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Molecular and Cellular Biology, November 1998, p. 6816-6825, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cytoplasmic Dynein Intermediate-Chain Isoforms with
Different Targeting Properties Created by Tissue-Specific
Alternative Splicing
Dmitry I.
Nurminsky,1,*
Maria V.
Nurminskaya,2
Elizaveta V.
Benevolenskaya,3,4
Yury Y.
Shevelyov,4
Daniel L.
Hartl,1 and
Vladimir
A.
Gvozdev4
Department of Organismic & Evolutionary
Biology, Harvard University, Cambridge, Massachusetts
021381;
Department of Anatomy and
Cell Biology, Tufts University School of Medicine, Boston,
Massachusetts 0211112;
University of
Missouri
Columbia, Columbia, Missouri
652113; and
Institute of Molecular
Genetics, Russian Academy of Sciences, Moscow 123182, Russia4
Received 13 April 1998/Returned for modification 5 June
1998/Accepted 14 August 1998
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ABSTRACT |
The intermediate chains (ICs) are the subunits of the cytoplasmic
dynein that provide binding of the complex to cargo organelles through
interaction of their N termini with dynactin. We present evidence that
in Drosophila, the IC subunits are represented by at least
10 structural isoforms, created by the alternative splicing of
transcripts from a unique Cdic gene. The splicing pattern
is tissue specific. A constitutive set of four IC
isoforms is expressed in all tissues tested; in addition,
tissue-specific isoforms are found in the ovaries and nervous tissue.
The structural variations between isoforms are limited to the N
terminus of the IC molecule, where the interaction with dynactin takes
place. This suggests differences in the dynactin-mediated organelle
binding by IC isoforms. Accordingly, when transiently expressed in
Drosophila Schneider-3 cells, the IC isoforms
differ in their intracellular targeting properties from each other. A
mechanism is proposed for the regulation of dynein binding to
organelles through the changes in the content of the IC isoform pool.
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INTRODUCTION |
Cytoplasmic dynein is a multisubunit
complex composed of two heavy chains, three intermediate chains (ICs),
several light ICs, and one light chain (11, 20). It acts as
a minus end-directed microtubule motor, participating in a number
of events including anterograde organelle movement (1, 6,
22), mitosis (25), nuclear migration (28),
slow axonal transport in nervous tissue (7), and transport
from nurse cell cytoplasm to oocytes in Drosophila ovaries
(12). These events call for binding of the dynein complex to
multiple target organelles in the cell. Regulation of this binding is
also required to enable relocation of the dynein between targets during
the cell cycle and development of the organism. One of the proposed
mechanisms implemented in the cell cycle-dependent regulation of dynein
binding is through the phosphorylation of the subunits of the dynein
complex (17).
Although the heavy chain comprises the catalytic dynein subunit and is
capable by itself of the ATP-dependent moving force production on the
microtubules (14), the presence of other subunits is
apparently required for dynein function in vivo. For one class of these
so-called accessory subunits of cytoplasmic dynein, the IC subunits, a
key role in linking cytoplasmic dynein to the intracellular targets was
suggested and then proved (20, 23). In particular, the
N-terminal part of IC is directly involved in binding to the organelles
(23) through the interaction with p150/Glued, the major component of the dynactin complex (26). Dynactin, also a multisubunit complex, is an activator of dynein in vitro
(9) and is required for dynein function in vivo (4, 15,
16). Dynein and dynactin are colocalized in the cell, and
overexpression of components of the dynactin complex disrupts dynein
binding to organelles (5, 8).
Considering dynactin as a dynein "receptor" or at least a modulator
of dynein binding, the interaction of dynein ICs with dynactin is
likely to be the point where the regulation of dynein binding takes
place. A number of IC isoforms were detected, and the content of IC
isoform pool is highly regulated (21). The complexity of
IC isoforms is due to the expression of a family of structurally
different polypeptides, some of which are further modified by
phosphorylation (21, 26). The structural differences are
limited to the N-terminal part of the ICs, in the region essential for
dynactin binding (26). The phosphorylation is strongly
suggested to occur in the same region which contains the serine-rich
domain. Thus, the observed complexity of ICs presumably
provides a diversity in dynactin-mediated dynein binding to organelles.
This means that changing the content of the IC isoform pool would
result in relevant changes in dynein targeting.
The mechanism for generating the structural complexity of ICs has been
unclear. Alternative splicing of a limited number of transcripts was
suggested (26) but never shown directly. In this paper, we
demonstrate that in Drosophila, the structural IC
isoforms are created by the alternative splicing of transcripts from a single-copy Cdic gene. The isoforms differ in the
polymorphic region located near the N terminus of IC. The exact
positions of the polymorphic regions differ in ICs from
Drosophila and rats, suggesting independent evolution of IC
isoform complexity in the ancestry of distant orders.
