-
Wild type and tailless CD8 display similar interaction with
microfilaments
during capping
PASCALE ANDRE1, JEAN GABERT2'*, ANNE MARIE BENOLIEL1, CHRISTIAN
CAPO1, CLAUDE
BOYER2, ANNE MARIE SCHMITT-VERHULST2, BERNARD MALISSEN2 and
PIERRE BONGRAND1
1Laboratoire d'Immunologie, Hdpital de Sainte-Marguerite, BP 29,
13277 Marseille Cedex 09, France2Centre d'Immunologie de Marseille
Luminy, Case 906, 13288 Marseille Cedex 9, France
* Present address: Institut Paoli-Calmettes, BP 156, 13273
Marseille Cedex 9, France
Summary
We examined the influence of the intracytoplasmicregion of CD8a
on capping and interaction withmicrofilaments. We used cell clones
obtained bytransfecting a CD4+ T-cell hybridoma with (a)
T-cellreceptor (TCR) a and fi chains from a cytolytic cloneand (b)
CD8« genes that were either native ormodified by extensive deletion
of the intracytoplas-mic region or replacement of the transmembrane
andintracytoplasmic domains with those of a class Imajor
histocompatibility complex gene (Letourneuret al. (1990). Proc.
natn. Acad. Sci. U.S.A. 87,2339—2343). Different cell surface
structures werecross-linked with anti-T-cell receptor, anti-CD8
oranti-class I monoclonal antibodies and anti-immuno-globulin
(Fab')2. Double labeling and quantitativeimage analysis were
combined to monitor fluor-escence anisotropy and correlation
between differ-ent markers. Microfilaments displayed
maximalpolarization within two minutes. The correlation
between these structures and surface markers wasthen maximal and
started decreasing, whereas theredistribution of surface markers
remained stable orcontinued. Furthermore, wild type and altered
CD8«exhibited similar ability to be capped and to induceco-capping
of TCR and MHC (major histocompati-bility complex) class I: the
fraction of cell surfacelabel redistributed into a localized cap
rangedbetween 40% and 80%. Finally, cytochalasin Ddramatically
decreased CD8 capping in all testedclones.
It is concluded that the transmembrane and/orintracellular
domains of CD8 molecules are able todrive the extensive
redistributions of membranestructures and cytoskeletal elements
that are trig-gered by CD8 cross-linking.
Key words: membrane, molecular motion, cytoplasmic
domain,cytoskeleton, CD8.
Introduction
Since the elaboration of the fluid mosaic model of theplasma
membrane by Singer and Nicolson (1972), it hasbecome clear that
many cell functions are affected bylateral movements of membrane
molecules. Cell adhesionis thus highly dependent on the lateral
mobility of ligandmolecules (Rutishauser and Sachs, 1974). Clearly,
theformation of strong intercellular bonds may require thatligand
molecules be concentrated in adhesion areas (Bellet al. 1984), and
this concentration was indeed observed inseveral experimental
studies using different method-ologies such as electron microscopy
(Singer, 1975), micro-fluorometry (Poo and McCloskey, 1986) and
conventional(Kupfer and Singer, 1989) or computer-assisted (Andre
etal. 1990a,b) fluorescence microscopy. Also, the microaggre-gation
of cell surface receptors was found to enhance theiradhesive
capacity (Detmers et al. 1987).
However, the mechanisms of molecular motion in theplasma
membrane remain incompletely understood.When the mobility of cell
surface proteins was studiedwith the fluorescence photobleaching
recovery (Schless-inger et al. 1976) or post electric field
relaxation (Poo et al.1978) method, the diffusion coefficient of
concanavalin AJournal of Cell Science 100, 329-337 (1991)Printed in
Great Britain © The Company of Biologists Limited 1991
'receptors' (Schlessinger et al. 1976; Poo et al. 1978; Smithet
al. 1979), class I histocompatibility molecules (Edidinand Zuniga,
1984; Bierer et al. 1987; Wier and Edidin,1986) or lymphocyte
surface immunoglobulins (Dragstenet al. 1979; Barisas, 1984) varied
between less than 10~12
and about 2xlO~9cm2s~1. The latter value was roughlyconsistent
with theoretical estimates for free diffusion in athick viscous
sheet (Wiegel, 1984). Thus, it appearedreasonable to view the
motion of cell surface proteins as ahindered-diffusion process. In
addition, these proteins mayundergo active displacements as
exemplified by thecapping process (Taylor et al. 1971; Bourguignon
andBourguignon, 1984).
