Download cDNA-derived molecular characteristics and antibodies to a new

Journal of Cell Science 104, 19-30 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
19
cDNA-derived molecular characteristics and antibodies to a new
centrosome-associated and G2/M phase-prevalent protein
Karsten Rothbarth1, Christian Petzelt2, Xiang Lu1,3, Ivan T. Todorov4, Gaby Joswig1,
Rainer Pepperkok5, Wilhelm Ansorge5 and Dieter Werner1,*
1Division of Cellular Biochemistry, German Cancer Research Center, P.O. Box 10.1949, W-6900 Heidelberg, FRG
2Laboratoire International de Biologie Cellulaire Marine, F-85 350 Ile d’Yeu, France
3Present address: Institute of Biological Sciences, National Research Council of Canada, Ottawa, ON, K1A OR6, Canada
4Institute of Cell Biology and Morphology, Bulgarian Academy of Sciences, G. Bonchev Street B-25, 1113 Sofia, Bulgaria
5European Molecular Biology Laboratory (EMBL), P.O. Box 10.2209, W-6900 Heidelberg, FRG
*Author for correspondence
SUMMARY
Differential screening of a murine RNA-based cDNA
library with cell cycle phase-specific transcripts released
a cDNA clone ( CCD41) to a mRNA (1.349 kb) which,
according to the mode of its detection, increases as
expected during the cell cycle. The molecular characteristics of the protein (27 103 Mr) encoded by this
mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein.
Immunofluorescence studies performed with PtK2 cells
revealed that the amount of the antigen specified by the
CCD41 sequence increases during the cell cycle out of
proportion with the DNA content. In G1 phase cells, the
antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes
also detectable in perinuclear vesicles that increase in
number and size, reaching a maximum in G2 phase cells.
The centrosomal location of the CCD41 antigen was
investigated in relation to another centrosomal antigen,
INTRODUCTION
One approach to obtain biological probes for proteins
involved in cellular structures or functions of interest is
based on differential screening of cDNA libraries. This
approach is designed to detect those mRNAs with altered
cytoplasmic abundances during phenotypic changes or
other biological events including cell cycle stages (Lu et
al., 1990). Principally, there exist three different ways of
regulation by which gene products actively involved in the
cell cycle control could achieve their optimal levels of
activities: (a) the gene is expressed at high level only during
the time when its product is required for cell cycle progression; (b) the mRNA is translated only when the protein is needed for special cell cycle events; (c) the (inactive) gene product is post-translationally modified, resulting
in an ‘active’ product when required. Of course, only those
centrosomin A. Since the latter antigen is detected by a
monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the
cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome
and to add new information about the general architecture of the organelle and its changes during the cell
cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the
centrosome, the CCD41-encoded protein appears to be
localized as a compact structure inside the centrosome.
Its epitopes are exposed throughout the cell cycle except
during a brief period immediately after the formation
of the daughter centrosome.
Key words: cell cycle, cell cycle phase-specific transcripts,
differential hybridization, cDNA, post-translational modification,
fusion protein, PtK2 cells, centrosome, CCD41, centrosomin A
genes that belong to group (a) are detectable by the differential screening approach.
The majority of the known cell cycle phase-dependent
and transcriptionally regulated genes are those required for
DNA synthesis, coordinating DNA replication tightly with
the cell cycle. The G2/M transition is another decisive
boundary marked by the activity of a family of genes,
termed ‘cyclins’ (reviewed by Nurse, 1990). For example,
human cyclin B has been reported to be regulated at the
level of transcription because its mRNA increases in G2
phase severalfold over the amount present in G1 phase
(Pines and Hunter, 1989). However, there must be other
cell cycle genes belonging to group (a) that have not been
discovered. Accordingly, we used the differential screening
approach in order to detect new genes differentially
expressed during different cell cycle stages.
In this paper we describe the characterization of a cDNA
20
K. Rothbarth and others
(CCD41) reflecting a transcript with cell cycle phase-dependent abundance. Moreover, we used recombinant DNA
techniques to obtain antibodies to the CCD41 product,
which were used to verify the cell cycle stage-dependent
expression of the corresponding protein and to obtain information about the protein’s localization and other characteristics, without biochemical isolation of the protein.
MATERIALS AND METHODS
Recloning and sequencing of the CCD41 insert
The insert of the recombinant phage λCCD41 contains an internal EcoRI recognition sequence. In order to reclone the complete
insert in the EcoRI-digested and alkaline phosphatase-treated
pBluescript vector (Stratagene), amplified phage DNA was only
partially digested with EcoRI. Inserts released from the phage
arms, but not cleaved at the internal EcoRI site, were extracted
from low melting point agarose gels and ligated with the EcoRI
and alkaline phosphatase-treated pBluescript vector. The insert of
the resulting plasmid pCCD41 was sequenced according to Sanger
et al. (1977) by means of the DNA sequencing kit from Pharmacia. The insert was sequenced in both directions using commercially available M13 as well as custom-made primers and reverse
primers.
