

migration in 3-D matrices First, a cell in a matrix

leading

as

the inter-

on hydrostatic

involve

less reliance on specific ond, biological matrices contain fibers of differing sitions,

such

a 3-D matrix can present cell must either deform

inhomogeneities directed by the

a barrier or degrade

to migrain order

It has

been

3-D

may

[16], able

monocytes to migrate

proposed

differ

that

the in

mechanism

2-D

of traction

[13-15].

in

Lymphocytes

[17], and neutrophils [14, 17] that are unon collagen-coated glass are able to migrate of collagen.

In addition, neutrophils lackeither through genetic defects or by treatment with 132 function-blocking integnins,

132

display but

that

from

no

profound

deficits

significant

in migration

differences

on

on

3-D

2-D

substrates

as measured by the distance penetrated by the advancing front of cells [18].

into Macno-

(polymonphonuclear (3-D) extracellu-

of force, and de-adhesion [3]. Adhesion to the ECM is largely mediated by members of the integrin superfamily I4] Integnins are heterodimenic transmembrane glycopro-

188

actin

but

How might surfaces?

of collagen, the matrix

[2],

via molecules

of myosin sure

through matrices ing functional in the I2 subunit

calcein

lan matrix (ECM) is critical for host defense against bacteria and is an important component of pathological inflammation. Neutnophils are rapidly locomoting cells that exhibit chemotaxis toward a chemoauractant source [1]. Much has been learned about neutnophil migration on two-dimensional surfaces

cytoskeleton

to pass.

INTRODUCTION

(2-D)

actin

York

7J and inside-out may be mediated

surfaces

migration of neutrophils through the three-dimensional

New

talin, and vinculin [5]. The link between the cytoskeleton is regulated by a complex

antibodies,

The directed leukocytes)

Surgeons,

action of outside-in [6, The generation offonce

to uti-

not present locomotion.

amnion

.

linked

a-actinin, ECM and

tion

1997.

foothold

and

morphology. The presence the cell to gain mechanical

Expansion in the ma-

and

R. Maxfield

of Physicians

substrate,

mem-

ofthese pores compared

through the openings. Neutroofthe elastic amnion matrix durpermanently altering the sub-

guidance was also

lize mechanical on 2-D surfaces,

J.

was

inby were

polycarbonate

the presence and migration

membranes without pores. distal to narrow constrictions

be used to pull cells phils deformed parts ing migration without strate.

across

0.8-.tm pores; cell polarization

Frederick

teins

human

a 3-D matrix of amresolution of 30-60 2 im

and

Volume

61,

February

1997

Abbreviations:

2-D,

acetoxymethyl

ester;

two-dimensional;

indacene-sphingomyelin;

ECM,

tg/ml

PBS,

extracellular

and

10%

contrast;

saline;

(v/v)

carboxytetramethyb-hodamine, interference

matrix;

FWHM,

phosphate-buffered

saponin

calf

full

fMLP,

width

PBS-S.

serum;

AM,

PBS

ester;

dimethyl

containing

250

5-(and DIC,

sulfoxide;

N-formyl-L-

at half-maximal

rhodamine/SE,

succinimidyl DMSO,

three-dimensional;

4,4-difluoro-4-bora-3a,4a-diaza-s-

methionyl-L-leucyl-L-phenylalanine; intensity;

3-D,

BODIPY-SM,

PET,

6-)-

differential polyethylene

terephthalate. Correspondence: try,

Cornell

NY

10021.

ber

Frederick

University

Received July 22, 1996.

29,

Medical 1996;

R.

Maxfleld,

College, revised

Department 1300

October

York 18,

of BiochemisAvenue,

1996;

New

accepted

York, Octo-

phages

migrating

served

into

fibrin

matrices

have

by scanning electron microscopy into the surrounding matrix that may

tions

to generate

traction

[19].

Contact

guidance,

or

also

been

ob-

shown to exhibit directed [23, 24]. Although contact ing through 3-D biological

137

alignment

and

migration

the

(3)

move

the

ically, or (4) stop moving. Without migrating cells within the matrix, can

only

be

inferred.

studied on texhave also been

phils

as

dynamic the final

Neutrophils

carry

roles [25]. Cell motility studies in three on experimental methods such says that do not permit direct [18,

26-28],

electron

often based migration asof cells during that

only static images [14, 17, 19], on differential contrast (DIC) and phase images of live cells stnicted

to a single

of the

matrix

of fluonescently phils during cally

focal

plane

[14-161.

We

labeled chemotactic

cells

relevant

3-D

The matrix of the structure

with

have

little

used

of

are

microscopy

provides

to no

to directly observe migration through

re-

resolution

[31],

enogeneous

matrix

in which

both

permissive

study,

and

and

contact

cuss

the

similarities

2-D

and

3-D.

to study the

guidance

from and

amnion a range

to cell

nature

Neutrophils

were

previously

described

matrix

structures,

form

differences

between

tumbled

Human

and

sodium lated lution

heparin by

blood

was

obtained placed

M

(Becton-Dickinson,

centrifugation

(1-Step-Polymorphs.

for

30

San

Jose,

mm at 400

Accurate

from

Chemical

CA).

g over and

tubes

F. R. Maxfield,

following

am-

in situ

im-

unpublished

fluorescent

Inc.,

markers

used

acetoxymethyl

Eugene,

temperature calf

pluronic

using

within

buffer 0.2%

were

stored

(AM)

cells

were

[incubation

Molecular

Cells

and

ester

Briefly,

serum,

F-127;

as indicated. medium,

OR).

in loading

fetal w/v

calcein/AM

then

over

dimethyl

sul-

Probesj

con-

rinsed

ice

in PBS,

until

use.

Cells

Probes

and

5 h of loading.

label prepared

suspended Lipids,

argon,

and

was

by the

in a 1:1

From

solution

Neutrophils

stock and

were

solution,

vesicles

in cold

PBS,

resuspended

after

labeling.

Ethanol a 100

for

BODIPY-SM (Avanti

was

tM

evaporated

then

and

mm.

were

solution

PBS,

ice

30

in cold

labeling

1351.

vesicles

against

to 95%

as described

GIBCO-BRL,

fJ.

7.4)

KCI,

contained

diameter)

then

washing

munofluorescence

pH

(PBS,

morphology.

amniotic inner

was

Amnions

preparations

lysis

saline

5 mM

Preparations

within

by gentle

ig/mL;

nion

NaCl,

prepared

(22-mm

U/mL;

hypotonic

membranes

was

extensive

(500

medium

matrix.

migration

rings

NH4OH.

