Article |
Address correspondence to Nobutaka Hirokawa, Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo 113-0033, Japan. Tel.: 81-3-5841-3326. Fax: 81-3-5802-8646. E-mail: hirokawa{at}m.u-tokyo.ac.jp
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Abstract |
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Key Words: kinesin; kinesin superfamily proteins; apical transport; cholesterol; annexin XIIIb
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Introduction |
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Among the aforementioned processes, the packaging, docking, and retention mechanisms have been vigorously examined. An increasing number of cytoplasmic determinants for basolateral targeting have been identified, and removal or inactivation of these signals is shown to cause aberrant destination of the molecules, reflecting their missorting and/or improper retention. Concurrently, t-SNAREs that are differentially distributed on the PM subdomain and their specific partner v-SNAREs have been identified. Moreover, the membrane-specific docking mechanism is now being elucidated at the molecular level (Yeaman et al., 1999). Clarification of the retention mechanism has also led to the identification of the endosomal localization signal and structural markers (Aroeti and Mostov, 1994; Mellman, 1996).
On the contrary, very little is known about the transport process. It is yet unclear whether a selective transport system exists for either apical or basolateral targeting. MT motors are expected to carry transport vesicles along the MT network. In fact, disruption of the MT network by nocodazole treatment significantly, though not completely, inhibits the delivery of newly synthesized membrane proteins to the PM. Furthermore, such treatment also causes missorting of some apical molecules to the basolateral membrane (Breitfeld et al., 1990; Grindstaff et al., 1998), suggesting the role of MT motors in the sorting process.
However, the degree of the transport machinery's contribution to the membrane sorting system is controversial at present (Salas et al., 1986; Drubin and Nelson, 1996), as there are several problems related to the detection system used. First, previous studies have evaluated the efficiency and accuracy of membrane sorting by measuring the final amount of targeted molecules in each PM subregion. This classical method has successfully identified many targeting signals of each membrane protein because it can sensitively and quantitatively assay the efficiency and accuracy of the entire membrane sorting system. However, this method is not sensitive enough to dissect each possible subprocesses, as it only monitors the final result: the amount of protein on the PM. For example, if the docking and retention mechanisms remain intact, the effect of missorting preceding these processes will be underscored (Grindstaff et al., 1998). As a result, this method is not sensitive for evaluating the contribution of the putative selective transport step.
To overcome this limitation, a direct evaluation of the membrane transport within a cell is important. Recent progress in quantitative confocal microscopy has enabled monitoring of the dynamic movement of fluorescence-tagged molecules in living cells (Lippincott-Schwartz et al., 1998; Nakata et al., 1998), and individual transported intermediate vesicles are visualized within the cells. This method has been successfully applied to quantitatively estimate the kinetics of membrane traffic in nonpolarized cells (Hirschberg et al., 1998). To estimate the real contribution of the putative selective transport system, possibly mediated by a specific MT motor in polarized cells, we adopted a method by which transport to the sub-PM region was evaluated based on the fluorescence intensity of the molecules in the sub-PM region by quantitative confocal microscopy. This method enables direct examination of the transport from the TGN to the apical PM.
The involvement of cytoplasmic dynein and kinesin superfamily proteins (KIFs) in apical transport has been reported (Lafont et al., 1994; Fath et al., 1997; Kraemer et al., 1999; Kreitzer et al., 2000); however, their role in selective transport is not yet established. Futhermore, research has not yet identified which KIF is involved in the apically targeted transport. In polarized epithelial cells, MTs usually display an apicobasal alignment with their minus ends oriented towards the apical region of the cells (Bacallao et al., 1989). Thus, the apically targeted transport should be conveyed by the MT minus enddirected motor whose directionality is opposite to that of most KIFs, including conventional kinesin. This suggests that some new minus enddirected KIFs may be involved in apically targeted transport.