The splicing pattern of Cdic and therefore the content of
the IC isoform pool appear to be tissue specific. In addition to the constitutive set, tissue-specific IC isoforms are present in
ovaries and neural tissue, where tissue-specific kinds of
dynein-dependent transport take place. The IC isoforms differ in
their intracellular targeting properties, thus providing the mechanism
for developmental regulation of dynein binding to organelles by
changing the content of the IC isoform pool.
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MATERIALS AND METHODS |
RNA isolation and Northern analysis.
Total RNA was isolated
from various developmental stages and from adult body parts of
Drosophila melanogaster and from adults of D. simulans with Trizol (Gibco-BRL). Poly(A)+ RNA was
purified from the total RNA preparations with Poly(A)-Tract magnetic
particles (Promega Corp).
For Northern analysis, 10 µg of total RNA or 2 µg of
poly(A)+ RNA was electrophoresed through a 1%
agarose-formaldehyde gel and transferred onto a Hybond N membrane in
10× SSPE (1× SSPE is 0.18 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA [pH 7.7]).
Single-stranded 32P-labeled RNA probes were generated by T7
RNA polymerase from the pTZ19R-based plasmid containing the sequence of
exon 6 (probe A in Fig. 2). Random priming with the Prime-It system
(Stratagene) was used to generate 32P-labeled DNA probes
from the same fragment A or from the fragment representing the first
680 bp of Cdic cDNA (probe B in Fig. 2). Hybridization
procedures were as described previously (19).
Southern hybridization with oligonucleotides.
Reverse
transcription-PCR (RT-PCR) products corresponding to IC isoforms
were separated in a 3% agarose-Tris-borate-EDTA (TBE) gel and
transferred to a Nybond N membrane in 0.5 M NaOH-1 M NaCl by capillary
blotting. The membrane was neutralized in 1 M ammonium acetate, air
dried, and baked for 90 min at 80°C. The following primers, covering
specific variable exon junctions, were synthesized (see Fig. 7): "iso2"
(5'-TTATTATGATGAATAC-3'), covering the v2/v3 junction
specific for Cdic2 and Cdic5; "iso2
(5'-CGGCGATGCTCATGCT-3'), covering the 4/v2 junction
specific for Cdic1, Cdic2, and Cdic5; "iso3" (5'-CGGCGATGATGAATAC-3'), covering the 4/v3 junction
specific for Cdic3; "iso4" (5'-CGGCGATGTGCTTGCA-3'),
covering the 4/v4 junction specific for Cdic4; and
"iso5" (TATATGGAGGACTGGT-3'), representing exon v1, specific
for Cdic5. The primers were labeled with 32P by
T4 DNA kinase and hybridized with the membrane in 4× SSPE-1% Sarkosyl, 0.1% each Ficoll-400, polyvinylpirrolidone, and sodium pyrophosphate for 2 to 14 h at the following temperatures: "iso2" at 42°C; "iso2 and "iso4" at 55°C; and "iso3" and "iso5" at
50°C. The membrane was washed in 100 mM sodium phosphate (pH
8.0)-1% Sarkosyl-1 mM EDTA three times for 15 min at room
temperature and then once in 4× SSPE-1% Sarkosyl for 20 min at
hybridization temperature.
DNA cloning and sequencing.
cDNA clones were obtained by
screening a 
ZAP cDNA library made from poly(A)+ RNA from
D. melanogaster ovaries (supplied by Stratagene Corp.). Individual lambda clones were converted into the plasmid form by in
vivo excision, and the inserts were transferred into the vector pSP72
and sequenced with an ABI 373A automated DNA sequencer after saturation
with gamma-delta transposon insertions (24). Sequence data
were analyzed with Sequencher software (GeneCodes Corp.).
The 5'- and 3'-RACE (rapid amplification of cDNA ends) PCR products
were generated with the Marathon system (Clontech), using
female
poly(A)
+ RNA as a template, and were sequenced after T-A
cloning in the
pCRII vector (Invitrogen).
A
D. melanogaster P1 genomic library was screened by a
PCR-based assay as described previously (
10). The P1 clone
containing
the cytoplasmic dynein IC genes was subcloned in the
SCAN
vector
(
18), and the subclone of interest was transferred
into the
vector pSP72 and sequenced as described above.
Plasmid constructs.
The green fluorescence protein (GFP)
fusion expression plasmids were made by inserting the Cdic
open reading frames (ORFs) upstream of the GFP ORF in pGreenLantern
(Gibco-BRL). ORFs containing the specific Cdic isoforms
were amplified by PCR with the corresponding cDNA clones as the
templates and with the primers DIC-F
(5'-GGTACCAGCTAATCGCCCCGAGAAATGGAT-3') and DIC-LL
(5'-GGCGGCCGCGTTCATCTTGATCTCGCTAAG-3'). The N-terminal domains were amplified with the primers DIC-F and DIC-R
(5'-GCGGCCGCACGCACCACGAACCGCTGGAAG-3'). PCR products were
cloned in the vector pCRII and transferred into the pGreenLantern as
KpnI-NotI fragments. Partial digestions with NotI were used when necessary.
