Proc. Nati. Acad. Sci.
USA
Vol. 81, pp. 788-792, February 1984
Cell Biology
Lysosomes are associated with microtubules and not with
intermediate filaments in cultured fibroblasts
(cytoskeleton/vimentin/double immunofluorescence)
MICHELE COLLOT*t, DANIEL LOUVARDt, AND S. J. SINGER*
*Department of Biology, University of California at San Diego, La Jolla, CA 92093; and
Contributed by S. J. Singer, October 21, 1983
ABSTRACT Double immunofluorescent labeling experi-
ments for lysosomes and either microtubules or vimentin inter-
mediate filaments in cultured well-spread fibroblasts show a
remarkable degree of superposition of the lysosomes and the
microtubules. Under two different sets of conditions where the
microtubules and intermediate filaments are well segregated
from one another, the lysosomes remain codistributed with the
microtubules. It is suggested that this specific association of
lysosomes with microtubules reflects some type(s) of linkage(s)
between them and that such linkages may play an important
role in the location and intracellular transport of lysosomes
inside cells.
Microtubules (MT) and intermediate filaments (IF) have
been increasingly recognized as playing important roles in
regulating the location and movement of intracellular organ-
elles (cf. ref. 1). In this article, we are specifically concerned
about the relationships between lysosomes and cytoskeletal
elements. Previous studies have suggested an involvement
of MT or IF or both in lysosome activities and functions. For
example, the saltatory movements of lysosomes and other
organelles have been correlated with MT integrity (2, 3), the
intracellular distribution of lysosomes in mouse macro-
phages has been correlated with the degree of MT and IF
extension into the cytoplasm (4), and the secretion of lyso-
somal enzymes in polymorphonuclear leukocytes was
shown to be affected by the colchicine-induced disruption of
MT (5). These effects, however, only indirectly suggest an
association of lysosomes with cytoskeletal elements, and
more direct evidence concerning a possible physical associa-
tion is desirable.
In principle, electron microscopy is the technique of
choice for the detection and characterization of any physical
linkages between specific organelles and cytoskeletal fila-
ments. For cells with an amorphous internal structure, how-
ever, transmission electron microscopy of random ultrathin
sections may be of only limited use for this purpose, because
it may be difficult to find linkage elements in any given field
of a section. Under these circumstances, double immunoflu-
orescence light microscopy may yield significant information
about organelle-filament associations. Despite the low reso-
lution of the method, it may reveal distinct spatial correla-
tions of a specific immunolabeled organelle with a particular
type of immunolabeled cytoskeletal filament throughout an
entire cell. If other filament types show no such spatial cor-
relations with the organelle in question, the suggestion is that
a specific association exists between the organelle and the
spatially correlated filament. We have used this method pre-
viously to provide evidence for an association of mitochon-
dria with MT (6, 7) in a variety of cultured cells. In this pa-
per, similar double, indirect immunofluorescence experi-
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788
hInstitut Pasteur, 75742 Paris, France
ments have indicated a strong spatial correlation of
immunolabeled lysosomes with MT in cultured fibroblasts,
but not with vimentin IF, suggesting that some kind(s) of
linkages exist between lysosomes and MT in these cells.
MATERIALS AND METHODS
Antibody Preparations. Antisera to lysosomes (8) were
raised in rabbits to a membrane fraction of lysosomes isolat-
ed from rat liver, and anti-lysosome antibodies were purified
by extensive absorption of the sera with other cellular frac-
tions (9). The anti-lysosome antibodies reacted with both pri-
mary and secondary lysosomes, according to immunoper-
oxidase labeling in electron microscopy, and occasionally
with some Golgi stacks and the plasma membrane (8). The
antigen recognized by these antibodies appears to be a
100,000-dalton protein, according to immunolabeling proce-
dures. The guinea pig antibodies to chick brain tubulin and to
vimentin isolated from baby hamster kidney cells, were af-
finity-purified monospecific antibodies previously character-
ized (10). Affinity-purified and cross-absorbed goat antibod-
ies to rabbit IgG and to guinea pig IgG and their conjugation
to fluorescein and rhodamine fluorophores were as de-
scribed (10).
Cells and Immunofluorescence Labeling. All experiments
were carried out with normal rat kidney (NRK) cells infected
with a temperature-sensitive mutant (LA23) of Rous sarco-
ma virus. The infected cells were from stocks kindly sup-
plied by P. K. Vogt of the University of Southern California.