Production of antigenic fusion protein and
preparation of antibodies
An expression vector was constructed which could be induced to
express a recombinant β-galactosidase/CCD41 fusion protein. For
this purpose, the insert of the plasmid pCCD41 was released by
HincII / EcoRI. The recessive EcoRI end was made blunt and this
fragment was inserted into the SmaI site of the prokaryotic
expression vector pEX2 (Boehringer). The orientation of the
CCD41 insert in the expression plasmid pEX-CCD41 was determined by the size of BamHI restriction fragments. The fusion protein was expressed in Escherichia coli pop2136 (Boehringer),
where it was deposited in inclusion bodies consisting essentially
of the fusion protein (not shown).
Pre-immune serum was taken from rabbits. This was followed
by four successive subcutaneous injections of 1 mg of protein contained in inclusion bodies at intervals of three weeks. The antigenic fusion protein was emulsified in Freund’s complete adjuvant (first injection) or Freund’s incomplete adjuvant in successive
injections. Immune serum was collected two weeks after the last
injection. The serum was pre-adsorbed by treatment with an acetone powder prepared according to Harlow and Lane (1988) from
inclusion bodies produced in E. coli after transfection and induction of the pEX2 vector without insert. This procedure reduced
the titer of antibodies directed against the β-galactosidase portion
of the fusion protein and other E. coli proteins.
Protein blots
SDS-polyacrylamide gels (12%, w/v) were loaded with 14Clabelled marker proteins (Amersham) and protein fractions from
Ehrlich ascites tumor cells grown in vivo. Nuclear and cytoplasmic fractions of these cells were prepared by the method of
Mamaril et al. (1970). Briefly, freshly harvested cells were swollen
in a hypotonic buffer and nuclei were released by 8-10 strokes in
a Dounce homogenizer. The nuclei were collected by centrifugation. The supernatant fraction was used directly to prepare the
samples. Nuclei were first digested with DNase I (4˚C) and the
residual material was dissolved in sample buffer. About equal
amounts of protein of the nuclear and of the cytoplasmic fractions
were loaded. Following electrophoresis a section of the gel loaded
with BSA-marker, nuclear and cytoplasmic protein fractions was
stained with Coomassie Blue. Another section of the same gel
loaded with 14C-labelled marker proteins, nuclear and cytoplasmic protein fractions was electro-blotted to nitrocellulose (Schleicher & Schüll, BA85). The blots were treated as follows: incubation in blocking solution (PBS containing 0.5% casein (Sigma)
and 0.1% sodium deoxycholate, 12 hours, 4˚C), incubation with
diluted serum (1:1,000 in PBS containing 1% BSA, 12 hours,
4˚C), three washes with PBS containing 0.05% Tween 80 (Serva),
incubation with blocking solution as described above, incubation
with 20 ml of PBS containing 1% BSA and 2 µCi of 125I-labelled
anti-rabbit Ig (Amersham) for 2 hours at room temperature, three
washes with PBS containing 0.05% Tween 80. The blots were
dried and exposed to Kodak X-Omat film. Contact prints of autoradiographs are shown.
In vitro translation of pCCD41 in vitro transcripts
and in vitro post-translational modification
The plasmid pCCD41 was linearized with SspI and transcribed in
vitro from the T3 promoter of the vector. The trancripts were either
used to stimulate reticulocyte lysates (BRL) without processing
fractions and reticulocyte lysates supplemented with different
amounts of microsomes from canine pancreas (Boehringer)
designed to induce co-translational processing of proteins (Blobel
and Dobberstein, 1975; Kurjan and Herskowitz, 1982; Rothblatt
and Meyer, 1986). Assay conditions were as recommended by the
manufacturers of the lysate and the microsomal fraction. The
translation assays were not treated with proteases before gel electrophoresis on 12% (w/v) SDS-polyacrylamide gels.
Cell culture and synchronization procedures
PtK2 cells were maintained in MEM-medium supplemented with
5% fetal calf serum, non-essential amino acids, and 30 mM
HEPES, pH 7.2, in a 5% CO2 atmosphere at 36˚C. To obtain a
high level of synchronously developing cells, semi-confluent cultures were shaken off, the resulting mitotic cells in the supernatant
were centrifuged (1000 g, 5 min) and resuspended in fresh
medium. Such a procedure gave better than 80% initial synchrony.
The cells were then seeded onto coverslips and processed for
immunofluorescence at various time intervals.
Immunofluorescence
Cells were first washed once in PBS (37˚C). For double immunofluorescence detection of centrosomin A and CCD41 cells were
permeabilized with buffer A for 5 min. For double immunofluorescence detection of CCD41 and microtubules they were treated
for the same time with buffer B. Such different treatments are
necessary because permeabilization of the cells with buffer A
destroys a substantial part of labile microtubules whereas the
staining of the centrosome remains unaffected in this buffer.
