Localization of Tctex-1, a Cytoplasmic Dynein Light Chain, to the Golgi Apparatus and Evidence for Dynein Complex Heterogeneity*

Andrew W. TaiDagger , Jen-Zen Chuang§, and Ching-Hwa SungDagger §

From the Departments of Dagger  Cell Biology and Anatomy and § Ophthalmology, Margaret M. Dyson Vision Research Institute, Cornell University Medical College, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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-gamma -adaptin mAb (Transduction Laboratories) (26), anti-alpha -mannosidase II mAb (clone 53FC3, BAbCO) (27, 28), anti-alpha -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 DH5alpha 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.

Since our GST-Tctex-1 fusion protein contained several irrelevant linker amino acids between the GST and Tctex-1 domains, we wanted to ensure that the observed immunostaining results obtained with our affinity-purified antibodies were not due to reactivity against this linker region. Therefore, we further blot-purified (34) our affinity-purified anti-Tctex-1 antibody against electrophoretically purified Tctex-1, lacking any extra amino acids, overexpressed in 293S cells. The blot-purified antibodies gave the same results on immunofluorescent staining as the original affinity-purified antibody (data not shown).

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 alpha -mannosidase, alkaline phosphatase, and glucosidase II enzyme activities by procedures described previously (37-39). The remainder of each gradient fraction was diluted with an equal volume of 10 mM Tris (pH 7.4) and centrifuged at 220,000 × gavg (TLA 100.3 rotor) for 40 min. The resulting membrane pellets were resuspended in 40 µl of PBS, and 15 µl was used for immunoblotting.

For S100/P100 fractions, 3T3 cells were metabolically labeled as described previously (40). The post-nuclear supernatant, prepared as described above, was centrifuged at 100,000 × gmax for 45 min (TLA 100.3 rotor), and the resulting S100 supernatant was removed. The crude pellet was washed by resuspension in 1 ml of homogenization buffer and centrifugation at 100,000 × gmax for 45 min to produce a P100 pellet. Triton X-100 was added to S100 to a 1% final concentration, and P100 was resuspended in homogenization buffer plus 1% Triton X-100; they were both incubated at 4 °C for 20 min and centrifuged at 100,000 × gmax for 20 min to remove detergent-insoluble material. The resulting supernatants underwent two-step immunoprecipitations. Two-step immunoprecipitations were performed as described previously (41). Briefly, the first-round immunoprecipitates were dissociated in 2% SDS, followed by dilution to 0.1% SDS and addition of Triton X-100 to a final concentration of 3%, and then were subjected to a second round of immunoprecipitation with fresh antibody-protein A-Sepharose beads. Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and quantitated with a Molecular Dynamics PhosphorImager.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Sequence of the bovine tctex-1 cDNA clone and its amino acid sequence alignment with human TCTEL1, murine Tctex-1, Drosophila Tctex-1, and human RP3 proteins. A, nucleotide sequence of the bovine tctex-1 cDNA clone including the full amino acid coding sequence; B, alignment of bovine Tctex-1 amino acid sequence with human, murine, and Drosophila Tctex-1 as well as human RP3 using the Jotun-Hein algorithm. Identical amino acids are boxed.

tctex-1 (t-complex testis-expressed) was originally identified as a candidate gene in the mouse t-complex for transmission ratio distortion (43). Male mice heterozygous for the t-haplotype transmit the mutant chromosome to over 99% of their progeny, the most extreme example of transmission ratio distortion known in vertebrates (45). More recently, partial peptide sequences of a cytoplasmic dynein light chain isolated from bovine brain were identified as Tctex-1 (16).

Additional proteins homologous to Tctex-1 have been reported, including another t-complex-encoded protein, Tctex-2 (46), that was identified as an axonemal outer arm DLC (47) and several nematode open reading frames identified in the Expressed Sequence Tag Data Base (47). A human protein, RP3 (48), has also been reported to be a cytoplasmic DLC (47) and is 52% identical and 75% similar to bovine and human Tctex-1 at the amino acid level. An alignment of bovine, murine, human, and Drosophila Tctex-1 and human RP3 polypeptides generated by the Jotun-Hein algorithm shows that the carboxyl-terminal portions of the Tctex-1 sequences are more highly conserved relative to RP3 compared with their amino-terminal portions (Fig. 1B).