LA23-infected NRK cells were grown on coverslips either at
39C, where they exhibit the normal phenotype, or at 33C,
where they are transformed. In some experiments, cyclo-
heximide (20 ,g/ml) was added to the medium 15 hr before
the end of the growth period; in others, nocodazole (10
,g/ml) was added 6 hr before. After a minimum of 2 days of
growth, the cells were fixed for 20 min with 3% formalde-
hyde and then permeabilized by a 5-min exposure to 0.2%o
Triton X-100. Immunolabeling was carried out as described
(7, 10). In some of the experiments, in order to visualize
more clearly the spatial relationships of lysosomes and MT,
optical leak-through of the rhodamine immunofluorescence
for tubulin into the fluorescein immunofluorescence for lyso-
somes was deliberately generated. This was achieved by ap-
propriately increasing the fluorescence intensity of tubulin
labeling and by longer photographic exposures, so as to pro-
duce the desired effects. This required calibration with each
different batch of primary and secondary antibodies used.
Immunolabeling was observed by epifluorescence with a
Zeiss Photoscope-III instrument with the filters CZ487710
for fluorescein fluorescence and CZ487714 for rhodamine.
Abbreviations: MT, microtubules; IF, intermediate filaments; NRK
cells, normal rat kidney cells.
tPresent address: Departement de Biologie Cellulaire, Facultes Uni-
versitaires de Namur, 5000 Namur, Belgium.
To whom reprint requests should be addressed.
 Cell Biology: Collot et aL Proc. Natl. Acad. Sci. USA 81 (1984) 789

RESULTS in double immunofluorescence experiments (10, 11). There-

In LA23-infected NRK cells grown at 390C and exhibiting
the normal phenotype, double immunolabeling for lyso-
somes (Fig. 1A) and for microtubules (Fig. 1B) in the same
cells revealed a high concentration of lysosomes in the vicin-
ity of the microtubule-organizing center (arrows) near the
cell nucleus. The exposure in Fig. 1A does not allow clear
visualization of the many lysomes out in the cell periphery,
which are shown in Fig. 1C. In this field, the immunolabeled
lysosomes are the bright dots of fluorescein fluorescence,
and the background filamentous labeling is due to the optical
leak-through of the rhodamine immunolabeled microtubules
that also were recorded separately in Fig. 1D through the
rhodamine optical filter. More than 80% of the labeled lyso-
somes in the cell periphery were superimposed on labeled
MT, and this number was increased under different photo-
graphic conditions that revealed other labeled MT not appar-
ent in Fig. 1C.
In such well-spread cells exhibiting the normal phenotype,
it is known that MT and IF show substantial codistributions
fore, in order to discriminate whether the lysosome associa-
tion was primarily with MT or IF, two sets of conditions
were used that result in an intracellular dissociation of the
two filament types (7). One of these conditions involved
growth of the LA23-infected NRK cells at 330C, at which
temperature the cells exhibited the transformed phenotype,
and the vimentin IF were retracted around the nucleus (Fig.
2D), while the MT remained extended to the cell periphery
(Fig. 2B). The labeled lysosomes in such cells with retracted
IF remained distributed out in the cell periphery (Fig. 2C), to
a large extent superimposed on the labeled MT (Fig. 2 A and
B). The second condition involved growth of the cells at 390C
in the presence of cycloheximide, which is known (12) to
cause a retraction of the vimentin IF around the nucleus
(Fig. 2H). Under these circumstances, the lysosomes were
still found in relatively normal amounts out in the cell periph-
ery (Fig. 2G), where they were largely superimposed on MT
(Fig. 2 E and F).
In the presence of nocodazole in LA23-infected NRK cells

I
FIG. 1. LA23-infected NRK cells grown at 39C (normal phenotype). Double indirect immunofluorescent labeling for lysosomes (bright dots
in A and C) and MT (B and D). Treatment with a mixture of rabbit anti-lysosome antibodies (40 Ag/ml, see text) and affinity-purified guinea pig
anti-tubulin antibodies was followed by treatment with a mixture of cross-adsorbed rhodamine- or fluorescein-conjugated affinity-purified goat
antibodies to rabbit and guinea pig IgG. (A and D) Rhodamine labeling. (B and C) Fluorescein labeling. Note the high concentration of lyso-
somes (arrow in A) in the vicinity of the MT-organizing center (arrow in B). Out in the cell periphery, most of the lysosomes (C) are closely
associated with individual MT (C and D). The background filamentous labeling in C is due to the rhodamine fluorescence of labeled MT that was
allowed to leak through the fluorescein filter, producing an effect of superimposing the two patterns. The longer time of exposure of C compared
to D allows the visualization of some additional labeled MT. All fields are at the same magnification. (Bar in B = 20 Am.)