Treatment with buffer A has been chosen because permeabilization results in a much reduced background staining. Buffer A:
0.1 M PIPES; 2 mM EGTA, 1 mM MgCl2, 0.1 % Triton X-100,
pH 7.2; buffer B: same, but without Triton. In both cases the pH
value was adjusted with KOH. Thereafter, the cells were washed
with buffer B and fixed in cold ethanol (−5˚C) for 7 min. After
two washes with buffer B the cells were incubated with 1% BSA
in PBS for 10 min to reduce unspecific staining, which was followed by incubation for 45 min at 37˚C in sequence with the
antibodies. CCD41 antibody: rabbit-derived polyclonal antibody,
working dilution was 1:500 in 1% BSA, PBS. Centrosomin A
antibody (Joswig and Petzelt, 1990; Joswig et al., 1991): affinity-purified mouse monoclonal antibody, working dilution was 1
µg/ml of 1% BSA, PBS. α-Tubulin antibody (Sigma): mouse
monoclonal antibody against α-tubulin, working dilution 1:1000
in 1% BSA, PBS. After several washes with PBS the cells were
Centrosome-associated G2/M phase-prevalent protein
incubated with a mixture of the two secondary antibodies, RITClabelled anti-rabbit antibody and FITC-labelled anti-mouse antibody (Jackson Lab., West Grove, PA), working dilution 1:300 in
1%BSA, PBS, for 30 min at 37˚C. After three washes with PBS,
5 min each, the cells were incubated for 10 min in 0.1 M Tris,
pH 9.2, to reduce further background staining, and treated briefly
with H 33342, 0.1 µg/ml, in order to label the DNA. After a final
wash in PBS the cells were mounted in 25 mg/ml 1,4-diazabicyclo(2,2)octan (Aldrich, Steinheim, Germany) dissolved in glycerol/PBS (9:1, v/v).
Single cell fluorometry and microscopy
The system for single cell fluorometry and quantification of DNA
contents will be described in detail elsewhere (Pepperkok et al.,
unpublished). Briefly, PtK2 cells were immunostained with the
CCD41 antibody and their nuclei with Hoechst dye 33258. The
images were recorded and digitized with an inverted microscope
(Zeiss) equipped with appropriate optical filters and a SIT low
light level video camera coupled to a DVS Realtimeboard (Hamahatsu), which allows real-time enhancement of the images by
recursive filtering, background subtraction or pseudocolour representation. For off-line improvement of the images and quantitation of fluorescence an IBAS Image Processing System (Zeiss)
was used. Images of the staining with antibody and with Hoechst
dye were loaded into two separate image buffers of the IBAS
system. Both images were corrected for background fluorescence
using the IBAS function BLOWGR (IBAS reference manual).
Examples of such images are shown in Fig. 2 (below). Cell nuclei
were detected and localized automatically in the image for
Hoechst dye staining by a segmentation procedure using a
dynamic threshold. For each image pixel the mean grey value of
its neighbourhood region was calculated using an average lowpass Filter (IBAS reference manual). This mean grey value plus
a constant offset determined the local threshold value. Image
pixels with grey values below the threshold were recorded as
background, while those with grey values above the threshold
were identified as objects, e.g. cell nuclei. The amount of Hoechst
dye per nucleus (integrated optical density) was calculated by multiplication of the average intensity with the area of cell nuclei,
which reflects the relative amount of DNA per nucleus (Crissman
et al., 1985). Antibody-stained dots were recorded by visual
inspection of the corresponding image.
Other immunostained preparations were viewed using the
AXIOVERT 405 (Zeiss) with a ×100 Plan-Neofluar. Photographs
were taken using either Kodak Tri-X 400 or Ektachrome 400 film.
Biocomputing
The program package HUSAR (Heidelberg unix sequence analysis resources), supplied by S. Suhai, German Cancer Research
Center, Heidelberg, FRG, was used for inspection of nucleotide
and amino acid sequences. Especially, the fast search program
FASTA of Pearson and Lipman (1988) was applied to search for
similar sequences in nucleotide databases (EMBL release 12/91
and GenBank release 9/91) and in protein data bases (PIR, release
9/91, SwissProt, release 9/91). Putative post-translational modification sites were detected by means of the comparison tables of
A. Bairoch, Medical Biochemistry Department, University of
Geneva, Switzerland. Signal sequence analysis was performed
according to Heijne (1986).
RESULTS
Origin and characteristics of the recombinant
cDNA CCD41
The cDNA clone λCCD41 was detected in a murine RNA-
21
based λgt10 cDNA library by differential screening with
cell cycle phase-specific probes whose preparation and significance has been described previously in detail (Lu et al.,
1987; Lu and Werner, 1988; Lu et al., 1990). Briefly, cDNA
prepared from RNA of Ehrlich ascites cells grown in vivo
and separated into fractions of phase-synchronous cells by
centrifugal elutriation (Lu et al., 1987) was cloned into the
in vitro transcription vector pBluescript (Lu and Werner,
1988). In vitro transcripts of such cell cycle phase-specific
cDNA libraries were used to screen the λgt10 library (Lu
et al., 1990). The λCCD41 plaque was selected because it
showed a significantly stronger hybridization signal with
the G2/M phase-specific transcripts than with the S phasespecific transcripts. Only weak hybridization was observed
with the radiolabelled probe prepared by in vitro transcription of the G1 phase-specific cDNA library (Lu et al., 1990).
These results and appropriate controls with first-strand
cDNA probes published previously (Lu et al., 1990) indicate that the λCCD41 insert reflects a transcript whose copy
number per Ehrlich ascites tumor cell is low after mitosis
(G1-phase) but increases during the cell cycle progression,
reaching its maximum in G2/M phase.
