From the Departments of Cell Biology and Anatomy and
§ Ophthalmology, Margaret M. Dyson Vision Research
Institute, Cornell University Medical College,
New York, New York 10021
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ABSTRACT |
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To date, much attention has been focused on the heavy and intermediate chains of the multisubunit cytoplasmic dynein complex; however, little is known about the localization or function of dynein light chains. In this study, we find that Tctex-1, a light chain of cytoplasmic dynein, localizes predominantly to the Golgi apparatus in interphase fibroblasts. Immunofluorescent staining reveals striking juxtanuclear staining characteristic of the Golgi apparatus as well as nuclear envelope and punctate cytoplasmic staining that often decorates microtubules. Tctex-1 colocalization with Golgi compartment markers, its distribution upon treatment with various pharmacological agents, and the cofractionation of Tctex-1-associated membranes with Golgi membranes are all consistent with a Golgi localization. The distribution of Tctex-1 in interphase cells only partially overlaps with the dynein intermediate chain and p150Glued upon immunofluorescence, but most of Tctex-1 is redistributed onto mitotic spindles along with other dynein/dynactin subunits. Using sequential immunoprecipitations, we demonstrate that there is a subset of Tctex-1 not associated with the intermediate chain at steady state; the converse also appears to be true. Distinct populations of dynein complexes are likely to exist, and such diversity may occur in part at the level of their light chain compositions.
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INTRODUCTION |
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Cytoplasmic dynein has been shown to be involved in a wide range
of intracellular motile events, including microtubule ()-end-directed organelle movement (1), endosomal transport (2), centrosomal localization of the Golgi complex (3), anaphase chromosome segregation
(4), mitotic spindle alignment (5, 6), and nuclear distribution (7, 8).
The ATPase and motor activities of each multisubunit dynein complex
reside exclusively in two ~530-kDa heavy chains
(DHCs)1 (9, 10).
Each complex also contains two or three 74-kDa intermediate chains
(DICs), which have been proposed to anchor dynein to its target
membranes (11) via its interaction with the dynactin complex (12, 13),
which is required for cytoplasmic dynein-mediated in vitro
organelle movement along microtubules (14). Several smaller
polypeptides have also been described as components of the dynein
complex, namely, a group of light intermediate chains (~52-61 kDa)
(15) and three recently described light chains (DLCs; 8, 14, and 22 kDa) (16, 17). However, the functions of these subunits remain poorly
understood.
Independent lines of evidence have indicated that the cytoplasmic dynein pool is heterogeneous. First, at least four different DIC isoforms have been identified in neurons despite an estimated stoichiometry of only two or three DIC subunits per dynein complex (18). Similarly, three DHC isoforms have been found (19), even though there are only two DHC molecules per complex. Second, the subcellular localizations of individual dynein subunits appear to differ from one another within the same cell type, such as normal rat kidney (NRK) fibroblasts. Punctate staining throughout the cytoplasm has been described using various anti-dynein and anti-DHC antibodies (19-21). The DHC2 isoform localizes predominantly to the Golgi apparatus in interphase NRK cells and forms complexes with a significantly lower sedimentation coefficient than conventional cytoplasmic dynein (19). DIC, on the other hand, has been reported to be localized to lysosomes in NRK cells by immunofluorescence (22, 23). In polarized enterocytes, DIC and DHC have been found on trans-Golgi network (TGN) membranes (24). The intracellular localizations of dynein light intermediate chains and DLCs have not been described to date.
We have isolated a bovine ortholog of Tctex-1, which has previously been demonstrated to be a cytoplasmic dynein light chain (17), in a search for proteins that interact with the carboxyl terminus of rhodopsin using a yeast two-hybrid system. Further data on the interaction between rhodopsin and Tctex-1 will be presented elsewhere.2 In this report, we examined the intracellular localization of Tctex-1 in mammalian fibroblasts and found that Tctex-1 was predominantly localized to the Golgi complex of interphase cells by both immunocytochemical and biochemical methods. A subpopulation of DIC was also found to be localized to the Golgi apparatus by immunofluorescence, although the bulk of DIC labeling appeared on other cytoplasmic membranous structures. Sequential immunoprecipitations with anti-DIC and anti-Tctex-1 antibodies revealed the existence of free Tctex-1 not associated with DIC at steady state and possibly the existence of DIC-dynein complexes not associated with Tctex-1. We propose that diversity of cytoplasmic dynein complexes is therefore likely to exist not only at the level of DIC and DHC, but also at the level of their light chains. This may provide further insight into the ability of dynein to mediate temporally and spatially distinct functions.