790 Cell Biology: Collot et aL Proc. NatL. Acad Sci. USA 81 (1984)

FIG. 2. (Legend appears at the bottom of the next page.)

 Cell Biology: Collot et aL
.,.4.
:7. ..l.
A.
FIG . 3. LA23-infected NRK cells grown at 39.C (normal pheno-
type). Double indirect immunofluorescent labeling for lysosomes
(A) and vimentin IF (B) after 6 hr of treatment with nocodazole at 10
A.g/ml. For rhodamine labeling of lysosomes and simultaneous fluo-
rescein labeling of IF, see Figs. 1 and 2, respectively, for proce-
dures. Note that lysosomes are randomly distributed in the cyto-
plasm, to some extent apparently in patches (large bright dots in A),
but show no spatial correlation with the IF collapsed near the nucle-
us (B). MT are completely disrupted under these conditions (not
shown). (C) Same field in Nomarski optics. Cell edges are indicated
by arrowheads. (Bar = 20lttm.)
Proc. NatL Acad Sci. USA 81 (1984) 791
grown at 390C, the MT were disrupted and vimentin IF were
condensed to a perinuclear distribution, as has been ob-
served by others in similar systems (cf. ref. 13). In such no-
codazole-treated cells, the lysosomes appeared to be aggre-
gated to some extent (large bright spots in Fig. 3A) but other-
wise to be distributed randomly in the cytoplasm and
showed no spatial correlation with the collapsed IF (Fig.
3B). In particular, there was no longer a concentration of
lysosomes near the nucleus, as observed in Fig. 1A, in these
cells in which the microtubule-organizing center was disrupt-
ed.
DISCUSSION
We have provided evidence by double immunofluorescence
light microscopy that lysosomes are to a remarkable extent
codistributed with MT inside cultured fibroblasts. The anti-
lysosome antibodies used in this study (8) did not discrimi-
nate between primary and secondary lysosomes, so that this
conclusion presumably applies to both types. The codistri-
bution of lysosomes with MT is manifested in several cir-
cumstances.
(i) A concentration of lysosomes is spatially coincident
with the concentration of MT that exists in the vicinity of the
microtubule-organizing center (Fig. 1 A and B).
(ii) A large fraction of the lysosomes that are located out
in the periphery of the cell are closely associated with the
dispersed and convoluted MT that are also present there
(Fig. 1 C and D).
(iii) Because the interpretation of this last observation is
complicated by the fact that MT and vimentin IF are codis-
tributed with one another in LA23-infected NRK cells grown
at 390C (10), it is especially interesting that under two quite
different sets of conditions where the vimentin IF are retract-
ed to a perinuclear distribution away from the MT, a large
number of lysosomes remain out in the cell periphery codis-
tributed with the MT still located there. This is true for
LA23-infected NRK cells grown at 33TC exhibiting the trans-
formed phenotype (Fig. 2 A-D) and for the cells grown at
390C in the presence of cycloheximide exhibiting the normal
phenotype (Fig. 2 E-H).
(iv) When the MT are disrupted with nocodazole and the
IF become aggregated into perinuclear bundles (Fig. 3B), the
lysosomes appear to be randomly dispersed (14pand no long-
er tethered (Fig. 3A).
These results indicate that lysosomes are extensively as-
sociated with MT but not with vimentin IF inside fibroblasts.