All the PCR fragments used for plasmid construction were generated with
a polymerase mixture possessing proofreading activity
(Elongase;
Gibco-BRL), and their sequence was checked after cloning
in the pCRII
vector.
Cell culture transfections.
The D. melanogaster Schneider-3 cell culture was maintained in
Drosophila Shields and Sang M3 medium (Sigma) supplemented with 10% insect medium supplement (Sigma) at room temperature. For
transfection, the cells were plated on chamber slides (Falcon) at a
density of 5 × 105 to 7 × 105
cells/ml and the next day were transfected with the Lipofectin reagent
(Gibco-BRL). A 1.5-µg portion of DNA and 9 µl of Lipofectin were
used per 5 × 105 to 7 × 105 cells.
After 6 to 12 h, the medium was changed to M3 supplemented with
10% insect medium supplement; the cells were then allowed to grow for
another 1 or 2 days and fixed for 10 min with 3.7% paraformaldehyde at
room temperature. The cellular content was stained with propidium
iodide. Alternatively, for Golgi-specific staining, the cells were
permeabilized with 0.2% Triton X-100 for 15 min at room temperature
and incubated with 20 µg of rhodamine-labeled Lens
culinaris lectin per ml (Sigma). Lysosome-specific staining was
obtained by in vivo incubation of cells with 50 nM LysoTracker DND-99
(Molecular Probes) for 2 h before fixation. The cells were mounted
in Permount medium and imaged in a laser scanning microscope (Axiovert
100TV; Zeiss). The images were processed with Adobe Photoshop software.
Nucleotide sequence accession numbers.
All sequences were
deposited in GenBank and are available under accession no. AF070687 to
AF070699.
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RESULTS |
Multiple RNAs code for the cytoplasmic dynein IC proteins.
In
a previous study, we characterized a tandem repeat in cytological
region 19F of D. melanogaster (2). The unit
of this repeat is 7.2 kb long and contains a fragment of the
annexin X gene. It also contains a long ORF coding for a
polypeptide with high similarity to the dynein IC proteins. Using a
fragment of this ORF as a probe (probe A in Fig. 2), we were able to
detect two bands on a Northern blot, one of 2.4 kb and one of 2.8 kb (Fig. 1).
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FIG. 1.
A. Dynein IC transcripts in Drosophila. Samples
(10 µg) of total RNA isolated from various developmental stages of
D. melanogaster, as indicated at the top, along with
RNA samples from the heads of D. melanogaster adults or
D. simulans adults, were separated in a 1%
agarose-formaldehyde gel. Hybridization with probe A (Fig. 2) revealed
two major bands, corresponding to the Cdic and
Sdic transcripts. Only the Cdic transcripts were
detected in D. simulans. (B) Control hybridization with
the probe for the constitutively expressed gene oxen
(1a) shows sufficient RNA loading on all lanes. The numbers
on the right indicate the sizes of transcripts in kilobases.
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Using the same probe, we isolated numerous cDNA clones from a
D. melanogaster ZAP library. Six overlapping clones
were sequenced,
and the sequences were aligned, resulting in a
composite cDNA
sequence possessing an ORF for dynein IC polypeptide
(Fig.
2).
This composite sequence,
however, obviously lacked both the 3'
and 5' ends of the transcript,
since neither a poly(A) tail nor
a methionine initiation codon was
detected. The 3' end of the
RNA was unambiguously mapped by performing
3'-RACE and sequencing
several cloned PCR products. Mapping the 5' end
by 5'-RACE led
to the description of two major classes of RNAs
suggested from
Northern analysis. The sequences of the 5'-RACE products
could
easily be sorted in two subsets, the long and short subsets.
Aligning
the short 5' ends with the composite cDNA resulted in a
2.4-kb
sequence, apparently representing a 2.4-kb RNA. The
same alignment
with the long 5' ends produced a 2.8-kb sequence
corresponding
to the larger RNA.
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FIG. 2.
Cloning and characterization of Cdic and
Sdic cDNAs. ZAP clones are indicated by thin black
bars, and RACE products are indicated by shaded bars. In the composite
cDNAs, coding regions are black. The positions for the DIC-U and
DIC-LL primers, used to amplify cDNAs for Cdic
isoforms, and for the DICr-U and DIC-LL primers, used
for Sdic, are shown. A and B are the
Cdic/Sdic-specific and Cdic-specific probes,
respectively.