Verification of the G2/M phase prevalence of the
protein encoded by the recombinant phage
CCD41
Since the expression of a protein is not necessarily correlated to its mRNA level, experiments were designed to
verify the overproportional increase of CCD41 mRNAencoded protein during the cell cycle. For this purpose, the
insert of the recombinant phage λCCD41 was recloned in
the prokaryotic expression vector pEX2 resulting in the
plasmid pEX-CCD41. E.coli pop2136 cells were transfected and induced to produce the recombinant fusion protein which was used to prepare antibodies directed against
the protein specified by the CCD41 insert.
Using immunofluorescence techniques, it was found that
the antigen is not species specific. For example, the antigen could be serologically detected in very different systems including sea urchin sperm (Fig. 1) and in PtK2 cells
(Figs 2-7).
With respect to the expected cell cycle phase-specific distribution of the antigen, it was found that PtK2 cell cultures
were composed of subpopulations of cells differing in their
antibody-dependent immunofluorescence patterns which
could be correlated with their DNA contents (Fig. 2, Table
1). Single cell fluorometry revealed that cells containing a
single antibody-stained dot belong to the group of cells with
G1 phase-specific DNA contents. A second group of cells
was identified with multiple bright fluorescent dots in the
cytoplasm close to the nucleus. Cells containing more than
five of such dots showed the typical G2 phase DNA content. A third fraction of cells could be grouped with an intermediate DNA content, apparently S phase cells. Cells
belonging to the latter fraction exhibited 2-3 antibodystained dots per cell. The cells in mitotic stages showed the
brightest antibody-specific fluorescence, indicating that the
antigen apparently reaches its highest levels during the
G2/M phase, which correlates well with the increased levels
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K. Rothbarth and others
Fig. 1. Sperm of the sea urchin
Paracentrotus lividus. (A) Stained
with CCD41 antibody; besides a
weak staining of the sperm
membrane the centrosome is clearly
marked (arrow). (B) Stained with
tubulin antibody. (C) Sperm nucleus
stained with Hoechst H 33342. Bar,
10 µm.
of the CCD41 sequence in G2/M phase cDNA libraries (Lu
et al., 1990).
Localization of the CCD41-encoded antigen in
PtK2 cells
Immunofluorescence techniques revealed that the antigen
encoded by the CCD41 sequence is associated with two
types of organelles. It could be localized in perinuclear vesicles that increase in number during the cell cycle (Fig. 2,
Table 1) and it forms an integral part of the centrosome
(Figs 1, 3-7).
Identification of the centrosome in PtK2 cells
The centrosomes of PtK2 cells can be accurately identified
and followed throughout the cell cycle by their immunoreactivity with the established monoclonal antibody to centrosomin A (Joswig and Petzelt, 1990; Joswig et al., 1991).
Accordingly, new centrosomal components, e.g. the
CCD41-encoded antigen, can be identified by the co-localization with centrosomin A. Moreover, the application of
the monoclonal antibody to centrosomin A together with
the polyclonal antibody to the CCD41-encoded protein
allows us to reveal the relative positions of the two antigens in the centrosome. Thus, by means of the two antibodies it became possible to investigate whether the two
proteins occupy the same part of the centrosomal structure
and whether the daughter centrosome shows the same composition as the mother centrosome.
Localization of the CCD41 antigen during the cell
cycle
The cell cycle of PtK2 cells lasts for about 22 hours. Their
centrosomes show a growth cycle which can be visualized
with the antibody to centrosomin A (Joswig and Petzelt,
1990). During the cell cycle progression the centrosome
becomes enlarged and changes its shape. For example, ringlike structures with appendices appear, and immediately
Table 1. Summary of the single cell fluorometry data
Fig. 2. Out of proportion increase in the CCD41-encoded protein
during the cell cycle progression of PtK2 cells. (A) Hoechst
33258-stained cells. (B) Same window as shown above but cells
in CCD41-specific staining. Single cell photometry shows that
cells containing only one single CCD41-specific dot (a) have a
DNA content typical of G1 cells. Cells containing more than five
CCD41-specific dots show a DNA content typical of G2 phase
cells (b). A summary of the data obtained by investigation of 69
cells is given in Table 1.
Number of CCD41-specific dots per cell
1
DNA content (arbitrary units; mean values) 154
Standard deviations
±50
Number of cells in the different groups
32
2-5
205
±50
17
>5
293
±125
20
Hoechst 33258 and CCD41 antibody-stained PtK2 cells of the kind
shown in Fig. 2 (n=69) were investigated with respect to their relative
DNA contents by single cell fluorometry. In addition, the CCD41-specific
dots were counted and registered. Cells showing mitotic figures are not
included in this table. The analysis of variance (F-test) confirmed that the
differences in the DNA contents of the cells grouped according the
number of CCD41 antibody-specific dots per cell are highly significant
(>99%).
Centrosome-associated G2/M phase-prevalent protein
23
Fig. 3. Double-immunolabelling of
centrosomes in PtK2 cells. (A), (C)
and (E) Centrosomes stained with the
centrosomin A antibody (arrow). It
should be noted that no other
structure is labelled in the cell.
(B), (D) and (F) The distribution of
the CCD41 antigen in the same cells.
(A) and (B) reflect stages
approximately 6 hours after mitosis.
