The structure of freeze-fractured gap junctions was studied by electron microscopy and subsequent pattern analysis using a computer controlled image processing system. Rat mammary tumor cells (BICR/WIR-k) which are permanently coupled via gap junctions when cultered as monolayers were used under different fixed and unfixed conditions. Active (coupling competent) gap junctions seem to be characterized by loosely packed connexons, whereas non-active (permanently closed) gap junctions may consist of tightly packed particles

Blanz, Wolf-Ekkehard

Hülser, Dieter F.

Laub, Gerhard

Müller, A.

Online Publikationen der Universität Stuttgart

PATTERN ANAlYSIS OF GAP JUNCTION PLAQUES 
11TH OPEN AND CLOSED PORES 
..... + + 0 A. MuLLER, E.I. BLAHl , G. LAUD , D.F. H LSER 
Unlverlltlt Stuttgart, 'Blologlsches Inatltut, Abt.Blophysik 
und +Inttltut fOr Physlkallsche Elektronlk, Stuttgart 
Pllper read at the 4th International SYJIIPOsl1JII on BIIJ'HYSICS a= CELL SLRFACE, 
Heringscb-f, OOR, OCtober Xl - NoveIIIber 1, 1985 
The structure of freeze-fractured gap junctions was studied 
by electron .icroscopy and llubaequent pattern analysis using 
a co.puter controlled I.age procestlng syste., Rat .a •• ary 
tu.or cells EBfCoL~NoJkF which are per.anently coupled via 
gap junctions when cultered as .onolayers were used under 
different fixed and unfixed conditions. Active (coupling 
co.petent) gap Junctions see. to be characterized by loosely 
packed connexons, whereas non-active (per.anently closed) gap 
junctions .ay consist of tightly packed particles. 
Introduction 
Infor.atlon can be exchanged between cells by different 
.echanis.s such as hor.onal interaction, neuronal signal 
transfer or direct intercellular co •• unlcatlon. In e.bryonlc 
develop.ent, this direct Junctional cell to cell diffusion of 
a.all aoleculea Is the first Infor.atlon transfer syste.. In 
fully developed organis.s, direct Intercellular co •• unicatlon 
plays an I.portant role for regulation processes in organs 
and tissues whereas a disturbed co •• unication .ay cause 
.allgnant growth /6/. The .e.brane channels which enable the 
passage of .olecules fro. one cell to its attached neighbours 
are alwaya aggregated to plaques which .ay consist of up to 
several hundred individual Channels: the gap Junction. A 
single gap junction channel consists of two he_i-channels, 
the connexons which are provided by each of the co •• unicating 
186 stwdia biopkysica 1E!.. (1985) No. 1-3 
cella. Structural data provide evidence , that a connexon is a 
hexa.er of intra.e.branous proteins, the s o-called connexin s 
/ 4/. The gap junctional contact Is characterized by a 3 n. 
gap between adjacent cells. 
Under the electron .I croscope, gap junction plaques ca n be 
visualized either In thin sections of embedded cells or by 
Freeze fracturing the cells. In replicas of freeze-fractured 
gap Junctions of vertebrate cells the proteins re.aln In the 
p-face-Ieaflet of the Me.brane wher eas the corresponding pits 
are seen on the e-face-Ieaflet /3/ as is de.onstrated in 
FIG. 1. 
FIG.l creezeJf~actured gap junction between co nventi onal l y 
fixed BICR/MlR-k monolayer - cells with p-face ( left ) 
and e-face (r ight ) areas. Bar: 0.25 f1m 
Cells can be either In a coupled or in an uncoupled state , 
depending on functional activities. whi ch are regulated 
/11/ . The 
by 
C
•• 
pH , 8 , 
question 
cAMP 
wh ether 
or transjunctional 
the morph ologica l 
voltage 
appearance of gap 
junctions in freeze-fracture replicas can be correlated with 
the phYSiological state of the ce lls is still open . Several 
i nve s ti gat! on s dealing wi th this problem based on 
glutaraldehyde fixed ce lls / 1 ,2,7,8,9/ . 
studia biophysica 1!2 (1985) No. 1-3 
Our electrophysiological experiments showed. however. that 
glutaraldehyde rapidly uncouples intercellular communication. 
confir.ing results reported by Berdan and Coveney /1/ as well 
as by Spray et al. /10/. Therefore. we investigated whether 
glutaraldehyde fixation alters the connexon arrangement in 
gap junctions and whether their morphological appearance 
could be correlated with the coupling status of the cells. 
Materials and Methods 
Rat .a •• ary tu.or cells (BICR/MIR-k) which are per.anently 
coupled via gap junctions when cultured as mono layers have 