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The 70-kDa polypeptides encoded by the long, 2.8-kb
mRNAs have extensive homology to the cytoplasmic dynein ICs
from rats
and
Dictyostellium discoideum. The major stretch
of homology covers
more than 400 amino acids in the C-terminal part
of molecule,
which is 61% identical (79% similar) to the rat homolog
and 50%
identical (69% similar) to the protein from
Dictyostellium discoideum.
Included in the C-terminal region
are four WD-40 repeats (see
Fig.
6; a fifth repeat, described in
reference
27, is very degenerate
and is not shown
here). This set of repeats is extremely strongly
conserved among all
dynein ICs and probably accounts for the interaction
with other dynein
subunits.
An additional feature characteristic for the cytoplasmic dynein ICs is
the presence of a coiled-coil domain at the N terminus
followed by a serine-rich domain. Both these domains were detected
in
the 70-kDa polypeptide. Analysis of the alignment of this polypeptide
with other cytoplasmic dynein ICs revealed another conserved
stretch
of amino acids in the N-terminal region, called PPE/TQT
(Fig.
3).
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FIG. 3.
Comparison of the sequence of the N-terminal regions of
Cdic and other known cytoplasmic ICs from rats (GenBank accession no.
U39046) and Dictyostellium (accession no. U25116). Conserved
amino acids are outlined and presented in the consensus line.
Coiled-coil domains, shown by solid boxes, were predicted with the
PAIRCOILS (3) and COILS (13) algorithms. The
serine-rich domain and PPE/TQT conserved block are outlined by boxes.
The positions of variable regions in the rat IC (var-1 and var-2) and
the beginning of the variable region in Cdic (var) are indicated. Cdic
isoform shown is Cdic5b.
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Based both on sequence homology and structural similarity to the
cytoplasmic dynein ICs from rats and
Dictyostelium
discoideum,
the 70-kDa polypeptide encoded by the long mRNAs
was defined as
the
D. melanogaster cytoplasmic
dynein IC (Cdic). In contrast,
the 60.4-kDa polypeptide encoded
by the short 2.4-kb mRNA lacks
the N-terminal coiled-coil and
serine-rich domains characteristic
of ICs of cytoplasmic dyneins,
although it shares most of its
sequence with the Cdic polypeptide. It
was shown to represent
a novel sperm-specific IC subunit of axonemal
dynein and was called
Sdic (
17a).
Amplification and sequencing of full-length
Cdic cDNAs
revealed multiple transcripts that differ by small insertions and
deletions
in the N-terminal part of the ORF and apparently code for the
Cdic isoforms.
2.8-kb Cdic RNAs are transcribed from the
single-copy cytoplasmic dynein IC (Cdic) gene.
Although all dynein IC cDNAs were isolated with a fragment of
the 7.2-kb annexin-dynein repeat, the very 5' end of the
Cdic transcripts is not homologous to the repeated unit.
This sequence, represented by probe B specific for Cdic
transcripts (Fig. 2), was found to be unique in the genome on the basis
of Southern analysis and mapped by in situ hybridization in the site
19E, i.e., in close vicinity to the annexin-dynein repeat. Northern analysis demonstrated that, as expected, the same
Cdic-specific probe B hybridized with only the 2.8-kb
Cdic mRNAs and not with the 2.4-kb Sdic
mRNAs (data not shown).
When a P1 phage genomic library was screened for the
Cdic-specific sequence, three clones that also contained
the annexin-dynein
repeat were obtained. None of these three clones,
however, contained
the complete
annexin X gene located
at the end of annexin-dynein
tandem cluster (
2). Considering
the length of the tandem repeat
(about 10 copies at 7.2 kb each)
and the average length of a P1
clone (80 kb), these data suggest that
the
Cdic-specific sequence
is located in the vicinity of the
tandem cluster of annexin-dynein
repeats, at the opposite end from the
annexin X gene.
Cloning and sequencing of the corresponding genomic region
revealed the structure of the gene encoding the
Cdic
transcripts.
This
Cdic gene is located at the 5' end of
the tandem cluster,
and its 3' end is directly fused to the
initial 7.2-kb annexin-dynein
repeated unit. The exon-intron structure
of the 8.3-kb
Cdic transcription
unit was determined by
aligning the
Cdic cDNA sequences to the
genomic
sequence. A perfect match was obtained between
Cdic
cDNAs
and the exons of the
Cdic gene. A number of
significant differences,
however, were detected between the
Cdic genomic sequence and the
2.4-kb
Sdic
cDNA, indicating that, unlike 2.8-kb
Cdic cDNAs,
this
one does not represent the transcript from the
Cdic
gene. The
true origin of
Sdic transcripts was exposed, since
a perfect match
was achieved between the sequences of the
Sdic cDNA and the annexin-dynein
repeat (Fig.
