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Am J Physiol Cell Physiol
279: C369–C374, 2000.
Clathrin in mitotic spindles
CURTIS T. OKAMOTO, JEANA MCKINNEY, AND YOUNG Y. JENG
Department of Pharmaceutical Sciences, School of Pharmacy,
University of Southern California, Los Angeles, California 90089-9121
Received 10 August 1999; accepted in final form 23 February 2000
conventional and “muscle” isoforms of clathrin heavy
chain have been reported in various tissues (3). Together, these data suggest that clathrin may possess
diverse functions in a multicellular organism. Here we
show by immunofluorescence with two anti-clathrin
heavy chain monoclonal antibodies (MAbs) and anticlathrin light chain polyclonal antibodies that clathrin
is localized to the mitotic spindle of mammalian cells.
Clathrin heavy chain was also detected on Western
blots of isolated spindles. These results suggest either
a role for clathrin in mitosis or a cell cycle-dependent
regulatory mechanism for clathrin localization.
EXPERIMENTAL PROCEDURES
the role of clathrin
in the regulation of receptor-mediated endocytosis
from the plasma membrane and of budding of transport vesicles from the trans-Golgi network have been
made at the morphological, biochemical, cellular, genetic, and atomic structural levels (3, 16, 23, 25, 29,
32). Clathrin is comprised of two subunits, a heavy
chain of relative molecular mass (Mr) of 170 kDa and a
light chain of Mr of 32 kDa. In mammalian cells,
several isoforms of the light chains have been identified. Three heterodimers assemble into three-legged
structures known as triskelions that, in turn, polymerize to form cages on budding membranes in vivo and in
vitro. Recently, clathrin has been localized to other
intracellular membranes, such as endosomes, and may
function to regulate trafficking at these sites as well (6,
8, 19, 28). Moreover, a second isoform of the clathrin
heavy chain has been cloned that is apparently expressed ubiquitously in mammalian fetal tissues, but
is predominantly expressed in skeletal muscle in the
adult (12). Alternative mRNA transcripts of both the
Cells and antibodies. Madin-Darby canine kidney (MDCK)
cells (strain II) of European Molecular Biology Laboratory
parentage were obtained from Dr. Keith Mostov (Univ. of
California, San Francisco). CV-1 cells were obtained from Dr.
Sarah Hamm-Alvarez (Univ. of Southern California). The
MAb X-22 hybridoma (2) was purchased from the American
Type Tissue Collection (Washington, DC), and the hybridoma
supernatants were used neat for immunostaining. Anticlathrin heavy chain MAb 23 and anti-␥-adaptin MAb were
purchased from Transduction Laboratories (Lexington, KY).
The rabbit polyclonal antiserum against the conserved region
of mammalian light chains was a kind gift from Dr. Frances
Brodsky (Univ. of California, San Francisco). Goat anti-mouse
IgG conjugated to indocarbocyanine (Cy3) or FITC and goat
anti-rabbit IgG conjugated to FITC secondary antibodies were
purchased from Jackson Immunological Research Laboratories
(Bar Harbor, ME). ProLong antifade mounting medium and
propidium iodide were purchased from Molecular Probes (Eugene, OR) and used according to the manufacturer’s instructions. BSA, fish skin gelatin, anti-␣-tubulin MAb, nocodazole,
and brefeldin A (BFA) were purchased from Sigma Chemical
(St. Louis, MO). Goat anti-mouse IgG secondary antibody coupled to horseradish peroxidase was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence
detection kit for horseradish peroxidase was purchased from
Pierce Chemical (Rockford, IL). Molecular mass markers for
SDS-PAGE were purchased from Bio-Rad Laboratories (Hercules, CA).
Immunofluorescence. Cells were plated at subconfluent
densities onto glass coverslips. Cells were fixed either in 3.7%
formaldehyde, diluted from a 37% stock solution (Fisher
Scientific, Pittsburgh, PA) in PBS for 20 min at room temperature, or in cold (⫺20°C) methanol. Formaldehyde-fixed
glands were permeabilized by 0.5% Triton X-100 in PBS for
Address for reprint requests and other correspondence: C. T.