(500

Na2HPO4,

mM

of human

epithelium

and

icillin

foxide

parallel to the basement membrane, which is itself composed primarily of a dense meshwork of laminin, fibronectin, and collagens type IV and V [13]. The amniotic basement membrane has been reported to be a barrier to

In this

ing

mM

characteristic

(obtained

2 h, followed

microscopy

we used was human amnion, which is typical of loose fibno-connective tissue that neutno-

neutrophil

tissue

Teflon

were

phils encounter in vivo [29]. The stromal amnion is composed primarily of fibnils collagens type I, III, and V [30] that are

ions,

for

8.1

7.4).

their

pieces

amniotic

by a 30-s

(150

of amniotic

Neutrophils

interference that are

confocal

across

liceman

a path through elastase, cato play major

dimensions as trans-filter observation

by

removed

in phosphate-buffered

N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic pH

membranes

The

KC1,

mM

glucose,

amniotic

cental

rinsed

medium

assessed

Human

were

then mM

20

mM

To summarize,

mechan-

armament

2.7

Preparation

observation of three options

an

NaC1,

CaCl2, 10

obstacle

were

in incubation

acid,

pnoteolytic enzymes well-suited for digesting a noncompliant matrix, of which neutrophil thepsin G, and proteinase 3 are considered

migration

mM

of

migration in aligned matrices guidance of neutrophils migrattissues has been inferred from

obstacle,

erythrocytes

Cells

pended

these studies, it has not been observed directly. Neutnophils that encounter a physical barrier in a matrix have four options: (1) remove the obstacle chemically, (2) around

Contaminating

1 mM

the

cells along anisotnopic substrates, has been tuned or grooved surfaces [20-22]. Cells

move

NY).

procedure.

to insert projecserve as anchors

isoso-

Westbury,

Mandevilte

Chemotaxis Amnions 1 h before

et at.

Neutrophil

were use.

chambers rinsed

and

Membranes

migration

soaked were

within

in incubation then

suspended

a three-dimensional

medium over

at 37#{176}Cfor> a smaller

Teflon

matrix

189

0-ring

(16-mm

nM

inner

St. t.ouis, with

MO)

medium,

in the

culture

and

fresh

then

medium were

used

into

rinsed

from

were

transferred

for

the

upper

for

for

to a humidified In some

imaging

of live

microscope

cases,

cells

stage

were

fixed

unlabeled

were

cells, the

were

were

previously

plated

the chemoattractant after

IMLP

placed

the

oil immersion Migrating

and

initial

tail

from

each

IWctifl,

dish

phils

fixed.

dishes

substrate

images

disk

of confocal

micros(’ope

(Dialux

CA) WaS

Leitz

an image

with

(CCD-72:

Dag--MTI,

(DSP-200:

Washington.

.

Inc.,

Dage-MTI).

images approa(-hing light source. Cells

j((tiV(

fieI(l

cording

ntxt

fO(LIS knob To obtain

were

obtained

moving sections,

of the

for

Zeiss, was

planes.

with

images

fitted

artifact

would

an

with

a spinning

upright Co.

confocality.

The

through

gelatin intensity

beads

Rasband,

by

available

use

of

to Internet

in the

z-axis

confocal

Briefly,

10%

half-maximal

zippy.nimh.nih.gov

disk

±

at

in

directory

resolution.

microscope

Optical

had

a FWHM

SE).

San

,

Inter-

video

video

camera

frame

permitted

Oberkochen, optically

Moving

step

sizes

per

they

acquired disks

190

of

to image was

from

one

ranging 25

roughly

little

a

10-.tm

have

moved

volume

frame. out

of the

1-s

intervals

and

(PMO-650;

Leukocyte

Pinnacle

Biology

tm

0.8

arc ob-

dichroic

mirror

a 525-

hence

of view.

was

digitized Irvine,

61,

disk.

uously

were

focal

dual

plane

The

confocal used

in these

confocal

0.1

.tm

0.3

±

im

(mean

±

in the

interference

and

using

a z-axis

plane

were

four

scans

on a magnetostepper

motor.

acquired

Occasional

cells

mode.

over

stored

contin-

adjustments

in the

using for

field

of view

or

beads

as

line

steps.

scans

range taken

Optical

ranged

fluorescent

the

of pinhole through

sections

from

aper-

individual

acquired

a FWHM

SE) for 3-D reconstructions, SE) for time-series acquired

±

detection,

Pores

averaging

calibrated

microscope

(mean

for red

detection.

reflection

calculated

z-axis

filter

green

by a 560-nm

of view.

from

at 0.2-.tm

scanning ±

was

tubes

images.

field

experiments

acquired

and

to keep

was

FWHM

mirror,

focal

moving

of the

microscope

above.

dichroic

acquired

necessary out

fluo-

a 527-nm

section)

a single

color

were

structures

described

were

through

were

filter;

Kalman

optical

laser.

matrix

excitation

for

with

each

Zeiss).

argon

bandpass

using

obtained

infinity1.2;

=

a 25-mW

emission

visualized

a 40x NA

from

scan-

Cambridge,

using

photomultiplier

filter

a laser

rhodamine-labeled

long-pass

frames

to construct

of the

and

two separate

s to acquire Volume

line

with

bandpass

images

on the

of 1.8 and

0.2

±

a FWHM

at a single

optical

plane.

Image processing Digitized

3-D

volume

images

passed

and

used

nal

noise

sity

was

viewed

inherent used

to

tions

1.5

cells, could

tions.

pixel’s

a summed-pixel

optical

the

following

onto

1997

the

color tial

stereo relationships

rotated

were

then

and

background.

to arbitrary

intenWe

surface

viewing These

a 3-D

sig-

a threshold

a solid

conditions.

first

to diminish

and

cells

to form

were

rendered

used image

angles

volume

animated

slice. monitor

images,

and projec-

sequence

Pairs

next

algorithm, Image

image

of

vertical

the

each

were Inc.

dual-labeled

slice

,

viewed Beaverton, samples.

(Vicom were

was

was

on OR)

to

Maynard,

compared

intensity pixel

projec-

Vis-

made

algorithm,

was

maximum

where

stereo transferred

processor

projections

(1) a brightest-pixel

stacks

(Tektronix,

were

Corporation,

of 2-D

either

and

as 3-D

data

Equipment IP9000

CA).

in the

between

for viewing

stereo (Digital

using

projection

(2)

filter

to generate

a Gould-Vicom

intensity

existing

be

of paired

ofimages

could

using

between

Fremont,

stack

imaging,

concatenated

with

of the

level

as serial

Images

cells.