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EXPERIMENTAL PROCEDURES |
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Reagents and Antibodies--
All reagents were obtained from
Sigma unless otherwise specified. The following antibodies were used:
anti-p58 mAb (clone 58K-9, Sigma) (25), anti--adaptin mAb
(Transduction Laboratories) (26), anti-
-mannosidase II mAb (clone
53FC3, BAbCO) (27, 28), anti-
-tubulin mAb (Amersham Pharmacia
Biotech) (29), TGN38 mAb (Affinity Bioreagents) (30), anti-DIC mAb
(clone 74.1, Chemicon International, Inc.) (31), rhodamine-conjugated
donkey anti-mouse antibody and fluorescein isothiocyanate
(FITC)-conjugated donkey anti-rabbit antibodies (used at a titer of
1:50, Jackson ImmunoResearch Laboratories, Inc.), and Alexa
488-conjugated goat anti-mouse and Alexa 594-conjugated goat
anti-rabbit antibodies (used at a titer of 1:500, Molecular Probes,
Inc.).
Cell Culture-- Madin-Darby canine kidney (MDCK) cells, normal rat kidney fibroblasts (NRK-49F; ATCC CRL 1570), and NIH/3T3 fibroblasts (ATCC CRL 1658) were grown in Dulbecco's modified Eagle medium (Mediatech, Inc., Herndon, VA) supplemented with 5% fetal calf serum, 10% fetal calf serum and 10% calf serum, respectively. 293S human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (1:1) plus 10% calf serum. All media were supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. All cell cultures were maintained in 5% CO2 at 37 °C.
Plasmids and Bacterial Fusion Protein Production-- Constructs expressing glutathione S-transferase (GST)-Tctex-1 and maltose-binding protein (MBP)-Tctex-1 fusion proteins were generated by fusing the EcoRI/XhoI- and BamHI/XhoI-digested full-length bovine tctex-1 cDNA fragment (isolated by two-hybrid screening) from pACTII 3' to the open reading frames of GST and MBP in EcoRI/XhoI-digested pGSTag vector (32) and BamHI-SalI-digested pMAL-cR1* vector (33), respectively. The eukaryotic Tctex-1 expression construct was generated by isolating and inserting the ClaI/XbaI-digested bovine tctex-1 cDNA fragment from pACTII into the ClaI/XbaI-digested pCIS vector (Genetech, Inc.) downstream of the cytomegalovirus promoter. The full-length rp3 sequence was polymerase chain reaction-amplified from human retinal cDNAs (forward, 5'-CGGAATTCGAGCCGGCGCTACCATGGAGGAG; and reverse, 5'-GCATCTAGACTCGAGGTCAGTTAAAGAACAATAGC) and inserted into EcoRI/XhoI-digested pGSTag to generate the GST-RP3 fusion expression construct, which was confirmed by sequencing. All GST (Amersham Pharmacia Biotech) and MBP (New England Biolabs Inc.) fusion proteins were produced and purified according to the manufacturers' instructions.
Antibody Generation and Purification--
Purified GST-Tctex-1
fusion protein was used as the immunogen to generate two independent
rabbit antisera (Cocalico, Reamstown, PA). The immune serum was passed
through three CNBr-activated Sepharose CL-4B columns (Amersham
Pharmacia Biotech) conjugated with Escherichia coli DH5
lysate, GST protein, and MBP protein, respectively. The final
flow-through fraction was affinity-purified by incubating with a
MBP-Tctex-1-Sepharose column and eluting with 0.1 M glycine
(pH 2.8). 1-ml fractions were collected and neutralized with 50 µl of
1 M Tris-Cl (pH 9.5), and the peak
A280 fractions were pooled. The
affinity-purified anti-Tctex-1 antibodies from both rabbits (CUMC24 and
CUMC25) yielded identical results and were used interchangeably in this
report.