In B, in addition to the centrosome,
only one small immunostained
vesicle is visible which points to the
low expression of the antigen at the
beginning of the cell cycle. (C) and
(D) Cells about 16 hours after
mitosis. (D) Numerous vesicles of
various sizes have appeared around
the nucleus while no change in the
staining intensity of the centrosome
can be observed. The separation of
the centrosomes marks the profound
difference in the centrosomal
localization of the CCD41-encoded
protein and centrosomin A. Whereas
in E the two centrosomes are clearly
marked with the centrosomin A
antibody, no such reaction is found at
the daughter centrosome when the
CCD41-specific antibody is applied
(F). See also Fig. 4 for a clear
demonstration of this phenomenon.
Bar, 10 µm.
before the breakdown of the nuclear envelope, the centrosomes are divided into the two daughter centrosomes that
migrate to the mitotic poles (Fig. 3A, C, E). Treatment of
PtK2 cells with the antibody to the CCD41 antigen shows
that this protein is another integral part of the centrosome
where it is present essentially throughout the cell cycle (Fig.
3B, D, F). The antibody to the CCD41 antigen does, however, not reveal the growth cycle of the centrosome in the
same clear way as can be observed with the antibody to
centrosomin A. The structure occupied by the CCD41 protein remains smaller and more compact throughout the
cycle than that marked by the antibody directed against centrosomin A. Are the two proteins superimposed in the centrosome? A comparison of the structures visualized by
application of the two antibodies shows that the two proteins occupy different regions of the centrosome with partial overlapping of centrosomal regions stained with both
antibodies. The CCD41 protein is located as a compact
24
K. Rothbarth and others
Fig. 4. In a number of cells the
centrosome can become quite large.
High magnification of this organelle
labelled with the two antibodies to
centrosomin A (A) and to the
CCD41-encoded protein (B) shows
the differences in the localization of
the two centrosomal proteins.
Centrosomin A appears as a
condensed fibrous structure whereas
CCD41 antigen apparently
comprises several compact subunits.
Bar, 10 µm.
body closer to the center of the centrosome than centrosomin A, which occupies a relatively larger space with filaments extending around the organelle (Fig. 4).
Localization of the CCD41 antigen during mitosis
During mitosis, at least until metaphase, the centrosome
appears in almost all cells as a compact body which can be
visualized by the appearance of its decoration with the antibody to centrosomin A (Joswig and Petzelt, 1990). During
the same period the CCD41-specific labelling is as well
located in a dense structure at the mitotic poles (Fig. 5).
However, the structure detectable with the antibody to the
CCD41-encoded protein does not show any shape changes
of this dense structure during all mitotic phases (not shown).
Although centrosomin A and the protein specified by the
CCD41 sequence are located close together at the site of
the centrosome, a profound and important difference is seen
when the separating centrosomes are examined with respect
to their compositions. From the beginning of the separation
process the two centrosomes can be permanently visualized
with the centrosomin A antibody. In contrast, the staining
with the CCD41-specific antibody remains restricted to the
mother organelle. No CCD41 is detectable within the newly
formed daughter centrosome at this stage (Fig. 6). Only
when the two centrosomes have reached the future mitotic
poles, does the daughter centrosome also become stained
with the CCD41 antibody, and no further difference in
CCD41-specific immunofluorescence intensities of the two
centrosomes can be observed. Therefore, for the first time,
a definite albeit brief period of centrosome ‘maturation’ is
demonstrated during which the two organelles are not alike.
Localization of the CCD41 antigen in non-centrosomal
compartments
In addition to its centrosomal localization, the CCD41 protein shows a pronounced periodic appearance in the cytoplasm. In early G1 phase cells the CCD41-specific staining
is virtually absent in all cellular areas other than the centrosome. After a few hours, additional staining appears
associated with small vesicle-like structures around the
nuclear envelope. These vesicles become abundant in G2
phase (Fig. 2), showing occasionally large lacunae of presumably fused vesicles. Their localization suggests a preference for the vicinity of the nuclear envelope. Perinuclear
vesicles of this type have been suggested as being involved
in sorting of proteins and in directing them to their proper
intracellular destination (Darnell et al., 1986). In mitotic
cells, after the dissolution of the nuclear envelope, the cell
is filled with a vesicular staining but still the two centrosomes are the most prominently stained structures. No preferential reaction of the CCD41-specific antibody with the
mitotic spindle could be observed (Fig. 5). The cell in mitosis remains positive for the CCD41 antibody until late
telophase, even at the time when the two daughter nuclei
have already re-formed. However, as soon as cytokinesis is
terminated, the major CCD41-specific staining of cytoplasmic structures disappears, only the centrosome continues to
react with the antibody to the CCD41 antigen.
CCD41-encoded protein and the microtubular
cytoskeleton
A fraction of polyploid giant cells is frequently found in
PtK2 cell populations. Cells belonging to this fraction are
permanently arrested in their cell cycle. Although the cell
cycle stage of such cells cannot be determined precisely,
they show, in addition to the CCD41-specific centrosome
decoration, multiple vesicular structures marked by the
CCD41-specific antibody around the nucleus (Fig. 7). In
this case, the centrosome was identified by the convergence
of the microtubules at this site. Again, no apparent relationship can be observed between the localization of the
vesicle-like structures containing CCD41 protein and the
distribution of the microtubules.