Okamoto, Dept. of Pharmaceutical Sciences, School of Pharmacy,
Univ. of Southern California, Los Angeles, CA 90089-9121 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Madin-Darby canine kidney cells; CV-1 cells; brefeldin A
SIGNIFICANT ADVANCEMENTS IN DEFINING
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C369
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Okamoto, Curtis T., Jeana McKinney, and Young
Y. Jeng. Clathrin in mitotic spindles. Am J Physiol Cell Physiol
279: C369–C374, 2000.— Subconfluent cultures of Madin-Darby
canine kidney (MDCK) and CV-1 cells were immunostained with
two monoclonal antibodies (MAbs), MAb X-22 and MAb 23,
against clathrin heavy chain and with polyclonal antiserum
against a conserved region of all mammalian clathrin light chains.
In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular
and perinuclear staining pattern. In mitotic cells, all three anticlathrin antibodies strongly stained the mitotic spindle. Staining of
clathrin in the mitotic spindle was colocalized with anti-tubulin
staining of microtubular arrays in the spindle. Staining of the
mitotic spindle was evident in mitotic cells from prometaphase to
telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the
mitotic spindle was not affected by brefeldin A. On Western blots,
clathrin was detected, but not enriched, in isolated spindles. The
immunodetection of clathrin in the mitotic spindle may suggest a
novel role for clathrin in mitosis. Alternatively, the recruitment of
clathrin to the spindle may suggest a novel regulatory mechanism
for localization of clathrin in mitotic cells.
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CLATHRIN IN MITOTIC SPINDLES
RESULTS
Two MAbs against clathrin heavy chain, MAb X-22
(2) and MAb 23, and a rabbit polyclonal antiserum
against clathrin light chain were used to stain two
Fig. 1. Indirect immunofluorescent labeling of mitotic spindles in Madin-Darby
canine kidney (MDCK) and CV-1 cells by
anti-clathrin heavy chain monoclonal antibody (MAb) X-22 and MAb 23 and anticlathrin light chain polyclonal antiserum.
a and b: subconfluent MDCK cells doublestained with propidium iodide for nuclear
and chromosomal staining (a) and MAb
X-22 for anti-clathrin heavy chain (b). c-e:
subconfluent MDCK cells stained with
MAb X-22. f: subconfluent MDCK cells
stained by anti-clathrin heavy chain MAb
23. g: CV-1 cells stained with MAb 23. h:
subconfluent MDCK cells stained with the
rabbit anti-light chain polyclonal antiserum. Bar ⫽ 5 ␮m.
types of cultured cells, MDCK cells and CV-1 cells, by
indirect immunofluorescence. MAb 23 recognizes the
NH2 terminus of clathrin heavy chain (the epitope is
within a fragment spanning amino acids 4–171), and
MAb X-22 binds nearer to the COOH terminus (17).
The polyclonal antiserum recognizes a consensus sequence found in all mammalian clathrin light chains
(amino acids 23–40) (1).
As shown in Fig. 1, immunofluorescent staining by
all antibodies results in the typical, well-documented
punctate intracellular staining pattern in interphase
MDCK and CV-1 cells (Fig. 1, b-d and Fig. 2a). In
addition, perinuclear staining is usually prominent;
this staining pattern is thought to represent staining of
the trans-Golgi network. In mitotic cells, punctate intracellular staining was also visible. However, unexpectedly, under the conditions employed here, mitotic
spindles in both cell types were also strongly immunoreactive with all three anti-clathrin antibodies: MAb
X-22 (Fig. 1, b-e), MAb 23 (Fig. 1, f and g), and polyclonal anti-light chain (Fig. 1h). In mitotic cells, the
anti-clathrin immunoreactivity in mitotic spindles was
significantly stronger than that observed on intracellular membranes. The pattern of anti-clathrin staining
of mitotic spindles is clearly distinct from that of chromosomal staining (Fig. 1, a and b), but is reminiscent of
staining of the microtubule array in mitotic spindles
(26). Anti-clathrin staining was evident from an early
stage of mitosis (late prophase, Fig. 1d) to later stages
(telophase, Fig. 1e). Thus, MAb X-22 and MAb 23,
MAbs that bind to distinct and widely separated
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20 min at room temperature. Nonspecific binding sites were
blocked by incubation with 3% BSA and 0.66% fish skin
gelatin in PBS. Cells were incubated with primary antibody,
followed by fluorescently labeled secondary antibody, and
mounted in ProLong antifade mounting medium. Cells were
observed and photographed on a Zeiss Axioskop epifluorescence microscope or images were collected using a Nikon
PCM Quantitative Measuring High-Performance Confocal
System attached to a Nikon TE300 Quantum upright microscope. Parenthetically, the manner in which the cells were
processed for immunofluorescence was important for detecting anti-clathrin immunoreactivity in the mitotic spindle,
particularly for MAb X-22. Anti-clathrin immunoreactivity in
mitotic spindles was not observed when cells were fixed and
permeabilized with cold methanol, a popular method of fixing
and permeabilizing cells for staining with MAb X-22 (data
not shown). For BFA experiments, cells were incubated with
BFA (20 ␮g/ml) for 20 min at 37°C and subsequently processed for immunofluorescence. Thymidine-nocodazole blocking of cultured cells to enrich for mitotic cells was performed
according to Zieve et al. (34).