Our recent systematic search for new KIFs (Nakagawa et al., 1997; Hirokawa, 1998a) demonstrated the existence of COOH-terminal motor domaintype KIFs (KIFCs) that are expected to be minus enddirected motors. KIFC3 is one of these new KIFCs and is abundant in the kidney, thus, it is a good candidate for the apically targeted transport motor in epithelial cells.
In this study, we cloned the full-length cDNA of KIFC3 and showed its property as a minus enddirected motor. KIFC3 is associated with apically targeted transport vesicles conveying annexin XIIIb and haemagglutinin (HA). The overexpression of KIFC3 and its dominant-negative form demonstrated the function of KIFC3 in apically targeted transport. The absence of the dyneindynactin complex on KIFC3-associated vesicles suggests the existence of at least two independent apically targeted transport pathways: KIFC3- and cytoplasmic dyneindynactin-dependent transport.
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Results |
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As shown in Fig. 1, E and F, the antibody against the recombinant KIFC3 rod fragment recognized three or four bands of protein in the total homogenate of the kidney (asterisks); these multiple bands were not detected in KIFC3 knockout mice (Fig. 1 F, right lane) (unpublished data). Because only the highest band was recognized by the anti-KIFC3 NH2 terminus antibody (unpublished data), the lower bands are NH2-terminally truncated proteins that may be produced by posttranslational modification or specific degradation pathway, as no splice variants were detected by the cap site hunting reverse transcriptionPCR (Fig. 1 G, arrow).
Minus enddirected motor activity of KIFC3
To estimate the in vivo function of KIF, it is important to analyze its motility characteristics (polarity, speed, and processivity), which are determined by the motor domain and its adjacent region, the so-called "neck" (Endow and Waligora, 1998; Sablin et al., 1998). Therefore, we expressed a motor domain construct of KIFC3 (C3S) that consists of an NH2-terminal polyhistidine tag and the reactive cysteine residue, followed by a short stretch of a coiled coil stalk, the conserved KIFC consensus neck (Saito et al., 1997; Hirokawa et al., 1998b; Sablin et al., 1998), and the COOH-terminal motor domain (Fig. 1 B).
The ATPase activity of C3S was activated >100-fold in the presence of MTs with Km (MT) values of 100 ± 30 nM. The kcat of the MT-activated ATPase activity of KIFC3 was 5 ± 1 /s per head.
To determine the motor activity of KIFC3, we directly measured the movement of a single fluorescence-labeled motor protein (Vale et al., 1996; Okada and Hirokawa, 1999) on the polarity-marked MTs (Hyman and Mitchison, 1991). The degree of error of the position measurement by centroid analysis was checked by the C3S protein tightly bound to MTs in the presence of adenylyl imidodiphosphate (AMP-PNP) (Fig. 2 C), or the C3S protein adsorbed on the cover glass surface (unpublished data). The root mean square error of the position measurement was 30 nm.
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Thus, the KIFC3 motor domain has an MT minus enddirected motor activity, which may be modulated or regulated by the NH2-terminal tail region. This issue needs more investigation with full-length KIFC3 protein.
Localization of KIFC3 in renal tubular epithelial and MDCK II cells
What is the function of KIFC3 in vivo? As the first clue to the intracellular functions of KIFC3, we examined the localization of KIFC3 in the kidney. Immunocytochemical staining of renal tissues showed that KIFC3 is localized in the distal tubules (Fig. 3 Aa, D) and loops of Henle (unpublished data), but not in the proximal tubules or the glomeruli (Fig. 3 Aa, P and G, respectively), with stronger staining in the apical area of these epithelial cells (Fig. 3 Ab, arrowheads).