Centrosome-associated G2/M phase-prevalent protein
25
Fig. 5. Double-immunolabelling of PtK2
cells with α-tubulin antibody and CCD41
antibody. During mitosis, the CCD41
antigen is found in a very compact
structure at the mitotic poles. In addition,
vesicular staining of the cytoplasm is
seen. No preferential labelling of the
mitotic spindle can be observed. (A) and
(C) show the distribution of microtubules
while (B) and (D) exhibit the localization
of the CCD41 antigen. Bar, 10 µm.
Sequence data
In order to obtain sequence data, the λCCD41 insert was
recloned in the pBluescript vector (pCCD41) and
sequenced.
the nucleotide sequence coding for the initial 18 amino acid
residues showed a high degree of identity with the initial
sections of several cDNAs which code for hydrophobic or
membrane proteins (not shown).
Sequence characteristics of the pCCD41 cDNA
Fig. 8 shows the nucleotide sequence of pCCD41. It reveals
that the insert of the plasmid pCCD41 reflects a complete
cDNA. The ATG start-codon is preceded by a 5′ non-translated region of 88 nucleotides with two in-frame stop
codons. The coding region has an open reading frame which
encodes 244 amino acids. The stop codon (TAA) at the 3′
end of the coding sequence is followed by an additional
475 nucleotides of untranslated cDNA. The poly(A) tail is
preceded by sequences that reflect putative polyadenylation
signals (ATAAA, ATTAAA). Neither the complete
nucleotide sequence nor longer sections of the sequence
were found in the most recent data base releases, which
indicates that the cloned sequence specifies a protein whose
cDNA has not yet been isolated and sequenced before. Only
Amino acid sequence characteristics of the
polypeptide encoded by pCCD41
The nucleotide sequence of the open reading frame of the
pCCD41 recombinant codes for a 27.14 × 103 Mr precursor polypeptide. The N-terminal section of the deduced
polypeptide exhibits a sequence motif that is typical of
secretory or transmembrane proteins (Perlman and Halvorson, 1983). The three domains characteristic for a transmembrane leader peptide are found in this section. The
short N-terminal domain is four amino acid residues in
length and ends with a basic residue (K). This domain is
followed by the hydrophobic core (amino acid positions 513) and the third domain with the putative signal sequence
cleavage site begins with the helix-breaking residue (P).
Based on the rules and probability tables of Heijne (1986),
26
K. Rothbarth and others
Fig. 7. Double-immunolabelling of PtK2 cells with α-tubulin
antibody (green fluorescence) and CCD41 antibody (red
fluorescence). A comparison between the distribution of
microtubules and the CCD41 antigen does not display a specific
relationship between the localization of the CCD41-positive
vesicles and the arrangement of the microtubules. In contrast, the
cytocenter reacts positively with CCD41 antibody as expected for
a centrosome marker (arrowhead). (A) A cell 6 hours after
mitosis. The CCD41 label is almost exclusively associated with
the centrosome. Only two small additional vesicles are found in
this cell that are labelled with CCD41-specific antibody. In
contrast, (B) shows a PtK2 cell, 16 hours after mitosis, containing
numerous vesicles of various sizes that fill the cytoplasm and that
are arranged in the vicinity of the nucleus. It should be noted that
no relation between these vesicles and the arrangement of the
microtubules can be observed. As in (A) the cytocenter is stained
clearly with the CCD41 antibody (arrowhead). Bar, 10 µm.
portion of PEST residues in this region amounts to 56%
while the preceding and succeeding windows of equal
lengths contain only 33% and 10% PEST residues, respectively.
Fig. 6. Double-immunolabelling of PtK2 cells with centrosomin A
antibody (green fluorescence) and CCD41 antibody (red
fluorescence). Immediately after the separation of the two
centrosomes, just before mitosis, only the mother organelle
remains stainable with centrosomin A antibody as well as CCD41
antibody. The daughter organelle reacts only with centrosomin A
antibody (arrows). Bar, 10 µm.
the most likely cleavage site in this signal sequence is to
be expected between amino acid positions 17 (A) and 18
(F). Consequently, the mature polypeptide has an expected
Mr of 25.3 × 103. Since the protein shows no ER-retention
signal (Pelham, 1988), the possibility cannot be excluded
that a portion of this protein is secreted. However, the
immunofluorescence experiments (Figs 1-7) show that at
least a significant portion of the protein is retained in cells.
PEST residues
Another sequence feature of the polypeptide chain is of
interest with respect to the rapid degradation of the cellular protein during M/G1 transition. Regions that are flanked
by basic residues and rich in proline (P), glutaminic acid
(E), serine (S) and threonine (T) are considered to indicate
short half-lives, or stage-dependent conditional short halflives, of cellular proteins (Rogers et al., 1986). A region of
this type is found in the CCD41-encoded polypeptide
between amino acid positions 103 (K) and 131 (H). The
Post-translational modification predictions for CCD41
The amino acid sequence of the polypeptide encoded by
pCCD41 was investigated with respect to putative functional sites and with respect to consensus motifs for posttranslational modifications. No motifs were found that indicate the enzymic activity of the protein. However, a
number of sites were detected that reflect potential sites
for modification by phosphorylation. Protein kinase C
exhibits a preference for the phosphorylation of serine or
threonine residues that reside close to a C-terminal basic
residue (Woodget et al., 1986; Kishimoto et al., 1985).