MA)

each

directly

be

minicomputer

optically

light

ways: (Indec

acquired

1331.

convolution

different program

images

x

then

images

equipped

from

3 Gaussian

two

microVoxel

described

lighting/shading

generation

Computing,

volumes

algorithm

could

different then

3-D

3-D

following

as previously

rendering

of migrating For

3

a microVAX-Il ual

optical

x

in low

Alternatively,

per

The

to discriminate

under

a field

in the

to render

a 3

which

of the

visualized

stereo-viewing.

sections

through

plane

were

rendering

each

Each

February

514-nm

selected

were

Zeiss)

a 514-nm

a 600-nm

images 5-10

Alternatively,

coarse

cells

CA)

and

3-D

z-axis).

fewer fewer

were

filters

Digital optical

with

to 535-nm

polycarbonate

at a rapid

being

desired,

the

with

Microscience,

(C-Apochromat,

cells

between

a first-pixel

morphology

moving

Micro,

Volume

divided

were

by re-

frames

in the

as it was

field

they

was

frames

of cell

was and

migrated at

60

I tm

emissions

optical

volume

cell

resolution

rescence

confocal

focal

from

was

imaged

Systems)

to the

volume

distortion

diameter

simultaneously

obtained

(Axiovert; objective

or BODIPY-SM-labeled

averager

Germany).

neutrophils.

approximately

Calcein-

were Bio-Rad

microscope source

us to ac-

sectioned

attached

an inverted

illumination

and

images (MRC-600;

micro-

Videoscope

and

of migrating

temporal

acquired

magneto-optical

Journal

there (e.g..

higher

before was

mm

(1 speed

When

imaged

(mean

width

to measure

spinning

water-immersion

of 2.9

amniotic

used Instrument

motor

on

to 2.3

fluorescence

through

confocal

Carl

MA)

laser

device

frame, stepper

with

resolutions

were

server used

fluorescence

corrected,

tures

using a conventional mercury with a 2Sx water-immersion

offocal

se(Juentially,

of 5 jtm/min

seetiofls

tm

full

(Molec-

[38J.

suspended

by Wayne

FTP it was

attachment

to identify

phalloidin.

to acquire

IN),

disk

by a z-axis 3-D

Ex-

on three neutro-

the

confocal

beads,

(KS-1381;

City,

spinning

micros(’ope.

moving

and

1371

rhodamine

maximum

Michigan

0.8;

fibro-

preparations

Technical

intensifier

volume

a series

acquisition

sectioned).

0.2

±

(requiring

Control

described

migration

charge-coupled

=

or

achieved

to motion

speed

Cap-

beads

thickness

cross-sections

(written

anonymous

acquired

ning

their

recorded

image- series. A typical 3-D volume frame consisted of 60 optical which we- found to be a suitable compromise between temporal

spatial

At this

from

was

)lm.

NA

of (‘ells,

images

to tlw

Bio:

video rates were imaged

(Plan-Neofluar,

Iach

from

10 jig/mL

we primarily

DC), The

on

leading

condition.

amnion

Germany)

(K2

equipped lid.

the

of neutrophils of these cells.

during

Wetzlar,

apertures

each

with

were

1 h with

used

images,

attachment

5(1 to pinhole

natft)nal.

(ltiir( lamp

20;

optical

7 tm

with

were

moving

a Zeiss

recorded

at least

for

minutes

microscopy

microscopes 3-D

were

previously

confocal

chamber

both

for

stained

neutrophils

To obtain

(115k confocal

Systems,

fluorescent

vertical

of beads

The

from

program

on the

The

5 mm.

using

fields

in our

been

and

ofcalcein-labeled

scot)e

present has

permeabilized.

membrane.

JOSt.

used

as

calcein/AM

in which

sequential

coated

dishes

Germany)

images

to move

were

(Petri

with

monitored

as those

dishes

without

beneath,

Oberkochen.

resultant

Three

or

experimental

was

Zeiss.

observed

motility

spinning

tYpes

determined

of 3.6

at 37#{176}C.Five

were repeated with fresh preparations days. To determine the morphology

Nipkow Two

three

The

motility

35g.

on coverslip

neutrophil

were

(Indec

as de-

at 37#{176}C.After

maintained

defined

were s 18.

a physiological

p(-riments separate

was Image

slice

a field

steps.

to 37#{176}Cwith

with

loaded

added.

The

were

cell

and

plated

which

cell

objective.

in 200

were

was

(Carl

cells

of the

position

((‘115 were

users

z-axis

on the

mounted

for 5 mm

stage

microscope

x

e(Ige

software

300-nm

image

through

at 0.2-tm NIH

using

the

sections

dishes

coverslips

filters

(10 nM)

of fMLP.

inverted

videotape.

a 63

the

on a microscope

application

Axiomat

and

Neutrophils

onto

CA)

coverslip-bottom

bottom

1361).

and

Livermore,

onto

in the

described

was then

taken

Dual-wavelength (Poretics.

mounted

punched

as described

were

(w/v)

calibrated

chambers

in 2-D

membranes

holes

was

to determine

and

amnion

Constructor

Laser scanning confocal microscopy

pores

with

Volume

/pub/nih-image),

the

warmed

within

to

cells

images

the

allowed

unattached

confocal

Probes)

(FWHM)

or BODIPYand

Cells

time

with

available

CA).

ular of

replaced

studies,

itola, The

incubated

a gradient

was

commercially

chamber

was

Calcein/AM-

migration

at which

For

Boyden

chamber

staining.

mm,

20

Co.,

to establish

neutrophils.

chamber.

assays

0.8-.tm

section

106

40

Chemical

chamber

tray

immunofluorescent

matrix

Polycarbonate

due

The

upper

used

containing

below.

Motility

and

x

were

the

curtain.

scribed

in the

5

neutrophils

migrate

an air

medium

dish Sigma

a modified

chamber.

37#{176}Cwarm

containing

SM-labeled cells

the

creating

lower

mm on a humidified

30

fMLP.

l)(

in a tissue

(fMLP;

in incubation

chemoattractant

for

on

diameter)

formyl-methionyl-leucyl-phenylalanine

of

where with

that

retained,

added

a Tektronix to observe

or

to that

of

SGS625 the

spa-

Time-series ferred

sequences

to the

taken

from

microVAX-II/IP-9000

a single and

optical

viewed

plane

were

as dual-colored

trans-

grating

cells

moving

viously

described

images.

cells

degradation

amnions

were

a cocktail 60

(Sigma). ig/mL

by secreted

for

2 h in incubation

(Sigma).

without

rinsing

described

aprotinin

This

by

laser

the fol-

.tM

motile

100

is effective

against

directly

to mem-

added

Cells

and

migrating

in the pres-

confocal

microscopy

scanning

the

5%

±

calcein-labeled

spinning cells with

use

of pre-

motile cells

disk 5-10

further to 1 tM calcein. extensions were visible

cell volume

with

56

control (mean

±

confocal microscopy, RM calcein allowed

excitation

microscopy,

tration plasmic

antipain

(Sigma),

cocktail were

inhibitors.

imaged

10

focal

[37]:

light levels. For laser scanning conwe were able to reduce the dye concen-

us to minimize

containing

including

(Sigma),

Neutrophils

of proteinase

were

proteinases,

medium

inhibitors,

9 .tg/mL

inhibitor

of proteinases.

of inhibitors

neutrophil

fluoride

(Sigma),

trypsin

spectrum

branes

proteinase

leupeptin

soybean

ence

matrix

phenylmethylsulfonyl

10 .tM

a broad

as

of the

pre-incubated

tM

8%

±

For imaging using we found that loading

of low-molecular-weight

lowing:

60

glass

methods

SE).