Strong experimental evidence suggests that interac-tions between
cytoskeletal elements and membraneintrinsic proteins are dynamic
events (Flanagan andKoch, 1978; Burn et al. 1988) that may hinder
randomdiffusion (Koppel et al. 1981; Tank et al. 1982).
Further,active movements may be affected by cytoskeletal
inhibi-tors (Taylor et al. 1971; Bourguignon and Bourguignon,1984).
However, the precise molecular interactions in-volved in these
phenomena remain incompletely under-stood and it is not well known
whether cytoskeletalelements are bound to many membrane proteins
or
329
-
whether a few membrane molecular species mediateindirect
interactions between cytoskeleton and membranestructures, as
previously suggested (Bourguignon andSinger, 1977).
Recently, several authors used mutated membraneproteins to
assess the influence of cytoplasmic domains onlateral diffusion.
Thus, deletion of the intracellulardomains of class I
histocompatibility molecules (Edidinand Zuniga, 1984), epidermal
growth factor receptor(Livneh et al. 1986) or viral proteins
(Scullion et al. 1987)did not change lateral diffusion, whereas a
similarmutation resulted in tenfold increase of the
diffusioncoefficient of MHC class II molecules (Wade et al.
1989).
The present work was planned to evaluate the influenceof the
cytoplasmic domain of membrane proteins on theiractive movements
and interactions with cytoskeletalelements and membrane proteins.
We used a CD4+ T-cellhybridoma transfected with CD8cx genes bearing
alteredcytoplasmic domains (Letourneur et al. 1990). CD8molecules
were capped with specific antibodies and theredistribution of CD8,
T-cell receptor (TCR) and MHCclass I molecules as well as
microfilaments was studied bycombination of double labeling and
image analysis. It isconcluded that the extracellular and/or
transmembranedomains of CD8 molecules are sufficient to mediate
theextensive interactions observed between these moleculesand
microfilaments or membrane proteins.
Materials and methods
CellsKB5C20 (Albert et al. 1982) is an alloreactive
H-2Kb-specificcytotoxic T lymphocyte (CTL) clone of B10.BR origin
(H-2k). ACD4+ anti-ovalbumin T-cell hybridoma (Marrack et al. 1983)
wastransfected with a- and /3 chain genes encoding KB5C20
T-cellreceptor (Letourneur et al. 1990) and (i) a CD8
-
derivatized streptavidin. The intensity of biotin-associated
label-ing was 7 % and 6 % of that obtained with fluorescent (Fab')2
whenboth fluorophore combinations (fluorescein/rhodamine
andrhodamine/fluorescein) were used.
Although this weak artefactual labeling was inapparent onvisual
examination of fluorescent cells, it was important to knowwhether
this could significantly alter the correlation between
thefluorescence distributions corresponding to different antigens
ondoubly labeled cells. This was tested by computer simulation:
100double fluorescence distributions were randomly generated.
Themean standard deviation of sector relative fluorescences was
0.28.The mean normalized correlation (V3/2xln[(l+r)/(l-r)] wasthen
calculated assuming 0 %, 5 %, 10 % and 15 % contaminationof each
fluorescence by the other one. We obtained 0.09, 0.29, 0.51and
0.78, respectively. It was concluded that the
correlationcoefficients measured on doubly labeled cells (see
Tables) couldnot be ascribed to artefactual overlap between both
labels.