Because there is much evidence that the distribution of actin
microfilaments is not coordinated with either MT or IF in
these cells (cf. refs. 15 and 16), no significantly spatial corre-
lation of lysosomes with microfilaments is indicated. There-
fore, our results are not consistent with the suggestion of
Moore et al. (17) that there is an association of lysosomes
with microfilaments, as studied with polymorphonuclear leu-
kocytes. The specificity and extent of the lysosome-MT co-
distribution in the fibroblasts suggest that some kind or kinds
of direct or indirect linkages exist between lysosomes and
MT in these interphase cells that involve most of the lyso-
somes at any given time. Such linkages could play important
roles in lysosomal activities, including the saltatory move-

FIG. 2 (on preceding page). LA23-infected NRK cells grown at 33C (transformed phenotype) (A-D) and grown at 39C (normal phenotype)
(E-H) after 15 hr of treatment with cycloheximide at 20 pg/ml. Double, indirect immunofluorescent labeling for lysosomes (bright spots in A
and E) and MT (B and F) or for lysosomes (C and G) and vimentin IF (D and H). In the pairs A/B and E/F, lysosomes are fluorescein labeled
and MT are rhodamine labeled. The rhodamine fluorescence for tubulin was allowed to leak through the fluorescein filter in A and E. In the pairs
CID and G/H, lysosomes are rhodamine labeled. For IF labeling (D and H), affinity-purified guinea pig antibodies to vimentin were used,
followed by fluorescein-conjugated affinity-purified goat antibodies to guinea pig IgG. Note in each case the coordinate distribution of lyso-
somes (A and E) and MT (A/B and E/F). Both are concentrated in a perinuclear area, and lysosomes can be seen out in the cell periphery
superimposed on individual MT (small arrows, for example). In this region (large arrows in CID and G/H) lysosomes are not associated with
IF, which have retracted around the nucleus as indicated by the absence of vimentin labeling. Cell edges are indicated by arrowheads in E and
G. All fields are at the same magnification. (Bar in D = 20 ,um.)
792 Cell Biology: Collot et aL
ments of lysosomes inside cells that have been shown to stop
upon disruption of microtubules (2, 3) and the secretory
functions of lysosomes, which also appear to depend upon
the integrity of microtubules (5).
The evidence presented here for a preferential association
of lysosomes with MT does not exclude the possibility that,
in other cells and under other metabolic conditions, lyso-
somes may associate with IF. By means of immunofluores-
cence observations similar to those presented here, we earli-
er demonstrated (6, 7) an association of mitochondria with
MT and not with IF in cultured cells. However, although
there is evidence from several studies that mitochondrial-
MT associations are preferred in certain systems (1, 7, 18,
19), there is also evidence for mitochondrial-IF interactions
in other cases (20-23).
The present results make a specific contribution to the
growing body of evidence that most, if not all, intracellular
organelles may form direct or indirect linkages to one or
more cytoskeletal filaments in most eukaryotic cells. In
structurally well-organized nerve axons, regularly arrayed
linkages between MT or IF and various membrane-bounded
organelles have been observed by transmission electron mi-
croscopy (20, 24, 25), although specific reference to linkages
to primary or secondary lysosomes has not often been made
in these studies. It also is not evident that special cases such
as the nerve axon are of general relevance; the axon is spe-
cialized for the long-distance transport of membrane-bound-
ed organelles, and may exhibit cytoskeletal-organelle con-
nections for the purpose that are not necessarily characteris-
tic of other cells. However, the immunofluorescence
observations of lysosome-MT association reported in this
paper and similar evidence for mitochondrion-MT (6, 7, 18)
and for Golgi apparatus-MT (26) associations in amorphous
cultured cells as well as electron microscopic evidence for
associations of nuclei with IF in striated muscle (22, 23, 27,
28), of nuclei with MT/IF in other cells (29), and of secretory
granules with MT in cultured pancreatic beta cells (30) are
among the kinds of evidence for the existence of a wide
range of organelle-cytoskeletal filament interactions in eu-
karyotic cells, which very likely serve to locate these organ-
elles and to direct their movements inside cells.
The molecular bases for such organelle-cytoskeletal inter-
actions are only poorly understood. Whether different mem-
brane-bound organelles that interact with a particular cyto-
skeletal filament do so via a common recognition mechanism
or via specific mechanisms for different organelles is not
clear. In cases where MT are involved, the linkage elements
may include the high molecular weight MT-associated pro-
teins (MAPs) (31, 32), which project from microtubules (33)
and which appear also to associate with IF (34-36). In vitro
evidence implicating MT-associated proteins has been re-
ported in the case of MT-secretory granule interactions (37),
but further investigations along these lines are required with
a variety of organelle-cytoskeletal filament systems.
We are grateful to Dr. Adrienne Rogalski for very helpful advice
and discussions. We thank Mrs. Margie Adams and Mrs. Bracha
Yacobsen for their excellent technical assistance. M.C. was sup-
ported in part by a grant from the International Federation of Uni-
versity Women. These studies were funded by National Institutes of
Health Grant GM-15971 to S.J.S., who is an American Cancer Soci-
ety Research Professor.
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