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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Fig. 2.   Specificity of the affinity-purified anti-Tctex-1 antiserum. A-C, equivalent A280 amounts of purified GST-RP3 and GST-Tctex-1 were analyzed by SDS-polyacrylamide gel electrophoresis and stained by Coomassie Blue (A) or transferred and immunoblotted with either anti-Tctex-1 antibody (Ab) (B) or anti-GST antibody (C). Note that GST-RP3 can be recognized by anti-GST antibody, but not by anti-Tctex-1 antibody. The difference in apparent molecular mass between GST-RP3 and GST-Tctex-1 was due to a longer linker region between GST and RP3 compared with that in GST-Tctex-1 (see "Experimental Procedures"). The additional smaller molecular mass bands in the GST-Tctex-1 sample represent degraded fusion protein. D, anti-Tctex-1 immunoblot of a lysate of 293S cells transiently overexpressing Tctex-1 (lane 1), a rat retinal detergent lysate (lane 2), and an NIH/3T3 mouse fibroblast detergent lysate (lane 3).

Using this anti-Tctex-1 antibody for immunoblotting and immunocytochemistry, we have found Tctex-1 to be expressed in a wide range of cell lines derived from different tissues and species. These include rodent NRK and NIH/3T3 fibroblasts, MDCK cells, Chinese hamster ovary cells, and 293S human embryonic kidney cells.3 The ubiquitous expression of Tctex-1 suggests that it may serve a general role in a variety of cells. As a first step in elucidating the physiological function of Tctex-1, we determined its subcellular localization. We chose NRK fibroblasts because of their well characterized subcellular organization, their previous use in localizing other dynein/dynactin subunits, and the wide range of available antibodies against rat organelle marker proteins. The subcellular localization of Tctex-1 was visualized using anti-Tctex-1 antibody followed by FITC-conjugated anti-rabbit antibody in NRK fibroblasts that were briefly extracted in 0.2% Triton X-100 before PFA fixation. Anti-alpha -tubulin mAb followed by rhodamine-conjugated anti-mouse antibody were also applied to label microtubules in the same cells. In interphase cells, our anti-Tctex-1 antibodies labeled tubulo-vesicular structures in a juxtanuclear distribution suggestive of the Golgi complex (Fig. 3A). There was also punctuate cytoplasmic staining that appeared to colocalize with microtubules (Fig. 3B); this colocalization of Tctex-1 with microtubules was best seen with detergent extraction, which removes "background" soluble Tctex-1 that interferes with the visualization of microtubule-bound Tctex-1. To better document the colocalization of Tctex-1 with microtubules in this particular experiment, we omitted microtubule-stabilizing agents such as Taxol or glycerol during extraction because such agents greatly increased the density of microtubules and of Tctex-1 immunostaining, making it very difficult to clearly see the colocalization of Tctex-1 with individual microtubules.3 In addition to the Golgi-like and microtubule-associated Tctex-1 staining, we also observed variably intense punctate staining in the area of the nucleus and/or nuclear envelope (Fig. 3, C and D). The intensity of the punctate cytoplasmic and nuclear punctate staining relative to the Golgi-like staining was dependent on the fixation conditions. Much less intense punctate peripheral and nuclear envelope staining was observed in methanol-fixed cells (Fig. 4); however, the perinuclear Golgi-like staining was reproducibly prominent regardless of the fixation technique. Anti-Tctex-1 antibody brightly labeled nuclei in transiently transfected fibroblasts overexpressing Tctex-1 (data not shown), suggesting that the observed nuclear staining was unlikely to be due to nonspecific labeling. Staining of NIH/3T3 mouse fibroblasts yielded identical results (see Fig. 8). Immunoblotting assays showed that our anti-Tctex-1 antibody recognized a single band of the expected molecular mass in lysates from NIH/3T3 mouse fibroblasts (Fig. 2D, lane 3) and rat retina (lane 2), comigrating with Tctex-1 overexpressed in 293S cells (lane 1).