Isolation of mitotic spindles and crude clathrin-coated vesicles from brain. Spindles were isolated from mitotic CV-1
cells according to a protocol adapted from Zieve et al. (34).
Crude clathrin-coated vesicles were isolated according to the
protocol of Pearse and Robinson (20). SDS-PAGE was run
according to the protocol of Laemmli (11), and Western blots
were performed according to the protocol of Towbin et al.
(30).
CLATHRIN IN MITOTIC SPINDLES
C371
epitopes on clathrin heavy chain, and the anti-clathrin
light chain antiserum all immunolabel the mitotic
spindle. These results suggest that clathrin is present
in mitotic spindles.
To confirm that clathrin is localized to the mitotic
spindle, subconfluent MDCK cells were double-labeled
by immunofluorescence for clathrin light chain and
tubulin (Fig. 2, a and b). In mitotic cells, there is
substantial colocalization of clathrin light chain and
tubulin in the spindle. An additional experiment was
performed in which mitotic cells were enriched by
arresting cells with a thymidine-nocodazole block and
subsequently releasing them from the block (34).
Shown in the confocal microscopic images in Fig. 2, c
and d, are subconfluent MDCK cells after being released from a thymidine-nocodazole block and stained
for clathrin heavy chain. Anti-clathrin staining is
present in spindle-like staining patterns, and even
appears to stain spindles in cells with hemispindles
and multiple spindles. These results confirm the im-
munofluorescent localization of clathrin to the mitotic
spindle.
For some populations of clathrin-coated membranes
and vesicles, the association of clathrin and clathrin
adaptor proteins with their target membranes is regulated by the small GTPase ADP ribosylation factor
(ARF). BFA reversibly inhibits the function of ARF by
inhibition of GDP:GTP exchange factors for ARF (4),
and incubation of cells with BFA prevents the recruitment of ARF-dependent vesicular coat proteins, such
as the Golgi-associated coatomer protein complex
(COPI) and the clathrin adaptor proteins AP-1 and
AP-3, onto target membranes (7, 24, 31). The sensitivity of clathrin in mitotic spindles to BFA was assessed
in CV-1 cells that were treated with BFA and subsequently double-labeled for ␥-adaptin and clathrin light
chain (Fig. 3). Addition of BFA to CV-1 cells results in
a characteristic dispersion in Golgi-associated anti-␥adaptin staining in interphase cells (Fig. 3, a and c).
However, BFA did not affect the localization of clathrin
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Fig. 2. a and b: confocal micrographs of double immunofluorescent labeling of clathrin light chain (a) and
␣-tubulin (b) in subconfluent MDCK cells. Clathrin
light chain and microtubules in mitotic spindles are
colocalized. c and d: confocal micrographs of clusters of
mitotic MDCK cells labeled with anti-clathrin heavy
chain MAb X-22. Clusters of mitotic cells were produced
by releasing cells from a thymidine-nocodazole block as
described in EXPERIMENTAL PROCEDURES. Spindle staining is apparent in cells with hemi-, single, and multiple
spindles. Interphase cells are barely visible in the background. All bars ⫽ 5 ␮m.