Immunoblotting of Cell Lysates-- For preparing detergent lysates of NIH/3T3 fibroblasts, cell monolayers were rinsed with cold PBS, scraped into ice-cold lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM MgCl2, 1 mM EDTA, and protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.7 µg/ml pepstatin)), and dispersed by extensive pipetting. The suspension was rotated at 4 °C for 20 min and centrifuged at 13,000 × g in a microcentrifuge to remove nuclei and other insoluble material. For rat retinal lysates, neural retinas dissected from eyes obtained from CO2-asphyxiated Long-Evans rats were homogenized by shearing through a narrow-gauge needle, followed by lysis in the above buffer and centrifugation.
Lysates were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Immunodetection was performed according to the Proto-Blot system of Promega.Immunofluorescent Staining of Tissue Cultures--
To fix cells
in microtubule stabilization buffer, subconfluent monolayers of cells
(~18 h after plating) grown on coverslips were rinsed briefly in
PHEEM buffer (50 mM PIPES, 50 mM HEPES (pH
6.9), 0.1 mM EDTA, 2 mM EGTA, and 1 mM MgSO4), permeabilized in 0.2% Triton X-100
containing PHEEM buffer for 2 min at room temperature, and fixed
immediately in 2% paraformaldehyde (PFA) in PHEEM buffer for 20 min.
The cells were then quenched in 50 mM NH4Cl in
PBS plus 0.2 mM CaCl2 and 2 mM
MgCl2 (PBS-C/M) for 10 min. To fix cells in methanol, cells
were rinsed briefly with ice-cold PBS-C/M and then fixed and
permeabilized with cold methanol at 20 °C for 10 min. For both
procedures, cells were washed after fixation, blocked in 10% fetal
calf serum in PBS-C/M for 30 min, incubated with primary antibodies in
10% fetal calf serum and 0.15% saponin in PBS-C/M for 1 h and
then with the corresponding secondary antibodies in PBS-C/M for an
additional hour, and finally mounted in Vectashield mounting medium
(Vector Labs, Inc.) with 4,6-diamidino-2-phenylindole. All antibody
incubation steps were performed at room temperature. Affinity-purified
anti-Tctex-1 antibody was used at a concentration of 1 µg/ml, and all
other antibodies were used at the concentration recommended by the
supplier.
Drug Treatments-- Nocodazole and cytochalasin D were dissolved in Me2SO to stock concentrations of 33 and 1 µM, respectively. Brefeldin A (BFA; Epicentre Technologies Corp.) was dissolved in ethanol at 5 µg/ml. For all drug treatment experiments, Me2SO or ethanol was added to a 0.1% final concentration as a control. Cells were treated at 37 °C and 5% CO2. Drug-treated cells were immediately placed on ice and washed with ice-cold PBS-C/M prior to methanol fixation to avoid the reversibility of pharmacological effects. The fixed cells were then immunofluorescently labeled as described above.
Subcellular Fractionation and Marker Enzyme Assays-- Subcellular fractionation procedures were modified from Ref. 35. Briefly, the cell pellet collected from 15 15-cm dishes of confluent NIH/3T3 cells (800 × g for 10 min) was resuspended in 5 ml of homogenization buffer (10 mM Tris-Cl (pH 7.4), 0.25 M sucrose, 2 mM MgCl2, 1 mM EDTA, and the protease inhibitor mixture described above) and homogenized by two to three passes through a ball-bearing homogenizer (36). Over 90% of the cells were disrupted after homogenization as judged by trypan blue staining. The resulting post-nuclear supernatants after centrifugation (800 × g for 10 min) of the cell homogenate and two more washes were pooled and centrifuged again at 220,000 × g for 40 min (Beckman TLA 100.3 rotor) to obtain a total membrane pellet. The total membrane pellet was resuspended in 600 µl of homogenization buffer by pipetting, followed by five passes through a 25-gauge needle, overlaid onto a 20-50% (w/w) continuous sucrose gradient (in 10 mM Tris-Cl (pH 7.4), 2 mM MgCl2, 1 mM EDTA, and protease inhibitors), and centrifuged at 180,000 × gavg for 20 h (Beckman SW 41 Ti rotor). 11 fractions of ~1 ml each were collected from the bottom of the tube. The density of each fraction was measured by refractometry. All manipulations described above were carried out at 4 °C.