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Fig. 3.   Tctex-1 localization in interphase NRK fibroblasts visualized by immunofluorescent staining. NRK cells were briefly extracted with 0.2% Triton X-100 prior to 2% PFA fixation (see "Experimental Procedures"). A and B, NRK fibroblasts were double-labeled with anti-Tctex-1 (A) or anti-alpha -tubulin (B) mAb. Tctex-1 was observed at the nucleus and at juxtanuclear tubulo-vesicular, Golgi-like structures. Punctate anti-Tctex-1 staining also decorated microtubules. C and D, shown is a single NRK fibroblast stained with anti-Tctex-1 antibody and photographed in two different focal planes, illustrating regular punctate staining in the area of the nuclear envelope. Bars = 10 µm.


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Fig. 4.   Double labeling of NRK fibroblasts with anti-Tctex-1 antibody and four Golgi markers. Methanol-fixed NRK fibroblasts were double-labeled with anti-Tctex-1 antibody followed by FITC-conjugated secondary antibodies (A, D, G, and J) and with anti-alpha -mannosidase II mAb (B), anti-p58 mAb (E), anti-TGN38 mAb (H), or anti-gamma -adaptin mAb (K) followed by rhodamine-conjugated secondary antibodies. Double exposures are shown in C, F, I, and L. Bar = 10 µm.

To confirm the Golgi localization of Tctex-1 and to further determine the subcompartmental localization of Tctex-1, methanol-fixed NRK fibroblasts were double-labeled with anti-Tctex-1 and antibodies against different Golgi marker proteins. p58 is a marker for the pre-Golgi intermediate compartment as well as cis-Golgi elements (49); alpha -mannosidase II is a marker for medial-Golgi cisternae (50); and TGN38 and gamma -adaptin are markers for the TGN (26, 51). Methanol fixation was chosen in these experiments to highlight the Golgi localization of Tctex-1. In NRK fibroblasts, Tctex-1 (Fig. 4A) and alpha -mannosidase II (Fig. 4B) staining patterns were coincident. The virtually complete colocalization of Tctex-1 with alpha -mannosidase II was particularly apparent in a double exposure (Fig. 4C). Although Tctex-1 (Fig. 4, D, G, and J) also colocalized well with p58 (Fig. 4E), TGN38 (Fig. 4H), and gamma -adaptin (Fig. 4K), subtle differences in staining patterns could be reproducibly observed in double exposures (Fig. 4, F, I, and L). In addition to the Golgi staining, anti-p58 mAb also labeled some fine peripheral vesicles that were not stained by anti-Tctex-1 antibody. Anti-gamma -adaptin mAb, presumably labeling the TGN, also labeled punctate structures (Fig. 4K) different in morphology, size, and precise location from those labeled by anti-Tctex-1 antibody (Fig. 4, J and L). This suggested that the bulk of Tctex-1 staining in the Golgi complex was not located in gamma -adaptin-enriched regions of the TGN. Although p58, alpha -mannosidase II, and TGN38 have been reported to be localized to distinct Golgi subcompartments, their distributions could not be unambiguously distinguished by the limited resolution of our immunofluorescent light microscopic analyses.

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 alpha -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 alpha -mannosidase II (Fig. 5B) staining.


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Fig. 5.   Effect of nocodazole and cytochalasin D treatments on Golgi localization of Tctex-1. NRK fibroblasts were treated with 0.1% Me2SO (DMSO; A and B), 33 µM nocodazole (C and D), or 1 µM cytochalasin D (E and F) for 1 h at 37 °C. Cells were then methanol-fixed and double-labeled with anti-Tctex-1 (A, C, and E) or anti-alpha -mannosidase II (Mann II; B, D, and F) mAb. Bar = 10 µm.

In contrast to treatment with nocodazole, treatment of NRK cells for 1 h with 1 µM cytochalasin D did not affect the juxtanuclear positioning and morphology of either Tctex-1 or alpha -mannosidase II staining (Fig. 5, E and F). Cytochalasin D treatment did cause extensive actin stress fiber collapse as assessed by rhodamine-phalloidin staining as well as cell retraction and arborization as previously reported (data not shown) (53). The lack of effect on the Golgi localization of Tctex-1 by cytochalasin D is consistent with previous reports that cytochalasin D does not change the centrosomal localization of the Golgi complex (56).