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CLATHRIN IN MITOTIC SPINDLES
light chain to the mitotic spindle (Fig. 3, b and d).
Anti-clathrin heavy chain MAb X-22 was also used to
stain cells treated with BFA. In the presence of BFA,
Golgi-associated staining was lost in interphase cells,
whereas staining of the spindle in mitotic cells remained intact (data not shown). Thus the association of
clathrin with the mitotic spindle appears to be mechanistically distinct from its association with Golgi
membranes and may be independent of ARF. Alternatively, if ARF is involved in the binding of clathrin to
the mitotic spindle, ARF may be regulated by a BFAinsensitive exchange factor (4).
Given the apparently significant amount of clathrin
immunoreactivity associated with the mitotic spindle,
one would predict to find anti-clathrin immunoreactivity associated with isolated mitotic spindles. Spindles
were isolated from mitotic CV-1 cells and were analyzed on Coomassie blue-stained gels and by Western
blot (Fig. 4). The profile of proteins associated with the
isolated spindles as analyzed by SDS-PAGE appears to
be similar to previously published results (Fig. 4A, lane
1) (34). Anti-tubulin immunoreactivity is shown in Fig.
4B, lanes 1 and 2, to confirm the presence and enrichment of tubulin, a major component of isolated spindles.
Although proteins are visible in the region where
clathrin heavy chain is expected to migrate (Mr of ⬃170
kDa), based upon the lack of a distinctly visible clathrin heavy chain band, clathrin heavy chain does not
appear to be a significant component of isolated mitotic
spindles (Fig. 4A, lane 1). A sample of crude-coated
vesicles from hog brain, in which clathrin heavy chain
is a major component, is shown for comparison (Fig.
4A, lane 2). However, by immunoblot analysis of isolated mitotic spindles (Fig. 4C, lanes 1 and 2), clathrin
is indeed detectable, although not apparently enriched,
in isolated spindles. In mitotic cells, the strong immunolabeling of mitotic spindles with anti-clathrin antibodies suggests that the spindle may represent a major
clathrin-containing organelle. However, the association of clathrin with the spindle may be quite labile,
particularly under the conditions used to isolate spindles, because clathrin does not appear to be enriched in
isolated mitotic spindles. Moreover, at this time, we
cannot rule out the possibility that clathrin in this
spindle preparation may represent clathrin that is
Fig. 4. A: Coomassie blue-stained SDS-gel of isolated mitotic spindles from CV-1 cells (20 ␮g; lane 1) and crude-coated vesicles from
brain (20 ␮g; lane 2). B: Western blot of total cell lysate (20 ␮g; lane
1) and isolated mitotic spindles (20 ␮g; lane 2) from CV-1 cells probed
with anti-tubulin MAb. C: Western blot of total cell lysate (20 ␮g;
lane 1) and isolated mitotic spindles (20 ␮g; lane 2) from CV-1 cells
probed with anti-clathrin heavy chain MAb 23. The positions of
molecular mass standards (in kDa) are shown.
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Fig. 3. Association of clathrin with the mitotic
spindle is not affected by brefeldin A (BFA).
Untreated (a and b) or BFA-treated (c and d)
CV-1 cells were fixed and double-labeled for
␥-adaptin (a and c) and clathrin light chain (b
and d) and viewed by confocal microscopy. The
perinuclear Golgi-associated localization of
␥-adaptin (arrows) is characteristically affected
by BFA. The localization of clathrin light chain
to the mitotic spindle is not affected by BFA
(arrowheads). The exposures for the images of
clathrin light chain have been adjusted to visualize spindle staining. Such images are indicators of the relative intensities of staining between interphase and mitotic cells. Bar ⫽ 10 ␮m.
CLATHRIN IN MITOTIC SPINDLES
associated with the detergent-insoluble cytoskeleton of
mitotic cells; in interphase cells, clathrin has been
shown to be associated with the detergent-insoluble
cytoskeleton (9).