4). The identity of 2.4-kb
Sdic mRNAs as the transcripts
from the annexin-dynein
repeat was further supported by the fact
that in
D. simulans, a close relative of
D. melanogaster that
does not have any repeated structure analogous to the annexin-dynein
repeat, no 2.4-kb
Sdic transcripts were found: the
only class
of dynein mRNAs detected corresponds to the 2.8-kb
Cdic mRNAs
(Fig.
1).
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FIG. 4.
Sequence comparison shows that Cdic cDNA
represents the transcripts from the Cdic gene and that
Sdic cDNA corresponds to the transcripts from the
annexin-dynein repeat. The entire Cdic gene sequence is
presented; for cDNAs and the annexin-dynein repeat, only the
differences are shown. Gaps introduced in the sequences are marked with
dots.
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Transcription of
Cdic changes throughout the development of
D. melanogaster (Fig.
1). The transcripts are abundant
in embryos
and adult flies and apparently are up-regulated in
the heads of
adult flies, but they are hardly detectable in larvae and
pupae.
Multiple Cdic isoforms are generated by alternative
splicing.
Previous data state that all Cdic mRNAs
are transcribed from the unique Cdic gene, even though
multiple variants of Cdic transcripts were detected.
Analysis of the exon-intron structure of the Cdic gene
demonstrated that these variants, coding for the Cdic isoforms, are
created by alternative splicing.
The transcription unit is 8.3 kb long and consists of 10 exons. The
first four exons (1 to 4 in Fig.
5) are
separated by relatively
small introns, as are the last three exons (5 to 7 in Fig.
5).
These two groups of exons are separated by 4.2-kb
"spacer" region
containing three "variable" exons, v1, v2, and
v3. Alternative
splicing of transcripts leads to the skipping of the
variable
exons, providing shortened versions of mRNAs apparently
carrying
the corresponding deletions in the dynein IC ORF (Fig.
5 and
6).
Additional
polymorphism of the mRNAs is created by using two alternative
splice acceptor sites preceding exon v1 and three alternative
splice
acceptor sites of the intron preceding exon d5, which also
results in
insertions/deletions in the same region of the dynein
IC ORF (Fig.
6).
Use of one of the acceptor sites preceding exon
v1 leads to the
frameshift and premature termination of translation
(isoform Cdic5a
in Fig.
6). Except for this one, as many as 10
full-sized Cdic
isoforms are generated by alternative splicing
of
Cdic
transcripts.
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FIG. 5.
Exon-intron structure of the Cdic gene. The
genomic sequence is shown at the top, with exons indicated by boxes.
Coding sequences are shown as solid boxes. The promoter is shown as
triangle. 1 to 7, constitutive exons present in all Cdic
mRNAs. v1 to v3, variable exons. Five classes of Cdic
transcripts are shown below the sequence.
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FIG. 6.
Sequence alignment of coding regions of the
Cdic gene with Cdic cDNAs. The genomic
sequence is at the top, with the gaps introduced in place of introns.
cDNA sequences are below, shown as a single line in
"constitutive" regions where they are identical and shown
individually in the variable region. A conceptual protein sequence is
shown at bottom; for the isoform Cdic5a, a frameshifted translation
of exon v1 is included (peptide 5a). Coiled-coil donains, outlined as
black boxes, were predicted with the PAIRCOILS (3) and COILS
(13) algorithms. In the N-terminal region, serine-rich
domain and the PPE/TQT conserved region are boxed. In the C-terminal
region, four of five WD-40 repeats (27) are underlined.
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The content of the Cdic isoform pool is tissue specific.
The representation of the Cdic isoforms in the total Cdic pool was
determined by two methods. First, RT-PCR was performed on the mRNA
isolated from the embryos and from the adult flies by using the primers
flanking the Cdic ORF, as shown in Fig. 2. PCR products were
cloned, and a number of randomly picked clones were sequenced,
resulting in the data presented in Table
1. Second, RT-PCR was carried out on the
RNA isolated from embryos and adult females and also from the
dissected female parts (ovaries, midguts, heads, and torsos) by using
the primers var-U (5'-GCAGGCTACGAACATTCCA-3') and var-L
(5'-GCAGGCCGTGGGTGAGATA-3'), positioned close to the variable region of the ORF. PCR products were purified and subjected to
10 cycles of mock sequencing with the Taq polymerase in the presence of deoxynucleoside triphosphates and primer var-L labeled with
32P by T4 DNA kinase. The reaction products were separated
in the sequencing gel and detected by autoradiography (Fig.
7A). The gel image was analyzed with
BioMax 1D software (Kodak), producing the quantitative data in Table
2. Females were used here to avoid interference with the testis-specific expression of the
Sdic. In males, similar representation of isoforms was
detected in the midgut, torso, and head.
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FIG. 7.
Tissue specificity of Cdic isoforms.
