Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089-9121
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ABSTRACT |
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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 anti-clathrin 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.
Madin-Darby canine kidney cells; CV-1 cells; brefeldin A
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INTRODUCTION |
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SIGNIFICANT ADVANCEMENTS IN DEFINING 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 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 anti-clathrin 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.
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EXPERIMENTAL PROCEDURES |
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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. Anti-clathrin 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 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 µg/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).
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RESULTS |
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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 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 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.
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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 immunofluorescent 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 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 BFA-insensitive exchange factor (4).
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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.
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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 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).
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DISCUSSION |
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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 anti-clathrin 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 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 cycle-dependent, 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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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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).
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: cokamoto{at}hsc.usc.edu).
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. §1734 solely to indicate this fact.
Received 10 August 1999; accepted in final form 23 February 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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[Abstract].
2.
Brodsky, FM.
Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.
J Cell Biol
101:
2047-2054,
1985[Abstract].
3.
Brodsky, FM.
New fashions in vesicle coats.
Trends Cell Biol
7:
175-179,
1997[ISI].
4.
Chardin, P,
and
McCormick F.
Brefeldin A: the advantage of being uncompetitive.
Cell
97:
153-155,
1999[ISI][Medline].
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.
ADP-ribosylation 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[ISI][Medline].
10.
Kaufman, SJ,
Bielser D,
and
Foster RF.
Localization of anti-clathrin 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[ISI][Medline].
11.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
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[ISI][Medline].
13.
Lowe, M,
Nakamura N,
and
Warren G.
Golgi division and membrane traffic.
Trends Cell Biol
8:
40-44,
1998[ISI][Medline].
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[ISI][Medline].
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[Abstract].
16.
Musacchio, A,
Smith CJ,
Roseman AM,
Harrison SC,
Kirchhausen T,
and
Pearse BMF
Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography.
Mol Cell
3:
761-770,
1999[ISI][Medline].
17.
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[ISI][Medline].
18.
Niswonger, ML,
and
O'Halloran TJ.
A novel role for clathrin in cytokinesis.
Proc Natl Acad Sci USA
94:
8575-8578,
1997
19.
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
20.
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[Abstract].
21.
Pypaert, M,
Lucocq JM,
and
Warren G.
Coated pits in interphase and mitotic A431 cells.
Eur J Cell Biol
45:
23-29,
1987[ISI][Medline].
22.
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[Abstract].
23.
Robinson, MS.
Coats and vesicle budding.
Trends Cell Biol
7:
99-102,
1997[ISI].
24.
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[ISI][Medline].
25.
Schmid, SL.
Clathrin-coated vesicle formation and protein sorting: an integrated process.
Annu Rev Biochem
66:
511-548,
1997[ISI][Medline].
26.
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
27.
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[ISI][Medline].
28.
Stoorvogel, W,
Oorschot V,
and
Geuze HJ.
A novel class of clathrin-coated vesicles budding from endosomes.
J Cell Biol
132:
21-33,
1996[Abstract].
29.
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[ISI][Medline].
30.
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[Abstract].
31.
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[Abstract].
32.
Ybe, JA,
Brodsky FM,
Hofmann K,
Lin K,
Liu S-H,
Chen L,
Earnest TN,
Fletterick RJ,
and
Hwang PK.
Clathrin self-assembly is mediated by a tandemly repeated superhelix.
Nature
399:
371-375,
1999[ISI][Medline].
33.
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
34.
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[ISI][Medline].
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