been investigated /5/. In order to gain information about the 
effect of glutaraldehyde we ~sed different preparation 
procedures for these cells before freeze fracturing thea 
at -150°C and at a vacuum of • 2 x 10-6 Torr in a Balzers 
8AF 301 instru.ent: 
- Conventionally fixed cells: 30 .in glutaraldehyde fixation. 
glycerInated for cryoprotection. frozen by freon dip. 
- Shortly fIxed cells: 5 .in glutaraldehyde fixation 
and i •• ediately frozen by freon dip. 
- Unfixed cells: frozen by propane jet. 
- UnfIxed cells: frozen by freon dip •. 
Fractured cells were replicated by platinum-carbon-
evaporation (45°. 2 n.) and carbon-evaporation (90°. 20 nm). 
Replicas were cleaned for up to 15 hours in sodiu. 
hypochloride and waShed 5 x in destilled water. Electron 
.icrograp~s were taken with a Zeiss EM 10 electron 
.Icroscope. 
For the analysis and quantitative evaluation of gap junction 
pictures obtarned with different preparations a co.puterized 
i.age processing.syste. was used (see TAB 1). The electron 
.icroscopical .icrograph negatives were digitized by an 
AID-converter, resulting In l.ages with a resolution of 256 
grey levels. The next operation .odes were framing of 
junctional areas and detection of centers fro. gap junctional 
particles or pits by thresholding, skeletonization and other 
188 studia biopkysica 1!£ (%985) No. %-3 
segmentation techniques. The data were recorded on tape and 
analyzed further on a VAX co.puter. The center-to-center 
spacings between all skeletonlzatlon points were .easured and 
classified by I n. stepa, but only spacings between 5 n. and 
14 nm were included for subsequent ,determination of frequency 
distributions and statistical analysis. These Dara.eters 
characterize the gap junction pattern in a co.puter-
applicable .ode. In add,ition nearest neighbour dJatances were 
determined fro. these collected data. 
determinstion of local grey level maxima 
I 
binary image by thresholding 
I 
ahrinkage of local maxima to single points 
I 
for.ation of a matrix containing all spacings between local 
.axima 
I 
formation of hiat~gra~ with spacings between 5 n. to 14 nm 
I 
statistical evaluation of histograms 
TAB. 1 Sche.e of image processIng procedure 
Results and DiscussIon 
In a'11 four preparation procedures junctional e-faces often 
exhibited a .ore regular particle pattern than p~facasI 
resulting in firat order spacings wIth more dIstInct peaks, 
a8 is shown in FIG. 2 for conventIonally fIxed cells. Our 
comparison of the different preparatIon methods revealed a 
ahift in the frequency diatribution to higher values when 
junctional particles were analyzed in cell preparatIons whIch 
were either unfIxed and frozen by propane jat or shortly 
stwdia biopkysica l!e (1985) No. 1-3 %89 
fixed and frozen by I •• erslon Into freon (FIG. 3). The .ean 
particle distance to nearest neighbour, however, was reduced 
In unfixed propane jet or shortly fixed preparations. 
Classification experl.ents based on .easured .tatl.tlcal 
para.eters according to the special features of the gap 
Junction appearances per.ltted a .ore detailed Inter-
pretation. The co.puter .e.orlzed distinct gap Junction 
classes (as defined by preparation procedure and fracture 
face of the analyzed replica) by the frequency distributions 
of their respective particles or pits. Thereafter, unkown gap 
junctions were attributed to the acquired cl •• s para8eter. 
(TAB. 2). Class A Includes p-face gap junctions of fixed and 
glycerinated cell preparations, class B accordingly prepared 
e-face gap Junctions. 
N/% 
ZO 
10 
r-
p-face 
N.99499 
~ 
~ 
r- ~~rJ-
r--
6 8 10 12 14 
particle dist. Ina 
N/% 
20 
10 
[ 
N • 46612 
-
--
r--
~ - ~~ 
r-
6 8 10 12 14 
particle dist. Ina 
FIG. 2 Frequency distribution of center to center spacings 
of gap junction particles (p-face) and pits (e-face) 
of conventionally treated BICR/MIR-k 8onolayer cells. 
Mean particle distance to nearest neighbour: 
6.85 n8 (p-face) a~d 6.43 n8 (e-face). 
studia biophysica 1!£ (1pBS} NO •• 1-3 
The relative unequivocal 
class B points at the 
reclassification 
fact that gap 
of class A 
junctions 
and 
froa 
conventionally treated preparations constitute a very 
homogeneous class. Interestingly, both preparations exhibit 
also some class F (loosely packed) gap junctions. However, 