Flow cytometryThe mean fluorescence of different populations of
labeled cells wasdetermined with a Profile flow cytometer (Coulter,
Hialeah, FL).
Results
Tailless CD8 molecules can be capped and interact
withmicrofilamentsIn a first series of experiments, we compared the
cappingproperties of wild-type and engineered CD8 molecules.
Typical fluorescence images are shown in Fig. 1. Cellswith
limited asymmetry of CD8 are displayed inFig. 1 A,C. A typical
redistribution of labeled molecules isshown in Fig. IE: in this
case, sector analysis shows thatthe mean CD8 fluorescence of the
right sector is about 3times higher than that of left CD8-depleted
sectors.
There was a need for quantitative parameters tomonitor CD8
capping. The standard deviation of therelative fluorescence of the
six sectors defined in testedcells (Fig. 1B,D,F) seemed a suitable
choice: this was 0.45on the capped cell (Fig. 1E,F) as compared
with 0.17 and0.13 of the two other cells (Fig. 1A,C).
The possible occurrence of a relationship between CD8and
microfilament distributions was studied by calculat-ing the
correlation coefficient between sector relativefluorescences on
doubly labeled cells. This is 0.97 on thecell shown in Fig. 2,
which is indeed indicative of a highlysignificant corrrelation
(P=0.001). Since the statisticaldistribution of the correlation
coefficient r in a randompopulation is expected to be strongly
non-Gaussian, weused its normalized transform (i.e.
V3/2xln[(l+r)/(l-r)])in order to allow simple averaging of
parametersmeasured on individual cells. This parameter was 3.62
onthe cells shown in Fig. 2. A value higher than 2 may beconsidered
as significantly different from zero at the 0.05significance
level.
The kinetics of fluorescence redistribution triggered
bycross-linking normal or engineered CD8 molecules isshown in Fig.
3. The anisotropy of CD8 and cytoskeletalfluorescences was nearly
maximal within 2-5 min. Wildtype and modified CD8 molecules
exhibited markedredistribution with comparable kinetics. However,
themaximum standard deviation of CD8 relative fluor-escences was
higher on DC41.1.4 clone bearing native CD8(0.87±0.06 S.E.) than on
DC142 clone with chimeric CD8(0.61+0.07) and DC 136 clone with
tailless CD8(0.52±0.07).
Also, with all tested cells, cross-linking CD8 induced amarked
redistribution of cytoskeletal elements, with amaximal correlation
between both fluorescence distri-
Fig. 1. Principle of sector analysis. Cells from the DC
41.1.4clone were labeled with anti-CD8 monoclonal antibodies,
thenexposed for 30 min at 4°C (A-C) or 37 °C (E-F) to
rhodamine-labeled anti-mouse immunoglobulin (Fab')2. Three
typicalfluorescence images are shown (A, C, E). These images
weredivided into six sectors of angle fc/3 and the
relativefluorescence of each sector is shown (B, D, F). The
asymmetryof each fluorescence distribution was quantitated
bycalculating the standard deviation of sector
fluorescences,yielding 0.17 (B), 0.13 (C) and 0.45 (F). The latter
value maybe considered as indicative of capping. Bar, 5 //m.
butions of 2.10±0.14 (DC142 clone) and 2.22±0.30 (DC136clone).
Hence, no alteration of the ability to interact withcytoskeletal
elements was detected in tailless CD8molecules.
Another finding was that the correlation between CD8and
microfilament distributions was nonzero in theabsence of any
cross-linking of surface molecules. Thismay be ascribed to
asymmetrical morphology (i.e. devi-ation from a spherical shape)
and permanent anisotropy(Andre et al. 1990a). This emphasizes the
interest ofquantitative methods of fluorescence analysis.
