Cytoplasmic dynein intermediate-chain isoforms with different targeting properties created by tissue-specific alternative splicing.

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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MOLECULAR AND CELLULAR BIOLOGY,
0270-7306/98/$04.00?0
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Nov. 1998, p. 6816–6825Vol. 18, No. 11
Cytoplasmic Dynein Intermediate-Chain Isoforms with Different
Targeting Properties Created by Tissue-Specific
Alternative Splicing
DMITRY I. NURMINSKY,1* MARIA V. NURMINSKAYA,2ELIZAVETA V. BENEVOLENSKAYA,3,4
YURY Y. SHEVELYOV,4DANIEL L. HARTL,1AND 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
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 Dro-
sophila, 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 iso-
forms 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 iso-
forms 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.
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-di-
rected 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. Reg-
ulation of this binding is also required to enable relocation of
the dynein between targets during the cell cycle and develop-
ment of the organism. One of the proposed mechanisms im-
plemented 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 cy-
toplasmic 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 dy-
nactin 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 colocal-
ized 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 regu-
lated (21). The complexity of IC isoforms is due to the expres-
sion 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 phos-
phorylation is strongly suggested to occur in the same region
whichcontainstheserine-richdomain.Thus,theobservedcom-
plexity of ICs presumably provides a diversity in dynactin-
mediated dynein binding to organelles. This means that chang-
ing 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 poly-
morphic region located near the N terminus of IC. The exact
positions of the polymorphic regions differ in ICs from Dro-
sophila and rats, suggesting independent evolution of IC iso-
form 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 ova-
ries 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 mecha-
nism for developmental regulation of dynein binding to or-
ganelles by changing the content of the IC isoform pool.
* Corresponding author. Mailing address: Department of Organis-
mic & Evolutionary Biology, Harvard University, 16 Divinity Ave.,
Cambridge, MA 02138. Phone: (617) 496-5540. Fax: (617) 496-5854.
E-mail: dnurminsky@oeb.harvard.edu.
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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 puri-
fied from the total RNA preparations with Poly(A)-Tract magnetic particles
(Promega Corp).
For Northern analysis, 10 ?g of total RNA or 2 ?g 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-stranded32P-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 generate32P-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 previ-
ously (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 mem-
brane 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 synthe-
sized (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?-CGG
CGATGATGAATAC-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 with32P by T4 DNA kinase and hybridized with
the membrane in 4? SSPE–1% Sarkosyl, 0.1% each Ficoll-400, polyvinylpirro-
lidone, 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 (Invitro-
gen).
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?-GGTACCAGCTAATCGCCCC
GAGAAATGGAT-3?) and DIC-LL (5?-GGCGGCCGCGTTCATCTTGATCT
CGCTAAG-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 pGreen-
Lantern 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 trans-
fection, the cells were plated on chamber slides (Falcon) at a density of 5 ? 105
to 7 ? 105cells/ml and the next day were transfected with the Lipofectin reagent
(Gibco-BRL). A 1.5-?g portion of DNA and 9 ?l of Lipofectin were used per
5 ? 105to 7 ? 105cells. 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 ?g of
rhodamine-labeled Lens culinaris lectin per ml (Sigma). Lysosome-specific stain-
ing 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.
RESULTS
Multiple RNAs code for the cytoplasmic dynein IC proteins.
In a previous study, we characterized a tandem repeat in cy-
tological region 19F of D. melanogaster (2). The unit of this re-
peat 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).
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, obvi-
ously 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 descrip-
tion 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.
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-ter-
minal part of molecule, which is 61% identical (79% similar) to
the rat homolog and 50% identical (69% similar) to the pro-
tein from Dictyostellium discoideum. Included in the C-termi-
nal region are four WD-40 repeats (see Fig. 6; a fifth repeat,
described in reference 27, is very degenerate and is not shown
FIG. 1A. Dynein IC transcripts in Drosophila. Samples (10 ?g) 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. Hybrid-
ization 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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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 dy-
nein ICs is the presence of a coiled-coil domain at the N ter-
minus followed by a serine-rich domain. Both these domains
were detected in the 70-kDa polypeptide. Analysis of the align-
ment of this polypeptide with other cytoplasmic dynein ICs re-
vealed another conserved stretch of amino acids in the N-ter-
minal region, called PPE/TQT (Fig. 3).
Based both on sequence homology and structural similarity
to the cytoplasmic dynein ICs from rats and Dictyostelium dis-
coideum, 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 axon-
emal dynein and was called Sdic (17a).
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.
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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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 dem-
onstrated 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, how-
ever, 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 re-
gion 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 struc-
ture 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).
Transcription of Cdic changes throughout the development
of D. melanogaster (Fig. 1). The transcripts are abundant in em-
bryos 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 splic-
ing. 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 splic-
ing.
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. Alter-
native splicing of transcripts leads to the skipping of the vari-
able exons, providing shortened versions of mRNAs appar-
ently 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 preced-
ing exon v1 and three alternative splice acceptor sites of the
intron preceding exon d5, which also results in insertions/de-
letions 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
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.
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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6820NURMINSKY ET AL.MOL. CELL. BIOL.