Proteinase inhibition To block

vs.

on fibronectin-coated

marker,

we also

To see whether using calcein,

imaged

cells

using

fine which

cytois a

the cell mem-

brane marker BODIPY-SM. Confocal imaging and 3-D rendening showed that all surface structures of cells labeled with BODIPY-SM were also visible with calcein labeling (not

above.

shown).

Antibody The

and

following

anti-as Inc.

phalloidin

antibodies

integrin (Ab1928;

lular

domain

(A2543;

CA).

rabbit

Co.

(Pierce).

and

body

integrin

rabbit

rhodammne-goat

polclonal

(Chem-

(Pierce

Chem-

secondary

anti-mouse

secondary

integrins

and

a 3-D

matrix,

through

(in

some

for

20

cases

mm

the

and

in the

(PBS

containing

250

cocktail

primary

PBS-S.

anti-

(Molecular

Fluorescent

labeling Labeled

neutrophils into

pre-labeled

caliserum.

Cells

then was

neutrophils

antibody

and/or

then

for for

in PBS-S

imaged

by

2 h

In addition

to amnions.

through

tissue

terephthalate

(PET)

polvcarbonate

neutrophils

culture inserts:

filters

with

purified filters rinsed

away. 3-tm

and

mm

Neutrophils

were with

primary

antibodies

in PBS-S. described rinse

with

by addition fixed

were

with

protease

+

for

above)

confocal

pores

laser

a i-h

con-

were

PBS.

1 h. as

stained

with

for 1 h. followed Labeled

neutrophils

first

cells 2%

migration

Corp..

Livermore.

precoated

with

were

induced

inhibitors

for

above.

secondary

10

to the

mm.

antibody

by a 10-mm then

on

to migrate

fMLP

described

CA). 1 mg/mL

cells

rinse imaged

and

stained

a 10-mm and

in PBS-S

and

by laser

to five cells In the absence

of the

with rinse

these (Fig.

)as 10-mm

did iB).

microscopy.

microscopy.

were cells

An av-

presence

neutnophils

observed remained

that

re-

to polarize on the sun-

of fMLP,

of pseudopods of the cell, but

unpolarized,

and

conditions

plane

In preliminary experiments we optimized the labeling conditions used for imaging live neutrophils within the amniotic matrix. Loading with up to 10 LM calcein had no effect on neutrophil migration as assessed by the percentage of mi-

tion

et

at.

cells

any extensions of pseudopods were seen to convert between

non-polarized/non-motile cells did not become whereas the majority

period,

states.

A small

motile exhibited

during some

(Fig.

migration was mainly parallel to the plane basement membrane even though the chemof chemoattractant was perpendicular to this 1, C and

that were slightly period. Despite net

occurred primarily on two major pro-

of migration.

Interestingly, ofthe amniotic ical gradient

LTS

one

seen on the lateral sides ofthe cells, to the direction of migration (Fig. trailed behind the cell body, and

of unpolarized

fraction

ilandeiitte

In the

not demonstrate Intermittently,

polarized/motile

scanning

degree

Labeling

morphology

matrix in response Over the course of of moving images

confocal

to the matrix unstimulated

amnion.

the observation

RESU

dynamic

in the image plane. of fMLP, none of the

jections were frequently roughly perpendicular iB). Knob-like uropods

mm.

phalloidin

the

lasted 15-25 mm and contained 5-10 cells. 39 series of moving images were acquired by confocal microscopy through a single optical series typically lasted 10-15 mm and contained

leading edge. Extension from the leading edge

chamber. for 5

After

the were

through

lower

to characterize

migrating cells were clearly polarized, with a leading edge and tail similar to those seen in 2-D (Fig. 1B). Cells migrating in a 3-D matrix appeared more cylindrical than those migrating on a 2-D surface, which often have a broad, flattened

across

plated

unattached

paraformaldehde

(wlv)

were

were

and

of 10 nM

migra-

polvethlene

during

Neutrophils buffer

during

124-well

and

matrix

neutrophils represented 15 ± 3% of cells in any given volume frame. After 3-D rendering, unpolarized cells appeared spherical, with some ruffling and protrusion ofpseudopods at the surface (Fig. lA) In contrast, actively

rinse

scanning

stained

pores

)Poretics

filters

experiments.

then

PBS-S

cells

in PBS.

rinsed

inserts

blocked

Dickinsonj

also

3-.tm

at 37#{176}Cin incubation

In some

pore

Becton assays.

albumin for 5

with

0.8-tm

For immunofluorescence

were

inserts

amniotic

nonmignating

microscopy.

tion

within

by spinning-disk

acquired

face

FL-

in PBS-S.

and

was

mained adherent or migrate. These

1 h in

BODIPY

(Sigma)

bs a 1-h rinse

were

proteinase.

rinsed

or rhodamine-phalloidin

followed

the

incubated

antibody

two

The 1 h in PBS-S

and

were

of secondary

mi-

rhodamine/SE) for

aim

erage series In addition, laser scanning plane. These

amnions

paraformaldehde.

permeablized

Primary

Probes)

with

(w/x)

lO%

above).

in PBS-S.

was

within to migrate

then

saponin.

by addition

phallicidin

focal

tg/mL

been

proteins allowed

1 h in 2% were

described

followed

in PBS.

for

amnion

antibody

were

had

fixed

contained

with

cells

amnions

then

cells inhibitor

cytoskeletal

first

was

antibody

(Chemicon).

grating

morphology

of neutrophils migrating in a 3-D amnionic to a chemoattnactant gradient of fMLP. these experiments, a total of 64 series

anti-a-actinin

antibody

isothiocvanate-goat

Our

doextracel-

antibody

anti-rabbit

Neutrophil

cytoplasmic integrin

secondary

secondary

polyclonal International.

anti-2

anti-rabbit

anti-rabbit

rabbit

Chemicon

anti-a5

Chemicon),

IL).

studies:

monoclonal

fluorescein-goat

fluorescein

To localize

the

polyclonal

(mAb1962;

Rockford.

.

in these (Ab1930;

mouse

fluorescein-donkey

ical

used

domain

Chemicon),

Sigma).

icon),

were

cytoplasmic

Temecula,

,

main

staining

migration of the

Neutrophil

D).

over longer chemoattractant

migration

Several

upward for at least the predominantly

within

times was gradient

cells

showed

pant of the horizontal

tnajectories observation movement,

downward in the direcas evidenced by the

a three-dimensional

matrix

191

D -

2

‘s_

C G)

a_-a.’-.

.

E

#{149}

I

.!