In order to assess the selectivity of cytoskeletal
proteinredistribution, we looked for a possible reorganization
oftubulin molecules after CD8 capping: as shown on Table 1,CD8
cross-linking induced no substantial redistribution of
CDS cytoplasmic domain and capping 331
-
B
Fig. 2. Principle of correlation studies. Cells from the
DC41.1.4 clone were labeled with an anti-CD8 monoclonal antibody,
thenexposed for 30min at 37 °C to rhodamine-labeled
anti-immunoglobulin (Fab')2 and fixed before cytoskeleton labeling
withNBD-phallacidin. The distribution of CD8 (A and B) and
NBD-phallacidin (C and D) was studied by quantitative
fluorescencemicroscopy. Cell images were divided into six sectors
of angle x/Z and the relative fluorescence of each sector was
calculated. Theasymmetry of each fluorescence distribution was
monitored by calculating the standard deviation of sector
fluorescences, yielding0.50 (A,B) and 0.25 (C,D). The correlation
between two fluorescence distributions was evaluated by calculating
the correlationcoefficient r between sector fluorescences and using
the normalized coefficient V3/2xln[(l + r)/(l-r)].
tubulin molecules, and no significant correlation wasfound
between CD8 and tubulin markers.
The capping of wild type and modified CD8 molecules isdependent
on microfilament integrityIt was important to determine to what
extent cytoskeletalreorganization was a cause or a consequence of
the cappingof normal and altered CD8 molecules. This point
wasaddressed by studying the influence of cytochalasin D, a
fairly selective inhibitor of actin polymerization
(Cooper,1987), on the capping of CD8. As shown in Table 2,
5minafter the onset of capping, cytochalasin D-treated
cellsdisplayed 3-4-fold decrease of the CD8 capping parameterin all
tested clones. Thus it was concluded that the cellcytoskeleton
plays an active role in CD8 redistribution.
In order to further explore the involvement of microfila-ments
in the capping process cells were labeled withNBD-phallacidin at
various times after the onset ofcapping. Mean cell fluorescence was
assayed with flow
332 P. Andre et al.
-
Correlation3-
0.5-
0 10
B S.D.
1-
0.5-
20 Time (min) 0
Correlation3-
10 20
1-
0 10
0.5
2 0 Time (min) °Correlation
3"
10 20
10 2 0 Time (min) ° 10 20
Fig. 3. Capping of CD8. Cells expressing intact CD8
(CD41.1.4clone, A) or CD8 modified by replacement of
intramembraneand intracytoplasmic residues with those of MHC class
I(DC142 clone, B) or by extensive deletion of
intracytoplasmicdomains (DC136 clone, C) were exposed to an
anti-CD8monoclonal and anti-immunoglobulin (Fab')2. They were
thenfixed at regular intervals and processed for
quantitativeanalysis of the distribution of CD8 and microfilaments.
Thestandard deviation of relative sector fluorescences of CD8
label(full lines) and microbilament stain (broken lines) is shown
onleftwards plots. The normalized correlation between both labelsis
shown on rightward plots. Each point represents a mean of10-30
determinations. Vertical bar length is twice thestandard error.
Table 1. Absence of substantial redistribution of
tubulinmolecules after cross-linking CD8
Cell treatment Control Ten minute capping
Standard deviation of sectorfluorescence
CD8Tubulin
Correlation between markers
0.13±0.014 (9)0.068±0.004 (9)0.66±0.18 (9)
0.55±0.03 (20)0.10±0.008 (20)
l.l±0.31 (20)
Cells from the DC41.1.4 clone were exposed to anti-CD8
monoclonalantibody and rhodamine-labeled anti-mouse immunoglobulin
(Fab')2 at4°C. They were then fixed immediately (control) or after
10minincubation at 37°C (capped cells) and stained for tubulin
withNBD-colcemid. The fluorescence distribution was determined for
bothmarkers and standard deviations of sector fluorescences were
calculatedas well as the correlation between both markers. Mean
values areshown±standard error of the mean. Number of studied cells
inparenthesis.