We also assessed the effect of BFA, a fungal metabolite that is known to disrupt the Golgi apparatus by a mechanism distinct from nocodazole (57-59), on the Golgi localization of Tctex-1. 10 µg/ml BFA treatment of NRK cells rapidly dispersed Tctex-1 juxtanuclear staining within 15 min into a fine punctate pattern against diffuse cytoplasmic background staining (Fig. 6C). alpha -Mannosidase II was also redistributed by BFA into faint, diffuse staining, although the punctate structures seen with anti-Tctex-1 antibody were not apparent (Fig. 6D). The BFA-treated Tctex-1 and alpha -mannosidase II localizations were significantly different compared with the nearly complete colocalization seen in control cells treated with 0.1% ethanol (Fig. 6, A and B). BFA-induced Golgi disruption is reversible (57, 58), and we tested whether the BFA-induced disruption of Tctex-1 staining was similarly reversible. Indeed, following BFA removal, reformation of the Golgi apparatus, as assayed by alpha -mannosidase II staining (Fig. 6F), was accompanied by a recovery in juxtanuclear Tctex-1 staining (Fig. 6E) that again colocalized with alpha -mannosidase II in double-labeled cells. This result provides further evidence that Tctex-1 is associated with the Golgi apparatus. Furthermore, it suggests that the interaction of Tctex-1 with the Golgi apparatus may be regulated, based on the disparity in staining between Tctex-1 and alpha -mannosidase II staining in BFA-treated cells.


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Fig. 6.   Disrupting effect of BFA on Golgi localization of Tctex-1 is reversible. NRK fibroblasts were treated with 0.1% ethanol for 15 min (A and B), 10 µg/ml BFA for 15 min (C and D), or 10 µg/ml BFA for 15 min, followed by recovery in the absence of BFA for 90 min (E and F), all at 37 °C. After treatment, the cells were fixed in methanol and then double-labeled with anti-Tctex-1 (A, C, and E) or anti-alpha -mannosidase II (Mann II; B, D, and F) mAb. Bar = 10 µm.

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 alpha -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 rho  = 1.13-1.14 g/ml (Fig. 7B). alpha -Mannosidase II activity was concentrated in fraction 8 with a buoyant density of rho  = 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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Fig. 7.   Subcellular fractionation of NIH/3T3 fibroblasts. A total cell membrane preparation was separated on a linear 20-50% (w/w) sucrose gradient (180,000 × gavg for 20 h), and 11 fractions were collected. Shown is a bar plot of alpha -mannosidase II and glucosidase II activities in each fraction (A). Enzyme activities were measured as described under "Experimental Procedures." Bar heights represent the fractions of total enzyme activity recovered from the gradient. The error bars indicate the S.D. from three independent fractionation experiments. Membranes collected from each fraction by high speed centrifugation were immunoblotted for Tctex-1 (B) and p58 (C).

The observed buoyant densities of Tctex-1-associated and mannosidase II-containing membranes were within the range of previously reported densities of Golgi membranes in a variety of cell types (35, 62). The buoyant densities of Golgi membranes increase from trans to cis, and this gradient of buoyant density continues through the intermediate compartment to the rough ER (35, 63, 64). Our results indicate that Tctex-1-associated membranes cofractionate with intermediate compartment and/or cis-Golgi membranes, based on their intermediate density between mannosidase II-containing medial-Golgi membranes and p58-containing pre/cis-Golgi membranes.

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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Fig. 8.   Comparative immunocytochemistry of Tctex-1 with DIC, DHC, and p150Glued in NIH/3T3 fibroblasts. A-D, methanol-fixed 3T3 fibroblasts were double-labeled with anti-DIC mAb 74.1 (A) or anti-DHC mAb 440.4 (C), followed by FITC-conjugated secondary antibody and anti-Tctex-1 antibody, followed by rhodamine-conjugated secondary antibody (B and D). Note the partial colocalization of DIC with Tctex-1 at the Golgi apparatus (A, arrow). E and F, 3T3 cells were first preextracted with 0.2% Triton X-100 for 2 min at room temperature and then fixed with 2% PFA. Double labeling was performed with anti-p150Glued mAb followed by Alexa 488-conjugated secondary antibody (E) and with anti-Tctex-1 antibody followed by Alexa 594-conjugated secondary antibody (F). The Alexa-conjugated antibodies were used because of their excellent resistance to photobleaching during photography. Bar = 15 µm.