DISCUSSION
vitro assay for the role of clathrin in the mitotic spindle
would greatly benefit from the ability to isolate spindles with clathrin bound to them. However, the biochemical data presented here suggest that the association of clathrin with the mitotic spindle may be
somewhat labile, given the inability to copurify significant amounts of clathrin with isolated spindles. This
lability may be the reason that clathrin appears to be a
minor component of isolated spindles and, therefore,
has not previously been reported to be a component of
isolated spindles.
A third possibility is that the anti-clathrin antibodies may be recognizing a closely related isoform of
clathrin with a novel function in the mitotic spindle.
Both of the anti-clathrin heavy chain MAbs bind to
regions of clathrin heavy chain that are highly conserved between the conventional and “muscle” isoform
of clathrin (3) and, therefore, could be recognizing an
isoform of clathrin with a distinct function in the mitotic spindle. However, one argument against the possibility that spindle clathrin is the muscle isoform is
that clathrin light chain is present in the spindle. The
muscle isoform of clathrin has amino acid substitutions
in the light chain binding region that make binding to
light chain highly unfavorable (32); thus the presence
of light chain in the spindle would not be expected.
The final possibility is that localization of clathrin to
the mitotic spindle might represent a novel, cell cycledependent, regulatory mechanism for the subcellular
localization of clathrin. The robust staining of clathrin
in the mitotic spindle suggests that the spindle may
contain a significant fraction of the total cellular clathrin in mitotic cells. One characteristic of mitotic cells is
that clathrin-mediated endocytosis is inhibited (21,
22). Although clathrin-coated pits are still present in
mitotic cells, early stages of clathrin-coated pits (shallow domes and wide necks) are abundant relative to
clathrin-coated pits in the later stages (i.e., narrow
necks) of formation (21). A massive recruitment of
clathrin to the mitotic spindle would, therefore, deplete
the cellular pool available for the completion of coated
pits and vesicles. This mechanism, working in conjunction with that of the inhibition of the interaction of
other components of coated pits at the plasma membrane, such as ␣-adaptin, Eps 15, and epsin, due to the
phosphorylation of Eps 15 and epsin during mitosis (5),
would reinforce a block in endocytosis. Alternatively,
clathrin may be recruited to the spindle during mitosis
to ensure a roughly equal division of clathrin between
the two daughter cells, similar to the process for the
mitotic division of the Golgi apparatus (13). In summary, all of these possibilities that may account for
clathrin localization to the mitotic spindle are highly
speculative, and further investigation is warranted.
The identification of clathrin in the mitotic spindle
suggests that clathrin may be a protein with multiple
functions or a protein with a localization that is subject
to cell cycle-dependent regulation. Its further characterization with respect to its role in the mitotic spindle
should lead to additional insight into clathrin function.
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The data presented here suggest that clathrin is
present in mitotic spindles. Interestingly, we are not
the first to report the presence of clathrin in the mitotic
spindle. Maro et al. (14), using their own anti-clathrin
polyclonal antibodies, observed immunofluorescently
labeled clathrin in the second metaphase spindle of an
unfertilized mouse egg and in mitotic spindles of cells
in early embryos. Thus we have confirmed and extended their seminal observations. The data from these
reports together suggest that clathrin localization to
the mitotic spindle may be a fundamental property of
dividing cells. To date, the only other report of clathrin’s involvement in mitosis was its role in the regulation of cytokinesis in Dictyostelium, where it was found
that a strain of Dictyostelium that lacks clathrin was
defective in cytokinesis (18).
The role of clathrin in mitotic spindles needs to be
defined at the morphological, cellular, and biochemical
levels. One possibility that may account for the distinctive and characteristic anti-clathrin staining of mitotic
spindles is that the anti-clathrin antibodies are staining clathrin-coated vesicles or membrane tubules that
are tethered to microtubules in the mitotic spindle.