Table 2. Effect of cytochalasin D on the redistribution
ofmicrofilaments and CD8 induced by CD8 cross-linking
Clone
DC41.1.4
DC142
DC136
Cytochalasin D
05/igml"1
05/igmr1
05/igml"1
Standard deviation ofsector fluorescence
Phallacidin
0.19±0.012 (60)0.18+0.014 (61)0.25±0.013 (57)0.17±0.019
(59)0.23±0.018 (37)0.16±0.016 (38)
CD8
0.80±0.039 (60)0.26±0.016 (61)0.79±0.034 (57)0.20±0.011 (59)0.91
±0.056 (37)0.23±0.019 (38)
Cells from the DC41.1.4 clone (expressing wild-type CD8ff), or
DC142(with hybrid CD8 bearing transmembrane and
intracytoplasmicdomains of MHC class I molecules) or DC136 (with
tailless CD8ff) wereexposed to anti-CD8 monoclonal antibodies and
fluorescent anti-immunoglobulin (Fab')2> then incubated for 5min
at 37°C with orwithout S^igml"1 cytochalasin D. They were then
assayed withfluorescence microscopy for quantitative determination
of the anisotropyof CD8 and cytoskeleton labeling. Five separate
experiments wereperformed and each value shown as a mean±standard
error of themean. The number of examined cells is shown in
parenthesis.
Table 3. Effect of capping on the microfilament
contentofDC41.1.4 cells
Mean relativefluorescence
Standard error
0
1.0
1
1.02
0.03
Time
2
1.08
0.03
(min)
5
1.12
0.06
10
1.13
0.06
30
0.94
0.08
Cells were exposed at 4°C to anti-CD8 monoclonal antibodies
andanti-mouse immunoglobulins. They were then warmed to 37 °C
andincubated for different periods of time before being fixed and
stainedwith NBD-phallacidin for determination of total fluorescence
with aflow cytometer. Values shown are means of 8 separate
experiments.
cytometry and quantitative fluorescence microscopy. Asshown in
Table 3, the former method suggested theoccurrence of a transient
and moderate increase of actinpolymerization following capping
induction. Fluorescencemicroscopy did not reveal any modification
of phallacidinstaining during the studied process (data not shown),
dueto the lower size of assayed cell populations and sub-sequent
higher dispersion of results.
Intact and modified CD8 molecules display similar
co-redistribution with other cell membrane proteinsThe simplest
explanation for the ability of tailless CD8molecules to interact
with cytoskeletal elements was thatthis association was mediated by
other cell membranemolecules that would interact with CD8 through
theirextracellular or intrabilayer domains and with microfila-ments
through their intracytoplasmic region in accord-ance with a model
suggested by Bourguignon and Singer(1977). The possibility of
extensive interactions betweenCD8 and other membrane molecules was
tested bytreating CD8 positive clones with anti-CD8 and studyingthe
redistribution of the TCR and MHC class I molecules.As shown in
Tables 4 and 5, a close correlation was foundbetween the membrane
distribution of CD8, TCR andMHC class I in all tested clones,
supporting the possibilitythat CD8 might drag cell surface
glycoproteins towards acell pole during the capping process, thus
allowing indirectinteractions between CD8 and microfilaments.
CD8 cytoplasmic domain and capping 333
-
Table 4. Co-capping of MHC class I molecules by anti-CD8
antibodies
Clone
DC41.1.4 (n=23)DC142 (n=14)DC136(n=14)
Standard deviation ofsector fluorescence
CD8
1.02±0.070.58±0.090.66±0.09
MHC class I
0.34±0.030.23±0.040.41±0.06
Correlationbetweenmarkers
1.56±0.301.85±0.503.23±0.34
Cells were treated with anti-CD8 monoclonal antibodies and
exposedfor 20min at 37 °C to fluorescein-conjugated
anti-murineimmunoglobulin (Fab')2- They were then cooled and
labeled withbiotinylated anti-MHC class I antibodies and
rhodamine-conjugatedstreptavidin. They were then examined with
quantitative fluorescencemicroscopy for determination of
fluorescence asymmetry (as expressedby the standard deviation of
sector fluorescence) and correlationbetween both antigens (as
expressed with normalized correlation). Meanvalues are shown ±
standard error. Number of analyzed cells inparenthesis.