Motifs meeting this prediction are located at amino acid
positions 145 (SFR), 167 (TLK), 207 (TGR) and 237
(SGR). Other sites of the protein meet the consensus pattern for casein kinase 2 (S or T)-(2X)-(D or E) (Pinna,
1990). Such patterns can be found at amino acid positions
89 (TQFE), 177 (TRYE) and 188 (TCVE). Finally, the protein comprises strings that reflect potential asparagine glycosylation sites. The consensus sequence for asparagine
glycosylation is (N)-(X, but not P)-(S or T)-(X, but not P)
(Marshall, 1972; Bause, 1983; Gavel and Heijne, 1990).
Asparagine residues that meet these conditions are found
at amino acid positions 72 (NVTI), 139 (NGSA), 165
(NFTL) and 175 (NRTR).
Evidence for glycosylation of CCD41
The consensus patterns for potential glycosylation sites
detectable in the amino acid sequence of CCD41 are of special interest because the cellular protein shows a higher
molecular mass than the polypeptide encoded by the
pCCD41 cDNA. Western blots probed with the CCD41
antibody result in a specific signal indicating a protein that
is as large as 50 × 103 Mr (Fig. 9). Interestingly, this mature
cellular protein is mainly detected in the ‘nuclear’ fraction,
which seems to conflict with the cellular distribution
detected by immunofluorescence techniques (Figs 2-7).
However, it is well known that many cytoplasmic components, including centrosomes (Mitchison and Kirschner,
Centrosome-associated G2/M phase-prevalent protein
Fig. 7
27
28
K. Rothbarth and others
Fig. 8. Nucleotide sequence of the cDNA clone pCCD41 and its
deduced amino acid sequence. Putative phosphorylation sites are
underlined. Amino acid residues of consensus sequences for
asparagine glycosylation sites are connected by dashes.
1984), remain rather firmly attached to nuclei and become
thereby co-isolated with nuclei. Since CCD41 is predominantly detected in centrosomes and in perinuclear vesicles
(Figs 2-7), it is not surprising that the majority of this antigen is co-isolated with nuclei. The apparent size of the cellular protein is best explained by a post-translational modification through glycosylation. This interpretation is
supported by the experimental data shown in Fig. 10. This
figure shows that in vitro translation of the cDNA transcripts results in a polypeptide of the expected size (27.14
× 103 Mr). Addition of microsomal fractions containing
signal peptide cleavage factors and other post-translational
modification factors including glycosylation activities
(Blobel and Dobberstein, 1975; Kurjan and Herskowitz,
1982; Rothblatt and Meyer, 1986) could not reduce the size
of the translation product. In contrast, the translation product increased in the presence of the co-translational processing fraction, which is most likely due to the potential
of these fractions to induce in vitro glycosylation. Although
the Mr of the in vitro glycosylated product is smaller than
that of the cellular protein found in Ehrlich ascites cells, it
is conceivable to conclude that glycosylation is the posttranslational modification event which explains the difference in size between the pCCD41-encoded polypeptide and
the cellular protein. Consequently, it is likely that CCD41
is a cellular glycoprotein.
DISCUSSION
Theoretically, the differential hybridization approach is a
Fig. 9. Estimation of the size of the CCD41-encoded protein in
Ehrlich ascites tumor cells. (A) Coomassie Blue-stained gel
section; M, BSA marker; N, nuclear protein fraction; C,
cytoplasmic protein fraction. (B) Contact print of the electroblot
of the gel section probed with CCD41-specific antibody and
125iodine-labelled anti-rabbit Ig; N, nuclear protein fraction; C,
cytoplasmic protein fraction. The positions of 14C-labelled marker
proteins on the blot (lane not shown) are indicated. The proteins
were analysed on a 12% (w/v) SDS-polyacrylamide gel.
useful method of identifying genes expressed differentially
during dynamic biological processes. However, limited
amounts of stage-specific mRNA and standardization of
stage-specific mRNA probes are major problems with this
technique. Previously, we proposed that the construction of
stage-specific cDNA libraries in in vitro transcription
vectors and the use of stage-specific in vitro transcripts,
instead of first-strand cDNA, could eventually overcome
these problems (Lu et al., 1990). By application of this
method we detected a recombinant clone whose distribution in cell cycle stage-specific cDNA libraries appeared to
be of special interest (Lu et al., 1990). From the distribution of the CCD41 sequence in phase-specific cDNA
libraries it could be suggested that this cDNA reflects a
mRNA encoding a translation product that is prevalent in
G2/M phase and rare in G1 phase. Since protein levels are
not necessarily correlated with their mRNA levels it was
of interest to verify the phase-prevalent distribution of the
translation product at the cellular level. Our results show
that this could be achieved by recombinant DNA techniques. The protein encoded by the CCD41 sequence could
be expressed in E.coli and antibodies directed against the
recombinant protein enabled us to verify that the level of
the cellular protein specified by the CCD41 sequence
increases out of proportion during the cell cycle progression of, e.g., PtK2 cells. This result suggests that the different levels of the CCD41 sequence in phase-specific
cDNA libraries reflect true changes of the corresponding
mRNA during the cell cycle. Consequently, this example
shows that in vitro transcripts of carefully amplified cDNA
libraries prepared in in vitro transcription vectors can
Centrosome-associated G2/M phase-prevalent protein
Fig. 10. In vitro translation of pCCD41 cDNA in vitro transcripts
in the absence and in the presence of microsomes from canine
pancreas. Samples were analysed by SDS-polyacrylamide gel
electrophoresis and autoradiography. The figure shows a contact
print of the autoradiography of the dried gel. M, 14C-labelled
marker proteins; (a) in vitro translation assay without addition of
microsomes; (b)-(d) in vitro translation assays with increasing
amounts of microsomes. The proteins were analysed on a 10%
(w/v) SDS-polyacrylamide gel.
replace stage-specific first-strand mRNA populations in differential hybridization experiments and that significant
clones can be obtained by this procedure.