We also compared the distribution of DHC1, the "conventional" DHC, with that of Tctex-1. Anti-DHC mAb did not stain the Golgi apparatus in methanol-fixed 3T3 fibroblasts, instead labeling vesicular structures throughout the cytoplasm (Fig. 8C). The intensity of the punctate staining was somewhat higher near the nucleus, decreasing gradually toward the cell periphery; this pattern of DHC staining is in agreement with previous observations (19, 21). A similar pattern of cytoplasmic vesicular staining was observed in PFA-fixed cells (data not shown). Although the patterns of vesicular DHC and DIC staining in PFA-fixed cells were very similar, we were unable to determine whether they labeled identical vesicular structures, as our anti-DIC and anti-DHC antibodies were both derived from the same species. No obvious colocalization of DHC with Tctex-1 (Fig. 8D) was detectable by immunofluorescence.

No Golgi-like staining was seen using an anti-p150Glued mAb in either methanol-fixed (data not shown) or PFA-fixed (Fig. 8E) cells. Interestingly, however, p150Glued was found to localize to filamentous structures suggestive of microtubules (Fig. 8E); a subset of the microtubule-like structures stained for p150Glued colocalized exactly with Tctex-1 microtubule staining in Triton X-100-preextracted, PFA-fixed cells (Fig. 8, E and F, arrowheads). Similar microtubule localization of p150Glued was observed in cells fixed with methanol or PFA without detergent extraction (data not shown), but such conditions did not give good Tctex-1 microtubule staining. In agreement with a previous report (14), we also observed p150Glued localization to centrosomes with all fixation conditions; the centrosome is outside the plane of focus in Fig. 8E, which was photographed to best reveal the microtubule localization of p150Glued.

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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Fig. 9.   Sequential immunoprecipitations of Tctex-1 and DIC from soluble (S100) and crude membrane (P100) fractions of [35S]cysteine/methionine-labeled NIH/3T3 fibroblasts. Detergent extracts of S100 (S) and P100 (P) fractions underwent immunoprecipitation (IP) with anti-DIC (A, first and second lanes) and anti-Tctex-1 (B, first and second lanes) antibodies. The depleted fractions were split into two equal portions. One-half underwent a second round of immunoprecipitation to demonstrate virtually complete immunodepletion of DIC (A, third and fourth lanes) or Tctex-1 (B, third and fourth lanes). The other half of the Tctex-1-immunodepleted fractions were immunoprecipitated with anti-DIC antibody (A, fifth and sixth lanes), whereas the other half of the DIC-immunodepleted fractions were immunoprecipitated with anti-Tctex-1 antibody (B, fifth and sixth lanes). Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected by phosphorimaging.

The DIC- and Tctex-1-depleted P100 and S100 fractions were then split into two equal portions. One-half was re-immunoprecipitated with the same depleting antibody to confirm that the immunodepletion was exhaustive (Fig. 9, A and B, third and fourth lanes). The other half was re-immunoprecipitated with the converse antibody to determine the amount of Tctex-1 remaining in the DIC-immunodepleted fractions and vice versa. As can be seen in the fifth and sixth lanes of Fig. 9, Tctex-1 was readily detectable in DIC-immunodepleted S100 and P100 (panel B), and DIC was found in Tctex-1-depleted S100 and P100 (panel A). Since anti-DIC mAb 74.1 has been shown not to disrupt the Tctex-1/DIC interaction (17),3 we conclude that there is a subset of Tctex-1 not associated with DIC-dynein complexes in both the soluble and membrane fractions of 3T3 fibroblasts. Similarly, there appears to be a pool of DIC-dynein not associated with Tctex-1 in both fractions, and this is consistent with our immunocytochemical findings that there is a substantial amount of DIC labeling not associated with Tctex-1. Preliminary anti-Tctex-1 epitope and DIC-binding site mappings on Tctex-1 have revealed that anti-Tctex-1 antibody, despite its polyclonality, recognizes a strongly dominant epitope within the first 24 N-terminal residues of Tctex-1, which is independent from its DIC-binding site (data not shown). Therefore, immunoprecipitation of Tctex-1 with anti-Tctex-1 antibody is unlikely to significantly disrupt its interaction with DIC.