The protein concentration of each fraction was measured by the Bradford assay (Bio-Rad). 10 µg of protein from each fraction was assayed for ![]() |
RESULTS |
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Cloning of the Bovine Ortholog of Tctex-1, a Cytoplasmic Dynein Light Chain-- We performed a yeast two-hybrid screen for proteins that interact with the carboxyl-terminal cytoplasmic domain of rhodopsin. The bait construct encoded a fusion protein containing a triple repeat of the carboxyl-terminal 39 residues of human rhodopsin fused to the GAL4 DNA-binding domain. This bait was used to screen a bovine retinal cDNA library directionally inserted downstream of the GAL4 transcriptional activation domain.
One lacZ+, his+ bovine cDNA clone chosen for further study contained a single open reading frame encoding a polypeptide of 113 amino acids with a predicted molecular mass of 12,450 Da and a predicted pI of 5.5 (Fig. 1A). The reading frame was flanked by 29 base pairs of 5'-untranslated sequence and a 363-base pair 3'-untranslated region containing a consensus polyadenylation signal 12 base pairs upstream of the poly(A) tail. A GenBankTM search using BLAST (42) revealed that our bovine cDNA clone was highly homologous to the murine tctex-1 (43) and human TCTEL1 genes (44), sharing 92 and 100% amino acid identity, respectively. The high degree of conservation between our clone and other Tctex-1 proteins unambiguously identified our clone as the bovine ortholog of Tctex-1.
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Colocalization of Tctex-1 with Markers of the Golgi Complex-- Although Tctex-1 was first thought to be testis-specific (43), we isolated Tctex-1 from a retinal cDNA library. This is consistent with results from other groups indicating that Tctex-1 is likely to be expressed ubiquitously (16, 44). To determine the distribution of Tctex-1, we first generated a polyclonal antibody directed against Tctex-1 by immunizing rabbits with purified GST-Tctex-1 fusion protein expressed in E. coli. The antiserum was depleted of GST and MBP immunoreactivity before purification on a MBP-Tctex-1 affinity column (see "Experimental Procedures"). The specificity of the affinity-purified anti-Tctex-1 antibody was confirmed by immunoblotting: it recognized GST-Tctex-1, but not the closely related RP3 (Fig. 2B). Note that GST was also not recognized. Roughly equivalent amounts of GST-Tctex-1 and GST-RP3 were loaded, judging by Coomassie Blue staining (Fig. 2A) and anti-GST immunoblotting (Fig. 2C).
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Microtubule-depolymerizing Drugs and Brefeldin A, but Not
Actin-disrupting Drugs, Disperse the Golgi Localization of
Tctex-1--
We further examined the relationship between Tctex-1 and
the Golgi apparatus by administering nocodazole and cytochalasin D,
pharmacological agents that are known to disrupt microtubules and actin
filaments, respectively (52, 53). Treatment of NRK fibroblasts with 33 µM nocodazole for 1 h dispersed the juxtanuclear Tctex-1 staining into dozens of discrete brightly staining
structures scattered throughout the cytoplasm (Fig.
5C). This pattern is characteristic of Golgi staining of nocodazole-treated cells (54, 55).
Notably, the colocalization of Tctex-1 with -mannosidase II
persisted even after Golgi dispersal with nocodazole (Fig. 5D). Control treatment of NRK cells with Me2SO
for 1 h had no effect on either Tctex-1 (Fig. 5A) or
-mannosidase II (Fig. 5B) staining.
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Subcellular Fractionation and Characterization of
Tctex-1-associated Membranes--
We then proceeded to further
investigate the association of Tctex-1 with Golgi membranes by
biochemical means. Total membranes from NIH/3T3 fibroblasts were
fractionated by centrifugation on 20-50% (w/w) linear sucrose
gradients, and the distribution of Tctex-1 was determined. Fractions
were assayed for -mannosidase II, alkaline phosphatase, and
glucosidase II, enzyme markers of the medial-Golgi membrane,
the plasma membrane, and the ER, respectively (37, 60, 61). Total
membranes from each sucrose gradient fraction were collected by high
speed centrifugation and subjected to immunoblotting for Tctex-1 and
p58. Membrane-associated Tctex-1 was found to be concentrated in a
single peak in fraction 7 with a buoyant density of
= 1.13-1.14
g/ml (Fig. 7B).