These vesicles may be remnants of fragmented Golgi
(26) or other clathrin-coated organelles, analogous to
mitotic fragments of Golgi membranes coated with the
COPI coatomer complex (15). However, the pattern of
anti-clathrin staining of the spindle is distinct from
that of anti-Golgi staining associated with the mitotic
spindle: the anti-Golgi staining is clearly found in
“clumps” that are clustered around the spindle poles
(24, 26). The absence of an effect of BFA on anticlathrin staining of the mitotic spindle (Fig. 2) also
argues against, but does not completely rule out, this
first possibility, because the association of clathrin
and, more directly, the AP-1 clathrin adaptor with
Golgi membranes is regulated by ARF (27, 33). In fact,
Robinson and Kreis (24) have shown that in mitotic
cells treated with BFA, the staining for COPI coatomer
and the AP-1 clathrin adaptor becomes diffuse, similarly to that observed for interphase cells.
A second possibility for the presence of clathrin immunoreactivity in mitotic spindles is that clathrin is
serving a novel scaffolding or mechanochemical function in the spindle. Such a function for clathrin might
be inferred from the distinct staining of muscle cell
sarcomeres by MAb X-22 (10). Clathrin may be involved in the assembly and/or maintenance of ordered
cellular structures such as the sarcomere and the mitotic spindle. If clathrin serves such a function, one
prediction would be that the mitotic spindles in clathrin-minus Dictyostelium may be abnormal; unfortunately, the morphology of mitotic spindles in this
strain of Dictyostelium was not investigated (18). An in
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CLATHRIN IN MITOTIC SPINDLES
We thank Dr. Sarah Hamm-Alvarez for helpful suggestions, we
acknowledge continual help from and generosity of Drs. Frances
Brodsky and Shu-Hui Liu from the Brodsky lab, and we thank Dr.
Francesca Santini for drawing our attention to the Maro paper.
We acknowledge the Confocal Microscopy Subcore for the Univ. of
Southern California Center for Liver Diseases, supported by National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK) Core Center Grant PO3 DK-48522. This work was supported by a Grant-in-Aid from the National American Heart Association and NIDDK 51588 (C. T. Okamoto).
REFERENCES
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
1. Acton SL and Brodsky FM. Predominance of clathrin light
chain LCb correlates with the presence of a regulated secretory
pathway. J Cell Biol 111: 1419–1426, 1990.
2. Brodsky FM. Clathrin structure characterized with monoclonal
antibodies. I. Analysis of multiple antigenic sites. J Cell Biol 101:
2047–2054, 1985.
3. Brodsky FM. New fashions in vesicle coats. Trends Cell Biol 7:
175–179, 1997.
4. Chardin P and McCormick F. Brefeldin A: the advantage of
being uncompetitive. Cell 97: 153–155, 1999.
5. Chen H, Slepnev VI, Di Fiore PP, and De Camilli P. The
interaction of epsin and Eps15 with the clathrin adaptor AP-2 is
inhibited by mitotic phosphorylation and enhanced by stimulation-dependent phosphorylation in nerve terminals. J Biol Chem
274: 3257–3260, 1999.
6. Dell’Angelica EC, Klumperman J, Stoorvogel W, and Bonifacino JS. Association of the AP-3 adaptor complex with clathrin. Science 280: 431–434, 1998.
7. Eng Ooi C, Dell’Angelica EC, and Bonifacino JS. ADPribosylation factor 1 (ARF1) regulates recruitment of the AP-3
adaptor complex to membranes. J Cell Biol 142: 391–402, 1998.
8. Futter CE, Gibson A, Allchin EH, Maxwell S, Ruddock LJ,
Odorizzi G, Domingo D, Trowbridge IS, and Hopkins CR.
In polarized MDCK cells basolateral vesicles arise from clathrin␥-adaptin-coated domains on endosomal tubules. J Cell Biol 141:
611–623, 1998.
9. Gaidarov I, Santini F, Warren RA, and Keen JH. Spatial
control of coated-pit dynamics in living cells. Nat Cell Biol 1: 1–7,
1999.
10. Kaufman SJ, Bielser D, and Foster RF. Localization of anticlathrin antibody in the sarcomere and sensitivity of myofibril
structure to chloroquine suggest a role for clathrin in myofibril
assembly. Exp Cell Res 191: 227–238, 1990.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.
12. Long KR, Trofatter JA, Ramesh V, McCormick MK, and
Buckler AJ. Cloning and characterization of a novel human
clathrin heavy chain gene (CLTCL). Genomics 35: 466–472,
1996.