#{149}#{149}#{149}..;4-..’

a

‘

4

-

1,

S

S

x Fig.

1

(40

nM

.

Three-dimensional fMLP)

was

volume-rendering

in the

spherical,

with

a trailing

uropod.

Pseudopods

migration

through

amniotic

basement in minutes.

membrane, toward Panel D indicates

bered primarily

192

cells.

extension

lower

The in the

Journal

starting x-y

of

plane,

Leukocyte

chamber,

and

retraction are

of calcein-labeled which of short

continuously

membrane

primarily

was

neutrophils

downward

pseudopods.

(B)

extended

from

occurs

in a plane

the source ofchemoattractant. the location of the centroids

location with

of the net

Biology

cells,

displacement

Volume

in the

either

at t

61,

migrating

view

shown

=

February

within

human

(A)

Non-migrating

here.

Migrating

cells

leading

edge,

whereas

to the

basement

the

parallel

display

Numbered arrows in panel (circles) at 1 -mm intervals

downward

displacement

0 or upon in the

1997

entering

z-axis.

Bars:

the (A,

amnion,

a polarized none

are

extended

membrane.

(C,

by D)

10

en not

face.

The

polarize

with from

Net

is indicated

B) 5 tm,

do

morphology

C indicate the locations of eight cells from the field,

viewed cells

the

migration

an

active

uropod. occurs

chemoattractant

and

remain leading

(C,

roughly edge

and

D) Neutrophil

perpendicular

to the

ofindividual cells; times are indicated sequence shown in (C), including numan tm.

open

circle.

Note

that

motion

occurs

accumulation membrane [29,

over

31].

much of the

of cells into the amnion near the 20- to 60-mm incubation

However,

greater than chemoattractant

Fig.

1D).

the

speed

the

net velocity (by roughly

As discussed

related

to the

of individual

below,

orientation

the basement time (Fig. iD) neutrophils

was

vector in the direction one order of magnitude;

the

horizontal

of matrix

movement

was

fibnils.

Subcellular localization of proteins found in adhesion plaques On

2-D

to the

surfaces, substrate

neutrophil [9, 39],

leading

and

they

edges

contain

focal adhesion complexes in other cell distribution ofthese proteins (e.g. F-actin, teins,

and

integrmns)

could

indicate

adhere proteins

types actin

which

tightly found

[9, 40]. binding

neutnophil

in The pro-

struc-

tunes, including leading edges, pseudopods, and uropods, are involved in cell adhesion and motility in our 3-D system. To study the distribution of these proteins, cells were fixed during

fMLP-stimulated

migration

through

the

matrix

and

stained for proteins connecting the ECM to the cytoskeleton. In addition to a subcortical distribution (Fig. 2A), F-actin was enriched in three major structures: leading edges, pseudopods,

and

pods

were

uropods

(Fig.

identified

pseudopods that could

were identified be distinguished

of F-actin the uropod

staining stained

grating

through

a-Actinin edge and

2, A-D).

Leading

monphologically

as protrusions from leading

is consistent for F-actin amnion

(i.e.,

co-localized pseudopods,

edges

and

in well-polarized from edges.

the cell body The pattern

with that in 2-D except more intensely in cells Fig.

2,

B and

with F-actin but it was not

unocells;

that mi-

D).

in both the found in the

leading uropod

(Fig. 2, B and C). Immunostaining for the cytoplasmic domain of the integrin a5 revealed a distribution similar to that of a-actinin; a co-localized with F-actin in the leading edges and pseudopods of migrating cells, but was not found in the ilar

unopods

(Fig.

distribution

2D).

to that

Integrmn of a5

a5

(data

not

was

found

in a sim-

Fig.

2.

and

shown).

F-actin,

a-actinin,

pseudopods,

man

amnion

but

neutrophils can in the matrix

utilize

physical

(red) ing

To investigate the dynamic role of neutrophil structures in 3-D cell motility, moving images of neutrophils obtained by spinning disk confocal microscopy were examined closely for examples ofpseudopod extension during migration. While migrating

nent

ci-actinin

(green) rows)

to extend

with

respect

and

rowhead).

is

in the

cell

Bars:

5 .tm.

body

(A),

pseudopods (B, with but

by indirect

complex

from

uropods.

(D)

is excluded

im-

in leading

arrows),

adhesion

in the from

leading the

hu-

F-actin

is enriched

C, thin

C) The

F-actin

edges

within

microscopy.

and (A,

excluded

co-localized

viewed

confocal

distribution

D arrowheads). (green)

is found

gradient,

scanning

arrows),

and constriction rings structures. Anchored pseudopods as lateral protrusions

(Fig. 3). In addition to these anchored pseudopods, neutrophils occasionally were observed to extend a pseudopod that continued to expand as the cell transferred its mass into the growing structure (Fig. 4, A and B). The connection between the pseudopod and the original cell body formed a constriction ring that remained stationary relative to the matrix (Fig. 4, A and B). This process resembled a balloon being squeezed thnough a hole and expanding on the

remained forward

other

currence

side.

(B,

fMLP

in leading migrating

and

trail-

compo-

a

Integrin

edge

trailing

(thick uropod

ar(ar-

through

seen

the matrix, neutrophils were frequently lateral pseudopods that remained stationary to the matrix as the cell body migrated past

in a cortical

(A, B, D, thick uropods

laser

co-localize

of neutrophils

to an

and

is found

edges

integrins

uropods,

in response

munofluorescence

Migrating structures

and

not

As discussed

below,

these

anchored

anchored

et at.

Neutrophil

closely

were defined from polarized,

associated

with

pseudopods

were

of footholds

was

migration

within

seen

in eight

greatly

a three-dimensional

moved These

different

disk confocal microscopy, imaging of the matrix. probably

matrix

in 3-D moving images migrating cells that

fixed with respect to the matrix as the cell past them in three consecutive volume frames.

through the use of spinning did not permit simultaneous

pseudopods

Mandevitte

were

cells which The oc-

underestimated

matrix

193

Fig.

3.

using

Neutrophils

spinning

relative

to the

due our

use

footholds

disk

confocal

matrix

as

in the

the

cell

from

and

Constriction rings were nections between the main remained fixed with respect as exhibiting a constriction

During

fMLP-stimulated

surface-rendered,

migrates

to the angle of viewing imaging system.

appeared

matrix.

microscopy.

the

the

and left

to the

temporal

right

(>

by 3-D focal

reconstruction

transferred

of cells

microscopy.

currence

As for

from

by use

the

of constriction

of

defined as narrow annular concell body and a pseudopod that to the matrix. A cell was scored ring only if the mass of the cell

to be continuously

the cell

of spinning

scoring

rings

was

body period times

disk

of footholds,

con-

the

underestimated

oc-

To determine rings

opposed media

whether

were

anchored

in fact

to positionally [41]), simultaneous

was performed

pseudopods

associated fixed

and

constnic-

a5

Staina comin both

and density, could represent

that olution

of light

oriented When

parallel viewed

images

lowing

ing),

were

maximal

and

ascertain ing through

microscopy.