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Association of KIFC3 with Triton-insoluble vesicles
For the biochemical characterization of these vesicular structures, we examined the behavior of KIFC3 using a flotation assay in combination with detergent extraction (Fig. 3 D). After extraction with a buffer containing 1% Triton X-100 at 4°C, the cell extract was applied at the bottom of a discontinuous sucrose density gradient (5/30%). The KIFC3-associated membrane was resistant to Triton X-100 and floated to the lighter fraction along with Fyn and annexin XIIIb, well-known components of unique Triton-insoluble, cholesterol-rich membrane subdomains (Brown and Rose, 1992; Lafont et al., 1998). The distribution of KIFC3-associated vesicles overlapped with that of KIF5B and the dynein intermediate chain (DIC), another candidate for a member of apical transport motor complex; the peak fraction of KIFC3 was lighter than that of DIC. Collectively, these data showed that KIFC3 exists in close association with the Triton-insoluble membranes (Fig. 3 D).
Specific transport of annexin XIIIbassociated vesicles by KIFC3
Both immunocytochemical and biochemical data suggest that KIFC3 is involved in the transport of apical Triton-insoluble membranes. Because the staining pattern of KIFC3 was similar to that of annexin XIIIb, a molecule apically transported from TGN to the PM (Lafont et al., 1998) (Fig. 3, Ba and Bc, green), we next examined the responses of KIFC3 and annexin XIIIb to perturbations known to affect such transport.
For the quantitative analysis of the perturbations, we adopted a quantitative immunofluorescent method. Because KIFC3 remains in the apical sub-PM region, the standard apical surface-labeling method cannot be applied. Therefore, we quantified the amount of the molecule in the apical region of the cell by measuring fluorescence intensity using quantitative confocal microscopy after immunofluorescent staining (Lippincott-Schwartz et al., 1998).
In both control and experimentally treated cells, the measured fluorescence intensities varied largely; however, their distribution was well fitted by the theoretically expected distribution, a logarithmic Gaussian distribution (Fig. 4 B) (accumulated 2 = 49.457; P < 10-128 by
2 test) (Kaplan and Meier, 1958). Therefore, in the following analyses we adopted parametric statistical tests (Student's t test, for example) and reported the logarithmic mean of the relative fluorescence intensity normalized by the corresponding control data sets, although nonparametric tests resulted in essentially the same conclusions.
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Necessity of motor function of KIFC3 for apical transport
The next question is whether KIFC3 actively transports its associated vesicles in vivo, and if so, how does this transport contribute to the transport of the apical PM protein? To answer these questions, we first disrupted an MT network using nocodazole. After overnight incubation with 10 µg/ml nocodazole, most MTs in polarized MDCK cells were depolymerized except for a small amount remaining in the apical area (Fig. 5, Ab and Ab', red). Such treatment significantly decreased the amount of KIFC3 in the apical sub-PM region (Fig. 5, Ab and Ab', green) (61 ± 2% with nocodazole), indicating that intact MTs are necessary for the apical localization of KIFC3.
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These perturbations altered the subcellular localization of annexin XIIIb (Fig. 5, B and C; Table II). In cells overexpressing headless KIFC3, the apical accumulation of annexin XIIIb decreased to 58 ± 1% (Fig. 5, Be, Be', and C). In contrast, in cells overexpressing full-length KIFC3, the apical accumulation of annexin XIIIb increased to 142 ± 1% (Fig. 5, Bf, Bf', and C). In either infected cell, the total amount of annexin XIIIb was same as that in noninfected control cells (unpublished data). Thus, the observed perturbation to the localization of annexin XIIIb implies that the apical accumulation of annexin XIIIb is dependent on the motor activity of KIFC3. The fact that annexin XIIIb is the cargo membrane protein of KIFC3 suggests that KIFC3 actively transports annexin XIIIbassociated vesicles to the apical region.