-Mannosidase II activity was concentrated in fraction 8 with a
buoyant density of
= 1.12 g/ml (Fig. 7A), immediately
adjacent to the peak Tctex-1-containing fraction. Glucosidase II
activity, a marker of the ER, was distributed in a broad peak among
denser fractions. Neither glucosidase II nor alkaline phosphatase (data
not shown) was enriched in Tctex-1- or mannosidase II-containing
fractions (Fig. 7A). Immunoblotting using anti-p58 mAb was
also used to determine the distribution of Golgi membrane subfractions.
Anti-p58 mAb revealed a reactive ~53-kDa band in fractions 4-7 with
a peak in fraction 6, corresponding to a buoyant density range of
1.13-1.17 g/ml (Fig. 7C). The distribution of this species
among the fractions was fully consistent with the expected distribution
of p58, which can be found in the ER, intermediate compartment, and
cis-Golgi elements, all denser than medial-Golgi
membranes.
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Partial Colocalization of Tctex-1 with Other Cytoplasmic Dynein and Dynactin Subunits-- We were interested in determining the distribution of Tctex-1 in fibroblasts in relation to several other dynein/dynactin subunits. Remarkably, we found that in methanol-fixed 3T3 cells, anti-DIC mAb 74.1 also stained what appeared to be the Golgi apparatus (Fig. 8A, arrow), overlapping with Tctex-1 Golgi staining in the same cell (Fig. 8B). Note that anti-DIC antibody produced a considerable amount of labeling of vesicular structures of varying sizes throughout the cytoplasm, especially when compared with the distribution of Tctex-1 in the same cell. This vesicular localization of DIC, which was observed in methanol- or PFA-fixed cells, was quite different from the microtubule-associated Tctex-1 staining seen in detergent-extracted, PFA-fixed cells (Figs. 3A and 8F). We did not observe any Golgi-like staining of DIC in PFA-fixed 3T3 fibroblasts (data not shown). It is worth mentioning that no Golgi-like staining of DIC was detected in NRK cells using either methanol or aldehyde fixation; under these conditions, only vesicular cytoplasmic staining was observed (data not shown).
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Biochemical Identification of Distinct Tctex-1 and DIC Subpopulations-- The overlapping but distinct distributions of Tctex-1 and DIC on indirect immunofluorescence can be most simply interpreted by the existence of several subpopulations of molecules: DIC-dynein molecules bound to Tctex-1, DIC not associated with Tctex-1, and Tctex-1 not associated with DIC. To test this model, a sequential immunoprecipitation assay using 35S-labeled 3T3 cell lysate was performed. Cytosolic (S100) and crude membrane (P100) fractions, prepared by high speed centrifugation of the post-nuclear supernatant, were extracted with 1% Triton X-100 before immunoprecipitation. Duplicate samples from the detergent-soluble extracts underwent immunoprecipitation with anti-Tctex-1 or anti-DIC antibodies. We found that significant amounts of DIC and Tctex-1 were present in the P100 fraction (Fig. 9, A and B, first and second lanes), ranging from ~27 to 48% and from 28 to 45% (n = 6) of total DIC and Tctex-1, respectively, upon quantitation by phosphorimaging.
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Tctex-1 Is Colocalized with DIC in Mitotic Spindles--
Several
reports have demonstrated the presence of several dynein and
dynactin subunits on mitotic spindles (14, 20, 21), and we therefore
examined whether Tctex-1 was also enriched in mitotic cells in this
area. 3T3 fibroblasts were extracted with 0.2% Triton X-100 prior to
PFA fixation to reduce cytoplasmic Tctex-1 staining and then labeled
with anti-Tctex-1 antibody (Fig. 10,
A, D, G, and J) in parallel
with anti--tubulin mAb (Fig. 10, B, E,
H, and K) and 4,6-diamidino-2-phenylindole DNA
staining (Fig. 10, C, F, I, and
L). In prophase cells, Tctex-1 was localized to discrete
perinuclear structures that presumably represented fragmented Golgi
elements and also strikingly decorated non-spindle microtubules (Fig.