The

to the basement by laser scanning obtained time

some

to

as well as a diffuse interstitial a hydrated gel of fibnils below

from

resolution

adjustment

whether constriction openings in the

ofthe cells and that constriction

matrix rings

in the

ECM,

through

which

D). Constriction

rings

of fibrils

a single

optical

and minimal of the focal plane rings matrix.

the

staining the nes-

membrane. confocal microscopy,

cell

were

(Fig.

10 times

imaging method. In addition, the opening could also stretch

pseudopods

matrix,

into

the

pores

(footholds)

through

4,

by this

but

large

enough

membranes.

pores

were

those

on filters

(Fig. face

(n

B);

surface =

pores

38% (n

Biology

Volume

61.

February

1997

more 5).

containing efficiently

In addition,

were

versus

the

than mor-

highly

9%

polarized

on

on the smooth

sun-

94).

exhibited

along contact

The fibnils in the amniotic parallel to the basement

is lim-

membranes

(Fig.

of cells 107)

=

Neutrophils migrate matrix fibrils Neutrophils

a small

in the absence on adsorbed

migrating on these porous surfaces was more that of cells migrating on the smooth surface

A and

the porous

on

significantly

fibnils). oriented

Leukocyte

by stain-

to accommodate

plated

to migrate without

ofcells than 6,

Cells

able

ical, deformation The observation

of

as seen

pseudopod. Motility assays were performed of any added proteins either in the medium

amnion

Journal

structures

conditions in sized holes matrix struc-

F-actin (data not shown). Cells were the 3-.tm pores. 2 integrmn stained

through

194

these

Polycanbonate membranes were used to directly test the hypothesis that neutrophils could productively use anchored pseudopods to facilitate migration. Cell migration assays in

phology polarized

expansion the matrix

rings

since

appropriate geometric presence of appropriately sufficient density ofthese

nion present an elastic barrier to neutnophil migration that can be circumvented by mechanical, as opposed to chemconstriction

5 .tm.

cell surface diffusely, whereas F-actin, a-actinin, and integrin localized to the expanding portion of the cell, distal to the constriction ring (Fig. 4, F and G). In general, F-actin tended to localize near cell-substratum contacts.

to the

to accommodate the cell (Fig. 4E). This deformation of the matrix directly demonstrates the exertion of mechanical force by the cell on the ECM. Therefore, some regions ofthe am-

of the matrix. offootholds and

imaged stationary

to crawl

(al-

by squeezimaging

migrating

of the

were

remains

mov-

optical plane showed by narrow openings was

Bar:

and

response to a uniform application of 10 nM fMLP (chemokinetic stimulation) were performed on polycarbonate membnanes with or without O.8-.tm pores, too small for the cell

photobleachwas necessary

observed

in minutes.

architecture

neutrophils

is extended

were

section

were caused Simultaneous

through a single were produced

C and

dual cell/matrix of the cell through

majority

is indicated

by the

calcein-labeled (arrow)

albumin-coated filters with 0.8- and 3-rim pore diameters, respectively. Although cells could not crawl through the O.8-.tm pores, they did migrate across them, inserting lat-

the

microscopy.

caliber

amnion,

tunes to be utilized by cells during the observation period and sufficient rigidity of the matrix against which neutnophils can exert locomotive force). To model footholds and constriction rings in a more simplified system, we employed

(as

confocal

structures

ited

Time

the

pseudopod

can only exist under the amnion (i.e., the on gaps in the matrix,

pseudopods projecting into the imaging of the cells and matrix

scanning

matrix

field.

A lateral

ing for l2 integnn and able to squeeze through

ing of the amnion with rhodamine/SE demonstrated plex network of interconnecting fibrils that varied

ing

by laser

with

through

face.

enal

in these

studies.

tion

en

of the

resolution

the pseudopod during the entire observation 5 mm). Constriction rings were observed seven

into

migration

viewed

(i.e.,

cell

and

distort

guidance

attachment

to and membrane membrane,

during

migration

migration

along

are generally perpendicu-

Fig.

4.

phils.

Expansion

viewed

fling

disk

en

confocal

labeled

constriction

appear

microscopy.

the

matrix

(red).

the

matrix

as the

coated

distal

exhibiting

microscopy.

neutrophils

confocal

of pseudopods face.

PET

The cell

the

lan to the migrating

Image

(red(.

the

panel

contains

is migrating

across

the

hole

simulating acquired

by

line-scan

both

stain

gradient neutrophils

during

distal

that

using

of chemoattractant. revealed that

appears (E)

plane

laser

to the

scanning

constriction

Images

by

and

i.e.

ring

leading

Dynamic images of migration occurred pri-

edge.

to the

attractant.

pulled

80_90%

to

migrate

The dynamic vicinity

imaging

neutrophils were along visible fibers. leading edge is a highly of

extensions in a groping

of the associated active

matrix with

structure.

the cells contacted fashion. This kind

many

ofgroping

showed and and fibers

while

(G)

Same

as

(A.

B. C.

E) Time

for

of a few

tended through

this

it;

difficult

and

pull

F.

pulling

and

but

is indicated

by spin-

(arrows).

Calceinscanning

a hole

(arrowheads)

in

elastic

deformation

of

pore

in an albumin-

of migration).

Indirect

is distributed

through-

stained

for

in minutes.

for neutnophils been suggested

actin

(green)

Bars:

and

5 tm.

migrating on to contribute

details in the environment [20]. wrapped around a fiber and distortion

is readily

to appreciate

(green)

neutro-

imaged by laser

a 3-tm

direction

112 integrin

reported it has

micrometers

but

through

panel

matrix

Note

arrow,

were

obtained

through

microscopy.

filter;

and

plane

migrating

detection ofstructural Frequently, the lamellipods

that

in still

of fibers

apparent images.

oven

distances

in moving This type

images of grab

behavior was seen continuously during migration all (n > 50) neutnophils imaged by laser scanconfocal microscopy, often with several fibers being

of virtually

in the behavior

tlande,ilte

edge).

in the

optical

migrating

of the

has been previously grooved surfaces,

of calcein-labeled cells

openings

(green)

neutrophil

(leading

B) Examples

a single confocal

surfaces

microscopy.

the

manly parallel to the basement membrane (Fig. iC), but with net downward migration toward the source of chemoSimultaneous

scanning ofa

bottom

constriction confocal

ring.

from cell

laser

(A.

Volume-rendered

through

are

BODIPY-SM-labeled

top

force. amnion.

of pseudopods

(F) Cross-section

(arrowheads. distal

human

red.

obtained

transmigration.

locomotive

through

by expansion

neutrophil.

localizes

to generate

migration

caused

optical

ring

to the

be used

amnion

a single

(arrows(

(red(

are

a single

a constriction

integrin

which

rings

may during

rhodamine-labeled

each

expands

rings (arrows)

D( Constriction and

cell

of a

cell.