13. Lowe M, Nakamura N, and Warren G. Golgi division and
membrane traffic. Trends Cell Biol 8: 40–44, 1998.
14. Maro B, Johnson MH, Pickering SJ, and Louvard D. Changes
in the distribution of membranous organelles during mouse early
development. J Embryol Exp Morphol 90: 287–309, 1985.
15. Misteli T and Warren G. A role for tubular networks and a
COP I-independent pathway in the mitotic fragmentation of
Golgi stacks in a cell-free system. J Cell Biol 130: 1027–1039,
1995.
16. Musacchio A, Smith CJ, Roseman AM, Harrison SC,
Kirchhausen T, and Pearse BMF. Functional organization of
17.
clathrin in coats: combining electron cryomicroscopy and X-ray
crystallography. Mol Cell 3: 761–770, 1999.
Näthke IS, Heuser J, Lupas A, Stock J, Turck CW, and
Brodsky FM. Folding and trimerization of clathrin subunits at
the triskelion hub. Cell 68: 899–910, 1992.
Niswonger ML and O’Halloran TJ. A novel role for clathrin in
cytokinesis. Proc Natl Acad Sci USA 94: 8575–8578, 1997.
Okamoto CT, Karam SM, Jeng YY, Forte JG, and Goldenring JR. Identification of clathrin and clathrin adaptors on
tubulovesicles of gastric acid secretory (oxyntic) cells. Am J
Physiol Cell Physiol 274: C1017–C1029, 1998.
Pearse BMF and Robinson MS. Purification and properties of
100-kd proteins from coated vesicles and their reconstitution
with clathrin. EMBO J 3: 1951–1957, 1984.
Pypaert M, Lucocq JM, and Warren G. Coated pits in interphase and mitotic A431 cells. Eur J Cell Biol 45: 23–29, 1987.
Pypaert M, Mundy D, Souter E, Labbé J-C, and Warren G.
Mitotic cytosol inhibits invagination of coated pits in broken
mitotic cells. J Cell Biol 114: 1159–1166, 1991.
Robinson MS. Coats and vesicle budding. Trends Cell Biol 7:
99–102, 1997.
Robinson MS and Kreis TE. Recruitment of coat proteins onto
Golgi membranes in intact and permeabilized cells: effects of
brefeldin A and G protein activators. Cell 69: 129–138, 1992.
Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu Rev Biochem 66: 511–548,
1997.
Shima DT, Cabrera-Poch N, Pepperkok R, and Warren G.
An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic
spindle. J Cell Biol 141: 955–966, 1998.
Stamnes MA and Rothman JE. The binding of AP-1 clathrin
adaptor particles to Golgi membranes requires ADP-ribosylation
factor, a small GTP-binding protein. Cell 73: 999–1005, 1993.
Stoorvogel W, Oorschot V, and Geuze HJ. A novel class of
clathrin-coated vesicles budding from endosomes. J Cell Biol
132: 21–33, 1996.
Ter Haar E, Musacchio A, Harrison SC, and Kirchhausen
T. Atomic structure of clathrin: a ␤-propeller terminal domain
joins an ␣-zigzag linker. Cell 95: 563–573, 1998.
Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proc Natl Acad Sci USA 76:
4350–4354, 1979.
Wong DH and Brodsky FM. 100-kD proteins of Golgi and
trans-Golgi network-associated coated vesicles have related but
distinct membrane binding properties. J Cell Biol 117: 1171–
1179, 1992.
Ybe JA, Brodsky FM, Hofmann K, Lin K, Liu S-H, Chen L,
Earnest TN, Fletterick RJ, and Hwang PK. Clathrin selfassembly is mediated by a tandemly repeated superhelix. Nature
399: 371–375, 1999.
Zhu Y, Traub LM, and Kornfeld S. ADP-ribosylation factor 1
transiently activates high-affinity adaptor protein complex AP-1
binding sites on Golgi membranes. Mol Biol Cell 9: 1323–1337,
1998.
Zieve GW, Turnbull E, Mullins JM, and McIntosh JR.
Production of large numbers of mitotic mammalian cells by use
of the reversible microtubule inhibitor nocodazole. Exp Cell Res
126: 397–405, 1980.