10, A-C). In metaphase cells, anti-Tctex-1 antibody clearly
stained spindle microtubules as well as punctate cytoplasmic structures
(Fig. 10, D-F). Tctex-1 also decorated the mitotic spindle in anaphase cells; particularly intensely labeled was the area of the
polar microtubules extending between the separating sister chromatids,
in agreement with functional studies implicating dynein in anaphase B
spindle elongation (Fig. 10, G-I) (4). Interestingly, however, the anti-Tctex-1 antibody labeling appeared to be considerably more intense than anti-
-tubulin antibody staining in this area. Finally, in cells in late telophase and cells undergoing cytokinesis, Tctex-1 appeared at the reforming Golgi apparatus adjacent to both
daughter nuclei as well as at the remains of the mitotic spindle
passing through the contractile ring at the midbody (Fig. 10,
J-L).
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DISCUSSION |
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Intracellular Localization of Tctex-1 and Its Proposed Functions-- Several previous reports, all performed in nonpolarized cells, have indicated that cytoplasmic dynein mediates multiple functions at the Golgi apparatus. The centrosomal localization of the Golgi apparatus is thought to be mediated by cytoplasmic dynein (3). Other roles for cytoplasmic dynein at the Golgi apparatus include ER-Golgi transport (65) and potentially the partitioning of the Golgi complex between mitotic daughter cells (66, 67). Further evidence for the role of cytoplasmic dynein in the maintenance of the Golgi apparatus comes from data showing that dynactin disruption also results in disruption of the Golgi complex and of ER-Golgi transport (68). Our observation that a large fraction of Tctex-1 localizes to the Golgi apparatus in interphase fibroblasts suggests that Tctex-1 may play a role in any or all of the above events.
In steady-state polarized enterocytes, DHC and DIC were found on TGN membranes, but not on Golgi stacks (24). However, DIC could bind Golgi stacks in vitro by interacting with a Golgi-associated peripheral membrane protein, and this interaction may drive vesicle budding (69). It would be of interest to test whether Tctex-1 is a candidate for this steady-state Golgi-associated peripheral membrane protein. In addition to the observed Golgi localization of Tctex-1, we also detected Tctex-1 labeling of microtubules and nuclei/nuclear envelope in interphase fibroblasts. The microtubule-associated Tctex-1 potentially represents another subset of Tctex-1 that is functionally distinct from soluble and Golgi-bound protein. Our immunostaining results indicate that this subset of Tctex-1 colocalizes with p150Glued. Cytoplasmic dynein has been immunolocalized to the nuclear envelope in developing mammalian spermatids and to the nucleus in Dictyostelium (70, 71). Genetic studies have demonstrated a role for cytoplasmic dynein in nuclear distribution in filamentous fungi (7, 8), in nuclear segregation in yeast (5), and in pronuclear migration in fertilized eggs (72). It is interesting to speculate that the stable positioning of the nucleus near the centrosome in the steady-state mammalian cell may also be mediated by cytoplasmic dynein binding to the nuclear envelope. Another possible role of dynein at the nuclear envelope is suggested by a report that cytoplasmic dynein mediates the rapid centripetal transport of herpes simplex virus capsids from the plasma membrane to nuclear pore complexes (73). Whether other transport events to the nucleus in uninfected cells also employ cytoplasmic dynein and whether the observed punctate staining of nuclei by anti-Tctex-1 antibody represents such activity remain to be confirmed. Finally, there may also be other sites of dynein activity regulated by Tctex-1 that cannot be visualized at the light microscope level. Since a single cytoplasmic dynein molecule is capable of translocating a latex bead on a microtubule protofilament (74), it is possible that there are additional populations of Tctex-1 molecules associated with active cytoplasmic dynein that cannot be visualized by immunofluorescence microscopy.Evidence for Distinct Populations of Dynein Subunits-- The surprising range of intracellular motile events ascribed to cytoplasmic dynein (see the Introduction and reviewed in Ref. 75) raises the obvious question of how such spatially and temporally distinct activities are regulated. The answers are beginning to emerge and, not surprisingly, appear to be complex. First, post-translational modifications, such as phosphorylation, have been described on several dynein subunits and appear to correlate with the state of activity of the complex (1, 18, 31, 76). Second, the concept of dynein as a single complex has been revised in recent years with the discovery of multiple DHC and DIC isoforms (18, 19). More recently, dynein subunit heterogeneity has also been described at the level of its light chains: at least two 14-kDa DLC subunits, Tctex-1 and RP3, have been identified (17, 47). At steady state, therefore, dynein complexes are likely to be heterogeneous in subunit composition. Third, it is possible that individual dynein subunits are not always associated with cytoplasmic dynein complexes at steady state. The regulated assembly of such "free" subunits into complexes, possibly by post-translational modifications, may represent another point at which cytoplasmic dynein function may be regulated. Moreover, free dynein subunits may be able to regulate other molecules on their own, as has been suggested for the 8-kDa DLC, which has also been shown to be a subunit of myosin V (77). These models are not mutually exclusive and probably all contribute to dynein regulation.