Q-actinin

rings

and

membrane.

immunofluorescence out

(C. green.

to constriction

ning

et at.

Neutrophil

migration

within

a three-dimensional

matrix

195

100

80

60

0

40

20

Fn Coated

Pores

No

Pores

Control

Fig.

5.

Neutrophils

ing

pores.

Cells

or

without

dishes

0.8-.tm

as a control.

counted

as motile.

each

condition

and

migrate were

were

more

plated

on

pores

or

on

Cells

able

to migrate

were

averaged

cells.

to be

significantly

on

membranes

data

than

7 tm

three

shown

different

filters

with

coverslip-bottom

more over

The

contain-

membrane

fibronectin-coated

Data

n > 150

found

efficiently polycarbonate

in 200

s were

experiments are

mean

from

each

and values

other

for

SE

±

by

t-test

(P < 0.05).

Fig.

6.

Cells

pores

have

face.

Cells

without and

pulled clung

toward the cell simultaneously. to the uropod ofthe migrating

ping back to its original In fields of heavy cell constant the

position density,

The matrix frequently neutrophil, finally snapafter the cell pulled the amniotic matrix

away. was in

Migration cell was

tortion

tended currently

to continue migrating,

by the grasping

Periodically, this tended pendicular parallel havior

along which

pseudopods

the fiber exhibited

than

did

a cell changed its direction to occur along an adjacent to the current fiber, rather

fiber (Fig. was observed

the

majority

the

observation

7, A and > 20 times

of cells

Germany) City,

did

parallel

fibers.

of migration, fiber that ran than switching

not change

and pento a

fibers

ized

morphology Bar:

fibers behind

tnacellular amnion, rhodamine shown).

In addition,

cells

were

matrix, and matrix density

noted

cells (data

to accumulate

in-

vesicles containing rhodamine, used to label the over the time course of migration even though the could not be detected in the media (data not These observations implied that neutnophils were

digesting the matrix intnacellularly.

during

migration,

either

extracellularly

on

196

Journal

of

Leukocyte

Biology

Volume

61.

a cooled

CCD

though

no

with

with

fMLP

proteins

(Leitz

(Dage-MTI

0.8-tm

sur(A)

or

fixed

immunofluorescence

Microscope camera

with

filters

stimulated Digital

0.8-.tm

a smooth

pores were

Wetzlar. Inc..

exhibited

present

Micha polar-

on

the

mem-

tim.

or generation the migrating

matrix. into

Fig.

We expected to observe pericellular proteolysis during neutrophil migration through the matrix because neutrophils express a number of pnoteinases, some of which are bound to the cell surface [42]. Confocal sections of cells within the matrix frequently revealed a slight penicellular halo of

not shown).

then Diavert

on membranes even

10

were

a Leitz

on

membrane

phalloidin.

with

containing those

However, time-lapse moving images evidence of active fiber degradation,

On

February

1997

the

pre-existing

weight

Extracellular proteolysis is not required for migration through amnion

than the surrounding found in areas oflower

Cells

than

of free cells.

contrary, areas

pnoteinase 8,

B and

did such

fiber ends, or cleaning cells

were

of low matrix

thermore, neutrophils were able the presence ofa high-concentration

during

time.

lower fluorescence were commonly

IN).

Cells

rhodamine with

membranes

polycarbonate

pores.

equipped

on

morphology

on

obtained

igan

migrate

on which less dis-

B). This fiber-switching bein 12 different cells, although

(n > 50)

plated

with

were

to

polarized

0.8-jim

stained

brane.

motion.

were

(B)

images

induced

a more

inhibitors

clearing of the often

density

any of

of tunnels penicellulan

seen (Fig.

to migrate cocktail

(see

not reveal as severing

to migrate 8A).

Fun-

into a matrix of low-molecular-

Materials

and

in

Methods;

C).

DISCUSSION Neutnophils

and

other

motile

animal

a complex, nescence

3-D matrix in vivo. microscopy to directly

behavior We

of neutnophils crawling found that many aspects

sue.

in a 3-D

matrix

are

functionally

cells

We have observe

migrate

through

used confocal the dynamic

fluomotile

through a biological tisof neutrophil migration similar

to those

on

a flat

surface but there are significant differences. The 3-D matnix can impede migration by presenting a physical barrier, such as the basement membrane [31], but neutnophils are also apparently capable of using the physical structure of the matrix to promote locomotion. The orientation of ma-

tnix fibers

was found

to be a major

range direction of cell tnix can be reversibly A central

theme

movement. deformed

to the

proposal

3-D matrix is different from that cells are able to use the architecture dition to specific ligand-neceptor force. niotic

determinant

Our direct observations matrix used in these

of the

that

motility

through

with

respect

amar-

unique pseudo-

relative to the substrate during inserted into footholds in the that contain a high density of

provide a variegated surface into which be extended. Movement of the anchored

rearward

a

a 2-D surface is that of a 3-D system in adinteractions to generate

support these ideas. The studies had a heterogeneous

pods that remain stationary cell translocation are indeed matrix. Areas of the matrix

ma-

on

chitectune in which a variety of motile cell behaviors to 3-D systems could be observed. We found that

fibnils could

short-

In some regions, the during cell migration.

to the

cell

body

will

pseudopods pseudopod propel

the

cell

forward with respect to the matrix, not unlike a climber using crevices to scale a wall. We also noted the expansion of pseudopods distal to constniction

which these

use

would

the

rigidity

too wide, tion non caliber

caused by narrow the cells migrated rings for locomotion,

rings,

through to utilize

be

dependent

of the provides a potential narrow

and

cannot migrate through, the opening, using the is somewhat

elastic,

to accommodate

of the

the

matrix

opening

of the

cell

can

is

to cell transmigraConversely, if the is rigid,

then

the

although it could migrate hole as a foothold. But if the

the

and

opening

distort

the

The

matrix

enough

observation

rings in amnion through a matrix

cell

across matrix of foot-

supports by use

the idea of simple

forces.