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-alpha -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-alpha -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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Fig. 10.   Indirect immunofluorescent staining of mitotic NIH/3T3 fibroblasts (A-L) and MDCK cells (M and N). ~18 h after plating, NIH/3T3 and MDCK cells were preextracted with 0.2% Triton X-100 for 2 min at room temperature and then fixed with PFA. A-L, Tctex-1 was visualized with anti-Tctex-1 antibody followed by FITC-conjugated secondary antibody (A, D, G, and J,); alpha -tubulin was visualized with anti-alpha -tubulin antibody followed by rhodamine-conjugated secondary antibody (B, E, H, and K); and DNA was visualized with 4,6-diamidino-2-phenylindole (DAPI; C, F, I, and L). In the prophase cell (A-C), the bright central Tctex-1 staining may represent dispersing Golgi elements. Note the prominent microtubule-associated staining. In the metaphase cell (D-F) and the anaphase cell (G-I), Tctex-1 staining overlapped with tubulin staining of the mitotic spindle, although the anti-Tctex-1 signal was more intense than the anti-alpha -tubulin signal in the region of the polar microtubules bridging the spindle poles. In late telophase/cytokinesis (J-L), Tctex-1 colocalized with reforming juxtanuclear Golgi elements as well as the microtubule bundles passing through the contractile body between daughter cells. Prominent nuclear labeling was also seen in this phase. MDCK epithelial cells were similarly extracted, fixed, and double-labeled with anti-Tctex-1 antibody followed by FITC-conjugated anti-rabbit secondary antibody (M) and with anti-IC74 mAb (clone 74.1; see "Experimental Procedures") followed by rhodamine-conjugated anti-mouse secondary antibody (N). The arrowhead points to anti-Tctex-1 juxtanuclear staining in the Golgi complex. The diffuseness of this staining relative to that of NRK fibroblasts is due to the spherical shape of MDCK cells compared with the flat shape of fibroblasts. Bars = 10 µm.

In our hands, attempts to immunolabel DIC in mitotic fibroblasts with two anti-DIC mAbs (clones 74.1 and 70.1), using the same procedures as for Tctex-1 staining, were unsuccessful. Nevertheless, in mitotic MDCK cells, we detected colocalization of DIC and Tctex-1 at the mitotic spindle during all mitotic phases, and an anaphase cell is shown as an example (Fig. 10, M and N). Perinuclear Golgi-like Tctex-1 staining was readily seen in adjacent interphase MDCK cells (Fig. 10M, arrowhead) in addition to some vesicular cytoplasmic staining.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 with alpha -mannosidase II in BFA-treated cells. One possible explanation is that the binding of Tctex-1 to the Golgi apparatus is regulated and that upon BFA treatment, Tctex-1 undergoes redistribution.

Lin and Collins (22, 23) reported that in NRK fibroblasts, an anti-dynein antibody primarily labeled lysosomes. Their polyclonal antibody was generated against the whole purified cytoplasmic dynein complex and reacted primarily but not exclusively with DIC on immunoblotting. The high intensity of juxtanuclear staining observed by Lin and Collins rendered it impossible to rule out a Golgi localization of DIC. On the other hand, we were unable to detect any obvious Golgi or lysosomal labeling using two anti-DIC antibodies in NRK cells under all fixation conditions tested. The reasons for this discrepancy are unclear.

It has been reported that there exists only a single pool of Tctex-1 in mammalian brain, all of which is associated with cytoplasmic dynein (17). This assertion was based on the finding that Tctex-1 quantitatively cosedimented with cytoplasmic dynein in a high speed supernatant prepared from brain cytosol upon Taxol-induced microtubule polymerization. The difference between this result and our own can be explained by several reasons. First, based on our results, it is likely that there was a pool of Tctex-1 associated with cell membranes, but not with other cytoplasmic dynein subunits, that was lost during the initial high speed centrifugation step. Second, the same work in fact reported that the ratio of Tctex-1 to DIC varies among different tissues (17); in some cell types, there may be an excess of Tctex-1 relative to DIC. Third, it is conceivable that the assembly of free cytoplasmic dynein subunits into a complex can be triggered by the nearly complete microtubule polymerization induced by Taxol.

Tctex-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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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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Abstract
Introduction
Procedures
Results
Discussion
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