Table 5. Co-capping of T cell receptor by anti-CD8antibodies
Standard deviation ofsector fluorescence
Clone CD8 T cell receptor
Correlationbetweenmarkers
DC41.1.4 (n=20)DC136(n=14)
0.60 ±0.060.76±0.04
0.58±0.050.34±0.02
3.75±0.282.83±0.16
Cells were treated with anti-CD8 monoclonal antibodies and
exposedfor 20min at 37 °C to fluorescein-conjugated
anti-immunoglobulin(Fab')2- They were then cooled and labeled with
biotinylated anti-T cellreceptor antibodies and
rhodamine-derivatized streptavidin. They werethen examined with
quantitative fluorescence microscopy fordetermination of
fluorescence asymmetry (as expressed by the standarddeviation of
sector fluorescence) and correlation between markers (asexpressed
with normalized correlation). Mean values are shown±standard error.
Number of analyzed cells in parenthesis.
Capping may involve an early microfilament-dependentand a late
micro filament-independent stage
As shown in Fig. 3A,B,C, the correlation between cyto-skeleton
and CD8 distributions was maximum withinabout 2 min after exposure
to the capping stimulus. Thiscorrelation then decreased, whereas
the anisotropy offluorescence distributions kept on increasing.
This sugges-ted that CD8 capping involved some kind of
associationbetween extra- and intra-cellular structures only
duringthe earliest phase of the capping process. Indeed,10-30 min
after the triggering of redistribution, weobserved cells displaying
redistributed CD8 and microfila-ment markers, with a different
intracellular localization(Fig. 4). It was of interest to know
whether these findingsapplied to other cell surface markers, in
addition to CD8.As shown in Fig. 5, when anti-TCR or anti-MHC class
Iantibodies were used to induce capping, a co-redistributionof cell
surface markers and microfilaments was also found,and the
correlation between extra- and intra-cellularmarkers was maximum
within two minutes, then de-creased, as was found with CD8.
Discussion
The purpose of our work was to assess the role of
theintracellular domains of membrane molecules on theactive
movements of these structures. Capping was chosenas a suitable
model for this study.
1-
0.5
Correlation31
S.D.
1-
10 2 0 Time (min)0
Correlation
10 20
0.51-
10 20 Time (min) 10 20
Fig. 5. Capping of TCR and MHC class I. Cells from theDC41.1.4
clone were exposed to an anti-TCR (A) or anti-MHCclass I (B)
monoclonal antibody and anti-immunoglobulin(Fab')2. They were
incubated at 37°C for various periods oftime, then processed for
quantitative analysis of thedistribution of TCR or MHC and
polymerized actin. Thestandard deviation of relative sector
fluorescences for TCR orMHC (full lines) or microfilament (broken
lines) is shown onleft plots. The correlation between both markers
is shown inthe righthand plots. Each point represents a mean of
20-30determinations. Vertical bar length is twice the standard
error.
A first requirement was to achieve a quanti tat ivedescription
of the tested phenomenon. The sector analysiswe performed was felt
a satisfactory way of monitoring thecapping process since in most
cases the value of thestandard deviation parameter matched the
intuitivefeeling of the extent of antigen redistribution provided
byvisual examination of fluorescence images (Fig. 1).
A second conclusion made clear by quanti tat ive fluor-escence
determinations is tha t only a limited fraction ofcross-linked cell
surface molecules were capped. Indeed,using the derivation shown in
the Appendix, the fractionof capped molecules was found to range
between 40 % and80% of total label. Obviously, it is difficult to
evaluatefluorescence intensities using a grey scale (Figs 1, 2).
Asshown in Fig. 4, a coded color display gives a much moreprecise
description of the distributions of light intensities.