The idea that used to generate

these force

by pseudopods

is supported

choned

size caliber

an obstacle of traction.

transmigration.

holds and constriction that cells could migrate mechanical

the

If the

neither source

it

is too

on

matrix.

openings in the matrix (Fig. 4). If cells are able we speculate that such

two

footholds and for locomotion

constriction through

the

rings are amnion

additional findings. First, these ancontain integrins, F-actin, and a-actinin

(Fig. 2), a molecule that links integnins to the cytoskeleton [43]. Therefore, the lateral pseudopods contain appropniate machinery to support traction. Second, cells were observed Fig. otic through optical

7. Neutrophils matrix. Images

exhibit contact of calcein-labeled

rhodamine-labeled plane by use of laser

is indicated leading

in seconds. edges

while

fibril tended

s). The

cell

(arrowhead. to the

does was

not detach migrating

began fiber

migrating (arrowhead,

amnion scanning

Pseudopods uropods

Cell to the left of the of thin fibrils (arrow, 150

switched 300 upper at the upward

fibrils s).

left

The along

10

fibrils within amni(green) migrating

(red) were acquired confocal microscopy, were

exhibited

panel was 0 s) and

migrating switched

continuously

little

change

a second cell in the a diagonal

time

parallel fibril

to the left (arrowhead,

to the right after switching onto 70 s). In the last panel. the cell

(A)

horizontal panel 0 s),

exbut

of the panel 0 s), and

a second, horizontal again changes fibers The cell that entered along a fibril that is panel (arrow, 400 s).

tm.

Mandevitle

the

matrix

during

both

retrograde

and while squeezing such deformation could

motion

through only be

holes medi-

ated

from

to the sheet (arrowhead,

right of the (arrowhead.

to deform

of lateral pseudopods in the matrix, and

a single the time

extended

to a different

lower fibril

to head downward. (70 s) was migrating in focus in the last

from and

in morphology.

to the left, to a vertical

tail. (B) The neutrophil along the vertical fiber

(arrowhead, 400 s) and begins the field from the lower right visible with the slight change Bars:

guidance along neutrophils

et at.

by transmission of force from the cell to the matrix. Because amnion provides chemical as well as mechanical interactions, we could not separate these to demon-

strate a role for mechanical interactions in facilitating migration. However, the studies with synthetic membranes cleanly indicate that both lateral pseudopods inserted into matrix footholds and squeezing through constriction rings can allow forward migration in the absence of specific adhesion proteins. The enhanced migration on polycarbonate membranes containing 0.8-.tm pores (Fig. 5) is particulanly informative because the enhanced migration could only be attributed in the substrate,

Neutrophil

migration

to mechanical (not the pores providing

within

chemical) footholds

a three-dimensional

differences into which

matrix

197

Fig.

8.

induced (roscopy.

A cocktail

of proteinase

to migrate

into

(A)

the- pa1.

The

migrating

in minutes.

the cells presence

architecture. amniotic

ment

membrane

chemoattractant ance appears

Journal

bottom

right

Bars:

could insert of pores also

along

does

of the

through 10

panel

Leukocyte

neutrophil in the

migrates

presence

downward,

a pre-existing

hole

demonstrating

the

anchored the cells

in the

pseudopods. to assume

the amniotic both in terms

Much of the motion fibnils that were oriented and perpendicular to gradient. Thus, in our to override chemotactic

of

prevent (red)

migration distorting

matrix

distribution

into

amniotic

of proteinase the

(arrows).

matrix.

inhibitors matrix

Images

of neutrophils

and

as

it moves;

were

taken

after

30-mm

Calcein-labeled imaged

laser

cell

enters

another from

neutrophils

using

a single

focal

migration

through

cells

progressively

the

at the

Time

matrix

were

confocal

field

plane. the

(green)

scanning

mitop

of

is indicated

in the

presence

.tm.

lateral allowed

(Fig. 6). to its elasticity, structure,

not

amnions

C) Stereo-projections

inhibitors.

gated shape In addition a heterogeneous

198

at the

to the

(B and

of l)re)ttinase

and

cell

inhibitors

rhodamine-labeled

Biology

The an elon-

matrix also has of composition

of neutrophils was parallel to the basethe direction of the system contact guidguidance on a local

Volume

61,

February

1997

scale.

However,

with direction

tnix

the ing dicate impart

since

time, net migration of the chemoattractant

chemoattractant a localized

binds haptotactic

still

occurs gradient.

to the matrix, gradient. Our

that the influence of the a directional preference

move

into

the

ma-

downward in the It is possible that thereby genenatobservations in-

chemoattractant when cells

are

may be to switching

from one fibnil to another. We observed that, during fibril switching, cells pnefenentially migrate along fibnils exhibiting the least deformation

(i.e. the greatest

tension/rigidity).

Although

tify this behavior, it appeared ing the tension in the network along

the

fibnil

may

most

stable

fibers.

be

related

to

we did

as though of fibers, This

recent

the cells favoring

not quan-

ACKNOWLEDGMENTS

were testmigration

This

work

preference

for the

stiffer

grants

observations

with

beads

Medical

was

supported

GM34770 Scientist

Training

in an optical trap as they bind to integrins [441. When the strength of the trap was increased, the resistance of the beads to displacements increased, possibly due to in-

(J.

creased linking

and/or crosscomponents

tions, Drs. Richik Ghosh croscopy, and Dr. Satyajit

through along

labeling.

polymerization of actin of surface receptors to the

of adhesion complexes

complexes. would favor

high-tension

matrix

in contact cell

with

Increased extension

fibrils,

stable

migration

[45,

adhesion migration

and

and

surfaces

filaments cytoskeleton

stabilization can

such rigid,

(Fig.

needed cilitate

8A),

through which

the

direction

and

one

of

proteinases

are

required

amniotic

[31].

In

if

4. 5.

tissues, idea may

neutrophil

between

Neutrophil

tems.

However,

gradients matrix ofthe can

are additionally matrix, which override

cell

with

forces

2-D

and

similar migrating

10.

differ-

3-D

sun-

1 1.

in both syson a flat

chemoattractant or neutrophils migrating

12.

haptotactic through

guidance

architecture

on a local of the

matrix

scale

a physiological

3-D

matrix

(Fig.

provides

that can be used to generate to 3-D (Figs. 3 and 4). We propose

through

9.

at the

a

13.

1).

14.

the

I 5.

traction that cells

generate

16.

trac-

through

interactions,

mined

a combination of chemical and mechanical the relative contribution of which is deterby the ECM and the adhesive state of the cells. Our

help to define tion of cells in 3-D matrices. techniques may be directed observations

behaviors has been

1 7. 18.

questions unique to the migraFuture studies using similar toward the quantitation of 3-D

during migration through biological matrices done for cells crawling on 2-D surfaces [41,

47]. The methods we have described to the study of other 3-D migratory cell

on

footholds

unique

migrating tion

the

8.

influenced by the physical structure provides contact guidance (Fig. 7) that

chemotactic

Furthermore,

migration

on

7.

specialized regions basement membrane

polarization appears while neutrophils

rely primarily for orientation,

surface

6.

out in the absence of sepnoteases. It is likely that

studies, the cells accumulated basement membrane and did not penetrate it. In conclusion, we have found both similarities and faces.

3.

faof

of cells through not support this

breaching such as the

2.

enlarge

prevent that pro-

carried serum

1.

in the matrix could active degradation

in our

ences

grant

and

5-T32GM07367

Kim and members of Dr. Samfor help with amnion preparaand Ken Dunn for help with miMayor for help with fluorescent

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