RT-PCR fragments were amplified across the variable region of
Cdic transcripts. (A) PCR fragments were labeled at one DNA
strand with 32P and separated in a 5% acrylamide
sequencing gel. The source of the RNA is indicated at the top. Lane M
contains marker fragments generated from the of cDNA clones
representing Cdic isoforms. (B) PCR fragments were
separated in a 3% agarose gel (1) and, after Southern
transfer, hybridized with oligonucleotides "iso2" (2),
"iso2 (3), "iso3" (4), "iso4" (5),
and "iso5" (6). The source of the RNA is indicated at the
top; individual cDNA clones Cdic1a, Cdic1b,
Cdic2a, Cdic3a, Cdic4, and
Cdic5b were used to generate the marker fragments in the six
right-hand lanes.
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The data presented in both tables are in good agreement, indicating
that the bulk of
Cdic mRNAs is represented by three
isoforms,
Cdic1a, Cdic2a, and Cdic2b. They usually make up over
70% of
Cdic mRNAs and are constitutively expressed in
flies and embryos and
in all tested body parts. The other eight
isoforms usually make
up to less than 30% of the pool. Only one of
these "minor" isoforms,
Cdic2c, is constitutively expressed at
low levels. All the others
demonstrate tissue specificity, being
differentially expressed
in the fly body. In particular, it appears
that the isoforms Cdic1b
and/or Cdic4, Cdic1c, and Cdic3a are
up-regulated in the ovaries
and Cdic5a and Cdic5b are overexpressed in
the head and, to a
lesser extent, in the torso. In these cases, the
representation
of some of the tissue-specific isoforms in the organ
(17% of Cdic1b/Cdic4
in the ovary and 23% of Cdic5b in the head) is
at the same level
as that of major constitutively expressed
isoforms.
Since isoforms Cdic1b and Cdic4 have exactly the same length
of variable region, they could not be resolved by gel
electrophoresis.
To discriminate between these two isoforms and to
confirm the
identity of the RT-PCR products seen in the sequencing gel,
they
were separated in a 3% agarose gel, transferred to the nylon
membrane,
and hybridized to the labeled oligonucleotides specific to
the
particular isoforms. As seen in Fig.
7B, Cdic4, as well as
Cdic3
and Cdic1b/Cdic1c, is abundant in ovaries, while Cdic1a and
Cdic2a/Cdic2b
are constitutively expressed. As expected, Cdic5a and
Cdic5b are
overexpressed in head and torso.
Cdic isoforms differ in intracellular distribution.
Structural variations between Cdic isoforms are limited to the
N-terminal region involved in the interaction with dynactin and binding
of the dynein complex to the organelles. We suggested that these
variations may impel the differences in the targeting properties of
Cdic isoforms. To check whether this may be true, Cdic isoforms
were expressed in Drosophila Schneider-3 cell culture, and
their intracellular distribution was analyzed. Since
Schneider-3 cells possess only the constitutive set of
endogenous Cdic isoforms (Fig. 7A), we excluded the tissue-specific
isoforms from this analysis. To visualize the molecules in the
cell, the polypeptides were fused at the carboxyl terminus to the green
fluorescent protein (GFP).
When overexpressed at high levels, fusion proteins
uniformly stained the cytoplasm. At low levels of expression,
however,
pronounced differences in the intracellular distribution of
the
fusion proteins were observed (Fig.
8). One of the ovary-specific
isoforms, Cdic1c, was still distributed more or less diffusely
throughout the cytoplasm, apparently reflecting the lack of
ovary-specific
target in the cultured cells. This isoform was
chosen as a control,
in contrast to the constitutive isoforms,
which should have the
relevant targets in the cultured cells, and
clearly demonstrated
different intracellular localizations.
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FIG. 8.
Localization of the Cdic-GFP fusion proteins in cultured
Schneider-3 cells. A schematic representation of the
full-size fusion and N-terminal fusion proteins is shown at the top.
Cells were transfected with plasmids expressing fusion proteins under
the control of the cytomegalovirus promoter and stained with propidium
iodide. Staining of the cellular content with propidium iodide was
detected in the rhodamine channel, and the image was converted to the
contour of the cell. GFP fluorescence was detected in the fluorescein
isothiocyanate channel and pseudocolored in white. IC isoforms are
indicated; for example, Cdic1a:GFP is a full-size fusion of Cdic1a
isoform with GFP, and N-Cdic2b and N-Cdic2c are the N-terminal
fusions. The localization of the GFP expressed alone is shown for
comparison.
|
|
Fusion proteins corresponding to the constitutively expressed
isoforms Cdic2a and Cdic2b possessed perinuclear localization,
which is tighter for Cdic2b. The aggregation of Cdic2b fusion
is
more proximal than the perinuclear structures stained for Golgi
(Fig.
9A) and most likely represents binding to
the nuclear envelope.
For the isoform Cdic1a, numerous small
aggregations were detected
and were distributed in the cytoplasm and
around the nucleus.
In the case of the isoform Cdic2c, the protein
accumulated in
one local area, as in Fig.