The immunocytochemical and biochemical data reported in this paper support the latter two mechanisms. On immunofluorescent staining, a subpopulation of DIC was found to colocalize with Tctex-1 at the Golgi apparatus, but a substantial fraction of DIC was located on vesicular structures throughout the cytoplasm and did not colocalize with Tctex-1. Moreover, a significant fraction of total Tctex-1 could not be co-immunoprecipitated with DIC using an anti-DIC mAb; conversely, a significant amount of DIC was not co-immunoprecipitated with an anti-Tctex-1 antibody. We conclude that individual cytoplasmic dynein subunits may not always be associated with one another in the steady-state interphase cell. RP3, which is another 14-kDa DLC closely related to Tctex-1 (47), is likely to share binding sites on the dynein complex with Tctex-1, and it is therefore likely that some dynein complexes contain Tctex-1, whereas others contain RP3. It will be of great interest to determine whether such distinct complexes in fact exist and whether they have different functions in the cell. In contrast to the partial colocalization of DIC and Tctex-1 in interphase cells, we observed extensive colocalization of Tctex-1 and DIC in mitotic cells, which is consistent with previous observations that DHC and DIC are localized at the mitotic spindle and kinetochore in multiple cell types (20, 21). This dramatic redistribution of dynein subunit localization is a good example supporting the notion that the assembly of cytoplasmic dynein subunits may be dynamic and regulated. Interestingly, we observed decreased colocalization of Tctex-1 withTctex-1 in Transmission Ratio Distortion and Male Sterility-- tctex-1 was originally cloned from the t-complex, a variant region of chromosome 17 in mice containing four non-overlapping inversions (43). Males heterozygous for a t-haplotype transmit the mutant chromosome to over 99% of progeny, an extreme example of the phenomenon called transmission ratio distortion (45). Complete t-haplotypes are usually lethal when homozygous, and males that carry two complementing complete t-haplotypes are sterile. Although the recessive t-lethal loci do not have any relationship to those that mediate transmission ratio distortion (45), it appears that the same set of genes mediates transmission ratio distortion in the dominant case and male sterility in the recessive case (78). tctex-1 is a candidate for one of these genes (43). The suggested role for Tctex-1 in meiotic transmission ratio distortion and male sterility in mice points to multiple roles for Tctex-1. Indeed, the recent report that Tctex-1 is a component of the flagellar inner dynein arm (79) is consistent with the proposal that individual cytoplasmic dynein subunits may have localizations and functions independent of the cytoplasmic dynein complex.
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ACKNOWLEDGEMENTS |
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We thank Drs. H. Xu and D. Cohen for advice on Golgi preparation and M. Alfonzo-Larrain for technical assistance. We thank Drs. E. Rodriguez-Boulan, F. Maxfield, and G. Gurland for helpful suggestions regarding the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY11307, the Foundation Fighting Blindness, and Research Preventing Blindness (to C.-H. S.); the Emil Holland Fund (to A. W. T.); and National Institutes of Health Tri-institutional Training Program in Vision Grant EY07138 (to J.-Z. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the GenBankTM/EMBL/DDBJ Data Bank with accession number AF067370.
¶ To whom correspondence should be addressed: Margaret M. Dyson Vision Research Inst., Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-2291; Fax: 212-746-6670; E-mail: chsung{at}mail.med.cornell.edu.
1 The abbreviations used are: DHCs, dynein heavy chains; DICs, dynein intermediate chains; DLCs, dynein light chains; NRK, normal rat kidney; TGN, trans-Golgi network; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PFA, paraformaldehyde; BFA, brefeldin A; ER, endoplasmic reticulum.
2 J.-Z. Chuang, A. W. Tai, and C.-H. Sung, manuscript in preparation.
3 A. W. Tai, J.-Z. Chuang, and C.-H. Sung, unpublished observations.
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REFERENCES |
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