Now, the most clearcut conclusion of our experiments istha t
wild type and altered CD8 chains were all able to beredistributed
in response to a cross-linking stimulus, andthey induced similar
redistribution of polymerized actin(Fig. 3). The simplest
interpretation for these findings isthat the redistribution of
cytoskeletal elements as well asTCR or MHC class I molecules
induced by bridging'tailless' CD8 on DC 136 clone was mediated by
molecularinteractions occurring in the bilayer or the
extracellularregion. This might involve some third-party structure,
inaccordance with a model previously reported by Bourguig-non and
Singer (1977). Direct CD8-cytoskeleton interac-tion did not play a
quantitatively significant role in thisrespect. This conclusion is
consistent with the finding that
334 P. Andre et al.
-
B
Fig. 4. Coded color display of immunofluorescence distributions.
Cells from the CD41.1.4 clone were exposed to anti-CD8monoclonal
antibodies and anti-immunoglobulin (Fab')2 for 5min (A and B) or
30min (C and D) at 37°C before fixation andstaining for
quantitative analysis of the distribution of CD8 (A and C) and
polymerized actin (B and D). Fluorescence intensitieswere
represented with coded color display using a 16-level scale. The
normalized correlation between both fluorescencedistributions is
0.81 (A and B) and -0.42 (C and D).
-
complement decay-accelerating factor, a lipid-anchoredmembrane
protein, could be made to cap and interact withcytoskeletal
elements as efficiently as CD3, CD4 or CD8(Kammer et al. 1988). It
must be emphasized that specificintermolecular associations may not
be required tomediate interactions between microfilaments and
cellsurface proteins, since a bulk displacement of a givenmolecule
may generate a lipid flow in the viscous bilayeror drive
extracellular glycocalyx elements entangled withthe oligosaccharide
chains of moving molecules.
Another finding is that capping CD8 molecules induceda
significant co-redistribution of TCR (Table 5) and, to alesser
extent, MHC class I (Table 4) molecules. It must bepointed out that
this co-redistribution was sometimeslimited (see 1st row of Table 4
and 2nd row of Table 5).Perhaps co-capping is not an all-or-none
phenomenon, asmight be suggested by qualitative observation. This
viewis not at all inconsistent with the finding that
antibody-induced co-capping of different cell membrane antigensmay
be dependent on the affinity and specificity of cappingantibodies
(Kupfer and Singer, 1988).
A final point is to understand the importance of
activecytoskeletal movements in the redistribution process. Asshown
on Table 2, the capping of CD8 measured 5 minafter exposure to
cross-linking antibodies was drasticallydecreased by cytochalasin
D, suggesting an involvement ofmicrofilaments in the first steps of
capping. Interestingly,capping-associated cytoskeleton
redistribution was quan-titatively less important and less
sensitive to cytochalasinD than that of surface molecules (Table
2). Two non-exclusive explanations may be offered for this
finding.First, only a minor part of cell microfilaments might
beinvolved in capping, as compared to the fraction of CD8molecules
affected by this process. Second, cytochalasinreduction of
microfilament length might at the same timeincrease their capacity
to rearrange and decrease theirability to drag cell surface
structures, which would beconsistent with a minimal overall effect
of cytochalasintreatment on microfilament distribution.
Since the redistribution of surface antigens went onafter the
peak of microfilament reorientation (Fig. 3)without any concomitant
increase of the correlationbetween extra- and intra-cytoplasmic
structures (Fig. 1),the simplest hypothesis would be that the
clustering ofstudied surface molecules was completed through
thermaldiffusion. This is consistent with quantitative
estimatesbased on known values of the diffusion constant ofmembrane
glycoproteins. As estimated by Berg andPurcell (1977), the mean
time required for a molecule withdiffusion constant D to be trapped
on a spherical cell ofradius a by a disc of radius s (representing
the cap) isabout:
t= 1.1 a2/'Dln(1.2 a2/d2).
Using 5xlO~10cm2s~1 as an estimate for the diffusioncoefficient
of the most mobile fraction of cell surfacemolecules (Dragsten et
al. 1979), 5,um for a and ljun for s,we find:
t = 30 min.