8, or in several patches
often clustered
in a sector of cytoplasm. In all cases, distribution of
the fusion
proteins was strictly different from the strong nuclear
localization
of GFP tag expressed alone (Fig.
8).
View larger version (64K):
[in this window]
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|
FIG. 9.
(A) Tight association of the Cdic2b fusion protein with
the nucleus. In this case, Golgi staining mostly reveals the
perinuclear elements. On the right, the GFP fluorescence image was
converted to the negative and overlaid on the Golgi staining image.
Note that the GFP fusion protein is located more proximal than the
Golgi elements. (B) Redistribution of the lysosomes in the cells
overexpressing the Cdic2c:GFP fusion protein. On the left, two
transfected cells are contoured. Staining with Lysotracker DND-99
revealed aggregation of the lysosomes in transfected cells, in contrast
to the random and mostly juxtranuclear distribution in nontransfected
cell in the left lower corner. On the right, the frequency of the
lysosomal aggregation in cells expressing Cdic2c:GFP (F) or
N-Cdic2c:GFP (N) versus nontransfected cells (C) is shown. Error bars
represent 95% confidence intervals.
|
|
In the cells overexpressing Cdic fusion proteins at a high level,
morphological changes which would result from competitive
inhibition of
dynein binding to organelles may be expected. In
particular, the
distribution of lysosomes in the cytoplasm was
checked for all
constitutive isoforms, and only in the case of
the Cdic2c fusion
was redistribution of lysosomes observed. This
effect has been proved
to be indicative of disruption of the dynein
IC-dynactin interaction
and, therefore, of inhibition of dynein
binding (
5). In 65%
of transfected cells, lysosomes were aggregated
in one local area, as
seen in Fig.
9B, or along the periphery
of one side of the cell. This
was found in only 12% of the untransfected
cells, in which much more
random and mostly juxtranuclear distribution
was usually observed.
Since interaction with dynactin seems to be limited to the N-terminal
portion of dynein ICs (
26), we tested whether fusion
proteins containing only this N-terminal part of ICs can possess
isoform-specific intracellular distribution. For
N-Cdic2b, perinuclear
localization was still observed, but it
was not as specific as
that with the complete DIC2b fusion (Fig.
8).
Overexpression of
the N-Cdic2c protein caused lysosomal aggregation in
30% of transfected
cells
somewhat greater than the 12% in
nontransfected cells, but
far less than the 65% in cells expressing
the complete Cdic2c
fusion (Fig.
9). This may explain the differences
in intracellular
localization between Cdic2c and N-Cdic2c; while the
first usually
accumulated in a local area of cytoplasm, presumably
binding to
the lysosomal aggregates, the localization of the other
suggests
binding to the normally distributed lysosomes (Fig.
8).
| |
DISCUSSION |
Alternative splicing of Cdic transcripts generates the diversity of
cytoplasmic dynein ICs.
Although the isoform complexity of
dynein ICs has been studied for a long time (21, 26), the
molecular basis for it has been unknown, since no gene has been
cloned to date. However, the suggestion has been presented that in the
rat, the structural isoforms are produced by alternative splicing
of transcripts from two highly homologous genes (26).
Our data directly demonstrate that in the
D. melanogaster species group, the Cdic diversity is created by
alternative splicing
of transcripts from a unique gene. Alternative
splicing of exons
and multiple splice acceptor sites are used,
resulting in both
length and amino acid sequence variability in the
short region
near the N terminus of the polypeptide. As many as 10 isoforms
were detected and characterized.
Comparison of the N-terminal part of
Drosophila Cdic
polypeptides with the cytoplasmic dynein ICs from rat revealed that the
presence of the variable region created by the alternative splicing
is
conserved. This does not apply, however, to the location of
this
region: there are two variable regions in the rat polypeptide,
flanking
the serine-rich domain, and only one variable region
in the
Drosophila polypeptide (Fig.
3). Moreover, the position
of
the single variable region in the
Drosophila protein does
not
correspond to the position of any of the two regions in rats,
since
these two are located on the different side of a well-conserved
PPE/TQT
block of amino acids. Therefore, it may be suggested that
the mechanism
of alternative splicing, generating the diversity
of the N termini of
dynein ICs, evolved independently in
Drosophila and rats.
This finding emphasizes the importance of IC heterogeneity
for dynein
functions and raises further questions about the implications
of this
heterogeneity.
The tissue specificity of splicing alters the content of Cdic
isoform pool.