p-face (class C) and e-face (class 0) gap junctions froa 
unfixed cells frozen by i.mersion into freon are more 
heterogeneous. Some of these gap junctions resemble those of 
fixed cells, whereas others are clearly different, with 
clustered or loosely arranged particles. Other examples for 
heterogeneity are class E which represents unfixed gap 
junctions frozen by propane jet and class F which consists of 
shortly fixed gap junctions frozen by immersion into freon. 
We conclude froG our electron micrriscoplcal investigations 
and computerized analysis of gap junction micrographs that 
glutaraldehyde fixation and glycerination alters the 
morphology of gap junctions. Unfixed cells frozen either by 
i.aersion into freon or by propane jet exhibit a 
heterogeneous appearance of gap junctions, whereas in 
conventionally fixed preparations mainly tight particle 
configurations are displayed. In addition, gap 
shortly fixed cells form clusters which may 
intermediate stage of 
glutaraldehyde fixation. 
the aggregation 
junctions 
represent 
following 
of 
an 
a 
The tight particle packing aay reflect the non-active state 
of the gap junction, when all pores are permanently closed. 
Active gap junctions are characterized by loosely packed 
particles, they may open or close their pores accordingly to 
functional necessities. Both active and non-active gap 
junction plaques can be present in BICR/MIR-k aonolayer cells 
under norGal eonditions but they are distinguishable only in 
shortly fixed or unfixed cell preparations. 
Up to now the question whether glutaraldehyde uncouples cells 
by direct action on gap junction proteins or by elevation of 
++ intracellular Ca is still open. Since glutaraldehyde 
causes a rapid uncoupling of cells, an investigation of open 
gap junctions is prevented by this fixation method. 
Glutaraldehyde fixation, however. entails an alteration of 
N/% 
20 
10 
10 
studia biopHysica 110 (lOBS) No. 1-3 
.... 
(a) 
r-
I-
--
, . I. 
N • 
p-face 
99499 
.... 
,..--
6 8 10 12 14 
pIIrtlcle dlst. Ira 
(c) p-f8Ce 
N. Z713J 
r-r-I--
r-
r- I-r-.... 
-,..-
6 8 10 12 14 
particle dlst. Ina 
N/% 
20 
10 
-
• 
N/% 
20 
10 
(b) p-face 
N = :n722 
r"'- -
r- -
,.... 
-
-
6 a 10 12 14 
pIIrtlcle dist. Ira 
(d) p-face 
N • 75084 
r-
r 
r-
r" 
I-
t-r" 
r 
r-
6 8 10 12 14 
partIcle dist. Ira 
FIG. 3 Frequency distributions of center to center spacings 
of gap Junction particles (p-face) of 8ICR/MIR-k 
.onolayer cells and .ean particle distance to 
neere.t neighbour. 
a) convent~onally fixed 
b) Ihortly' fixed 
c) unfixed propene Jet 
d) unfixed freon dip 
(6.85 n.) 
(6.43 n.) 
(6.52 n.) 
(6.82 n.) 
101 
studia biophysica !!2 (I98S) No. I-3 
gap junctional protein arrangement only after a 
( > 30 min) period , therefore , shortly (5 min) fixed 
preparations are still very helpful in distinguishing 
from non-active gap junction plaques. 
defined computer classified class 
class 
A B C D E F 
A 36 1 0 0 0 4 
B 2 24 0 0 0 5 
C 11 4 16 0 0 5 
D 0 2 1 0 0 1 
E 1 0 1 0 0 5 
F 7 0 1 0 0 16 
longer 
cell 
active 
TAB. 2 Classification of different gap junction preparations 
References 
/1/ 
/2/ 
/3/ 
Berdan, R.C., Caveney S.; Cell Tiss . Res. ~ (1985) 111 
Dahl, G., Isenberg, G.; J. MeMbrane Bioi. ~ (1980) 63 
Gilula, N.B.; Intercellular junctions and synapses. 
J. Feldman. N.B. Gilula, J .D. Pitts; ChapMan and Hall, 
London 1978 
/4/ Henderson, D., Eibl, H., Weber, K.; J. Mol. BioI. 132 
(1979) 193 
/5/ Hulser D.F., Demsey, A.; Z. Naturforsch. ~ (1973) 603 
/6/ Loewenstein, W.R.; Physiol. Rev. 21 (1981) 829 
/7/ Meda, P., Findlay, I., Kolod, E., Orci, L. 
Peterson, O.H.; J. Ultrastruct . Res. aa (1983) 69 
/8/ Page, E., Karrison, T. , Opshaw-Earley, J . ; 
Am. J. Physiol. ~ (1983) H525 
/9/ Peracchia, C.; J. Cell BioI . ~ (1977) 628 
/10/ Spray D.C., Harris, A.L., Bennett, M.V.L.; 
Biophys. J. ~ (1981) 108a 
/11/ Spray, D.C., Bennett, M.V.L., Ann. Rev. Physiol. ~ 
(1985) 281 
Prof. D.F. HilLSER, Department of Biophysics, Biological Instit 
University of stuttgart, Ulmerstr. 22.7, 0-7000 stuttgart-60, 


Pattern analysis of gap junction plaques with open and closed pores

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Pattern analysis of gap junction plaques with open and closed pores

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