Several characteristics of the protein identified by this
approach are remarkable: its greatly increased transcription
and translation during the G2 phase, and its continuous
localization at the centrosome throughout the cell cycle,
except for a very brief phase when the daughter centrosome
is formed. The latter quality suggests that the protein is
involved in a typical function of the centrosome whose central role in cell cycle progression (Maniotis and Schliwa,
1991) and the control of the three-dimensional structure of
the cytoplasm has been well established (reviewed by
Mazia, 1987; Bornens, 1991). However, if the CCD41encoded protein is only involved in a centrosomal function,
why do we find such an intense extra-centrosomal accumulation during the G2/M phase in the cytoplasm of PtK2
cells? It could be argued that the CCD41 protein is somehow related to other proteins whose levels increase greatly
during the cell cycle and which are known to activate periodically protein kinases (Murray and Kirschner, 1989;
Nurse, 1990). Proteins with these characteristics have been
termed cyclins. However, at present we have found no indi cation of a structural relationship between the CCD41 protein and the cyclins. The CCD41 protein contains, like the
cyclins, a PEST motif (Rogers et al., 1986), which is considered to be responsible for a stage-dependent short halflife. However, no further reasons for a closer structural relationship with known G2/M regulators could be detected on
the sequence level. Further studies are designed which
29
should reveal whether CCD41 is functionally related to
G2/M phase regulators; for example, whether the CCD41
protein is able to form complexes with protein kinases.
The available data add some valuable information to the
general architecture of the centrosome. It appears from our
results as well as from those of others (Mazia, 1987) that
the centrosome is a complex structure composed of several
different proteins. It undergoes shape changes during the
cell cycle, probably reflecting its control function in the
cell. However, not all proteins that are part of this structure exhibit the same shape changes of the organelle, which
points to the existence of core and surface proteins. Since
the antibody against centrosomin A indicates the shape
changes more clearly than the antibody to CCD41 it is
suggested that centrosomin A belongs to the centrosomal
surface proteins instead. In contrast, CCD41 seems to
reflect a more dense core component. In addition, the antibody to CCD41 points to the partial non-identity of the two
centrosomes just after the formation of the daughter centrosome.
It should be noted that several other proteins with known
or unknown functions have been identified which are either
components of the centrosome or are at least centrosomeassociated. γ-Tubulin has been shown to be present at the
centrosome in a broad range of eukaryotes (Aspergillus
nidulans: Oakley et al., 1990; Schizosaccharomyces pombe:
Horio et al., 1991; Drosophila: Zheng et al., 1991; Xeno pus and mammalian cells: Stearns et al., 1991) and its role
in nucleating microtubules is plausible (Oakley and Oakley,
1989; reviewed by Oakley, 1992). For example, it has been
shown that microinjected antibodies to γ-tubulin bind to
native centrosomes at all stages of the cell cycle and interfere with the regrowth of microtubules after artificial
depolymerization (Joshi et al., 1992). Cyclin B has been
convincingly demonstrated within the astral microtubules
in Drosophila embryos (Maldonado-Codina and Glover,
1992) as well as in a variety of other cell types. However,
a clear distinction should be made between components
accumulating in the vicinity of the centrosome, such as
cyclin B (Maldonado-Codina and Glover, 1992) or Ca2+transport vesicles (Petzelt and Hafner, 1986), and those
components located in the centrosome, such as γ-tubulin,
various antigens to which antibodies can be found in the
sera of patients with autoimmune diseases, centrosomin A
(Joswig and Petzelt, 1990), and CCD41 (this paper), respectively. Whereas the former antigens may reflect characteristics of the cytoplasmic environment necessary for the
function of the centrosome, the latter, as part of the structure itself, may provide more direct information on the various centrosomal functions.
So far, a detailed functional analysis at the genetic level
has been performed only with one centrosomal component,
γ-tubulin (Oakley et al., 1990; Oakley and Oakley, 1989;
reviewed by Oakley, 1992). The antibodies and the genetic
probes to the two new centrosomal proteins, centrosomin
A and CCD41, now provide the required tools for the functional analysis of other intrinsic centrosomal proteins.
This work was supported by grants of the Deutsche Forschungsgemeinschaft (Werner, We 589/2-2) and by the European Com-
30
K. Rothbarth and others
munities (Petzelt, Contract SC1*.CT91.0640). One of us (I.T.) is
grateful for a fellowship of the German Cancer Research Center.
The nucleotide sequence of full-length CCD41 cDNA published here has been deposited at the EMBL sequence data bank
and is available under accession number X63748.
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(Received 16 April 1992 - Accepted 23 September 1992)