Although at least 10 isoforms are created
by alternative splicing, only 4 are represented in consistent
amounts in all tested tissues. These are referred to as constitutive,
and in some tissues, for example in the midgut, or in cultured cells,
they make up all the content of isoform pool. This means that these
four Cdic isoforms provide at least the requisite set of the
dynein-mediated activities. It still is not apparently enough in the
other cases, requiring more specialized actions, since up-regulation of
other isoforms was observed in nervous tissue (in particular, in
the head) and ovaries. This up-regulation clearly reflects changes in
the splicing pathways and leads to a lowered percent representation of
constitutive isoforms, since the tissue-specific isoforms
contribute a significant proportion (up to a quarter) of the total
pool. This, however, does not seem to cause depletion of the
constitutive isoforms, since at least in the head it is compensated
for by the overall increase in expression of Cdic (Fig. 1).
In neurons, a specific and very active process of axonal transport
takes place, and it has been shown to be cytoplasmic dynein
dependent
(
7). In the ovaries, extensive transport takes place
from the nurse cell cytoplasm to the oocyte, and this transport
has also been shown to depend on cytoplasmic dynein
(
12). It
seems than wherever specific transport takes
place, specific Cdic
isoforms are added to the basic
set. Also, since the specificity
of these kinds of transport
mainly concerns the type of cargo
organelle to be moved, it seems very
likely that the specific
Cdic isoforms provide targeting of dynein
complex to these peculiar
cargoes.
Cdic isoforms differ in intracellular targeting.
Considering the multiplicity of intracellular targets for dynein
binding and the diversity of IC isoforms, it seems very likely that
different IC isoforms provide targeting of the dynein complex to
distinct organelles. If this were the case, the constitutive IC
isoforms would furnish the targeting to organelles regularly bound
to dynein, for example, lysosomes, Golgi complex, nuclei, and
mitotic chromosomes. Tissue-specific IC isoforms, in turn, would
provide dynein binding to cargo organelles that are specific to the
tissue and are not regularly present or are not bound with dynein in
other tissues.
When IC isoforms fused to the GFP at the C terminus were expressed
in the cultured cells, they possessed different patterns
of
intracellular distribution. Although it is not clear
whether
these patterns reflect the distribution of
endogenous ICs, it
is certain that they are due to the variable N
termini of ICs,
since all other parts of the expressed fusion proteins
are identical.
For the fusion proteins corresponding to the tissue-specific Cdic1c, a
uniform diffuse distribution in the cytoplasm or aggregation
around
small, unidentified structures was observed (data not shown).
This
pattern, which may reflect the absence of tissue-specific
targets
for these ICs in the cytoplasm of cultured cells, is clearly
different
from that observed for the constitutive ICs. An apparent
binding
to the nuclear envelope was detected for Cdic2b. Overexpression
of the
Cdic2c fusion protein caused relocation of the lysosomes.
Considering
that this effect was not detected for other isoforms,
Cdic2c could
be called the lysosome-specific IC isoform.
It has been demonstrated that targeting of the organelles by ICs
requires the N-terminal portion of the molecule (
23). We
checked whether this N-terminal portion of IC, fused to GFP, is
capable
of the specific targeting observed for the full-size IC
fusion. This
was somewhat true for isoform Cdic2b, although the
pattern was not
as well defined as with the full-size fusion protein.
Overexpression of
the N-terminal fusion for isoform Cdic2c had
little effect on the
lysosome relocation, compared to that of
the full-size protein. This
implies that the C-terminal part of
the IC molecule, which has
been considered to be a domain responsible
for interaction
with other components of the dynein complex, is
also important for the
organelle targeting. It is conceivable
that the incorporation of the
ICs in the dynein complex plays
a substantial role. Resolution of this
question requires additional
experiments that include localization of
endogenous IC isoforms
in fly tissues. This would also provide
additional data on the
specificity of organelle targeting by
isoforms.
The data presented here support a model in which alternative splicing
generates a number of cytoplasmic dynein IC isoforms
that
differ in their targeting properties. These IC subunits mediate
dynein
binding to organelles, enabling various types of minus-end
directed
transport along microtubules. The default splicing pattern
provides
isoforms necessary for dynein-mediated "housekeeping"
activities necessary for maintaining any cell or tissue, including
anterograde movement and positioning of lysosomes and Golgi complex,
mitosis, and nuclear migration. Tissue-specific alteration in
the
splicing supplies additional IC isoforms, capable of targeting
cargo organelles that are subject to unusual tissue-specific transport.
This mechanism provides a long-term modulation of the dynein binding
to
organelles in the development of organism, in addition to more
swift
regulation by phosphorylation of dynein subunits described
previously
(
17).
| |
ACKNOWLEDGMENTS |
Y. Y. Shevelyov and V. A. Gvozdev were supported by
grants 96-04-49015 and 96-15-98072 from the Russian Foundation for
Basic Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Organismic & Evolutionary Biology, Harvard University, 16 Divinity
Ave., Cambridge, MA 02138. Phone: (617) 496-5540. Fax: (617) 496-5854. E-mail: dnurminsky{at}oeb.harvard.edu.
| |
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Top
Abstract
Introduction