Hence, it seems reasonable to suggest that the reorgan-ization
of cell surface antigens was triggered by acytochalasin
D-inhibitable motile mechanism. This wouldresult in the formation
of a localized molecular clusterwithin a few minutes. Free
molecules might then gettrapped within this cluster when
encountering it onrandom diffusion. The decrease of the correlation
between
cell surface molecules and cytoskeletal elements observed5 min
after the onset of capping may be ascribed toindependent drift of
extracellular submembranar struc-tures. Hence, the main role of the
intracytoplasmic tail ofmembrane proteins such as CD8 might be
related totransmembrane signalling (Zamoyska et al. 1989)
orinternalisation (Boyer et al. 1989; Capps et al. 1989) ratherthan
lateral motility.
This work was supported by the INSERM (CJF 89-07).
AppendixSignificance of quantitative fluorescence indices
It was important to assess the significance of theparameters
used to describe fluorescence distributions.The basic difficulty is
that a measured fluorescencepattern depends on both the cellular
label distribution andthe relative orientation between the cell and
the opticalaxis of the microscope. This problem was addressed
byconsidering model fluorescence distributions with
randomorientation as shown below.
Significance of the standard deviation of relative
sectorfluorescenceLet us consider a labeled sphere with total
fluorescence T.We assume that: a fraction xxT (O^x^l) of total
labelingis concentrated on a point of the sphere. The
fluorescencemeasured on sector i (i=1...6) is kxTxpj, where k is
aconstant representing the fraction of emitted fluorescentlight
detected by the measuring apparatus and p{ valuesare positive real
numbers such that:
6
I P i = l .
A fraction ( l -x )xT of total labeling is uniformly spreadon
the sphere. The fluorescent light intensity falling onsector i is
kx(l-x)xT/6.
Hence, the relative fluorescence of sector i is:
The standard deviation of relative sector fluorescences
istherefore:
S.D. = x x (E(6pi - 1)2/5)J = xxf.
Hence, parameter S.D. is proportional to the fraction
ofredistributed label. In order to evaluate /"numerically,
weconsidered 4800 points uniformly spread on the surface ofa
sphere, and we used a previously described (Andre et al.1990a, b)
geometrical model of fluorescence images tocalculate the average
value of f. We found:
f= 1.23 ± 0.58 (standard deviation).
Significance of correlationThe significance of the correlation
between fluorescencedistributions was assessed with a similar
procedure to thatperformed for the standard deviation. Since the
corre-lation coefficient between two random variables x and y isthe
same as that between ajc+b and cy+d, where a, b, c andd are real
constants, the correlation between the fluor-escence distributions
of two spheres with a combination ofuniform label and a point
fluorescence (as described above)is only dependent on the location
of both concentrations offluorescent points. Thus, we considered
4800 points
CD8 cytoplasmic domain and capping 335
-
3-
1-
0-
-1
-0.5 0cos(A)
0.5
Fig. 6. Significance of the correlation parameter. Ten
thousandcouples of fluorescent spheres with a randomly located
pointfluorescence concentration were generated for determination
of(A) fluorescence correlation parameters of each couple
offluorescence distributions and (B) cosine of the angle betweenthe
directions of fluorescent points. Results are shown for 10classes
of cosine values (vertical bar length is twice thestandard
deviation of correlation parameter).
uniformly spread on a sphere. Since actual averaging ofthe
correlation coefficients between all couples of pointswould require
unreasonably lengthy calculation, we useda Monte-Carlo-like method
(Metropolis et al. 1953) tocalculate an average correlation: 10 000
random couples ofpoints on the sphere surface were generated (using
theBASIC RND function of an IBM-compatible desk com-puter). Sector
fluorescences were calculated in order toobtain the correlation
parameter. The average relation-ships between this parameter and
the cosine of the anglebetween the radius vectors of these points
is shown inFig. 6.
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(Received 4 March 1991 - Accepted, in revised form, 1 July
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