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Depletion of KIFC3 by acute efflux of cholesterol
Apical transport is reported to be closely related to lipid transport, particularly cholesterol transport (Keller and Simons, 1998; Heino et al., 2000). Consistently, KIFC3 is associated with a class(es) of Triton Xinsoluble, cholesterol-rich vesicles (Fig. 3 D). Therefore, we examined the effect of acute cholesterol efflux from the apical PM, induced by the methyl-ß-cyclodextrin (mßCD) treatment, which selectively and rapidly reduces the intracellular cholesterol content, and is expected to selectively disrupt the cholesterol-rich annexin XIIIb and HA vesicles (cargo vesicles of KIFC3) (Kilsdonk et al., 1995). Consistent with our expectation, a 60% reduction in the amount of free cholesterol in the apical PM occurred, as measured by the fluorescence intensity of combined filipin, dispersed KIFC3, and annexin XIIIb from the apical sub-PM region (Fig. 7, Ad and Ae); KIFC3 and annexin XIIIb reaccumulated rapidly after the addition of FCS for the replenishment of cholesterol. (Fig. 7, Ag and Ah). Through this process, the total amount of KIFC3 and annexin XIIIb remained constant (Fig. 7 C), indicating that the dispersion and reaccumulation of KIFC3 in the apical sub-PM region depend on the cholesterol content.
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Discussion |
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Multiple apical transport pathway supported by different MT motors
With this new assay system, we showed that the apical transport of annexin XIIIb and HA is inhibited by a dominantnegative KIFC3, and that the transport is activated by the overexpression of intact KIFC3 (Figs. 5, B and C, and 6, A and B). These results indicate that at least some part of the apical transport system depends on the activity of the KIFC3 motor, and that the efficiency of this transport (sub)system is limited by the amount of active KIFC3 motors.
It should be noted that inhibitory perturbations to KIFC3 activity only resulted in at most a 50% decrease in the rate of apical membrane transport. This suggests that about half of the amount of apical membrane transport in stationary MDCK cells proceeds through a process independent of KIFC3, which could explain the lack of gross abnormality in KIFC3 knockout mice (Yang et al., 2001, and unpublished data).
One good candidate for the KIFC3-independent apical transport pathway is the previously proposed pathway supported by cytoplasmic dynein (Fath et al., 1997; Lafont et al., 1994). The inhibition of cytoplasmic dynein activity by the anti-dynein antibody is reported to inhibit 70% of apically transported HA (Lafont et al., 1994). The discrepancy in the percentage of the contribution of KIFC3 and cytoplasmic dynein to the apical transport (50 vs. 70%) may not be very significant, considering that the experimental condition and the assay strategies differ significantly. Similarly, the discrepancy between the percentage of antiKIFC3-labeled HA vesicles in our immuno-EM experiment (10%) and that of KIFC3's contribution to HA transport (50%) will not be substantial, because the efficiency of the immunogold labeling is very low. Thus, we believe that half of the amount of the apical membrane transport of stationary MDCK cells is conveyed by KIFC3, and that the other half is conveyed by cytoplasmic dynein. Consistently, cytoplasmic dynein was undetectable on immunologically purified annexin XIIIbassociated vesicles with which KIFC3 was copurified. The possibilities remain that only an undetectably small amount of cytoplasmic dynein is required for the transport, or that cytoplasmic dynein can be easily lost during the purification process. However, the differences in the subcellular distribution observed by light microscopy and immuno-EM and in response to the cholesterol perturbations, strongly support the functional differentiation of these two motors.
Although KIFC3 is ubiquitously expressed in many different tissues, its expression level differs largely among different tissues. Even in epithelial cells, KIFC3 is highly expressed in the renal distal tubule epithelium, whereas it is barely detectable in the secretory epithelium such as that of the intestine and pancreas. Interestingly, the subcellular localization of cytoplasmic dynein differs between these two classes of epithelium. In KIFC3-rich epithelial cells such as MDCK cells, cytoplasmic dynein shows a diffuse pattern in the cytoplasm by immunostaining (unpublished data), whereas it is reported to localize apically in secretory epithelial cells such as intestinal and pancreatic cells (Fath et al., 1997; Kraemer et al., 1999). This implies that ubiquitous basal apical transport by cytoplasmic dynein is supplemented by a more specific apical transport by KIFC3 in some cells. Further analysis of the molecular mechanism of cargo binding by MT motors and identification of diversified transport markers are expected to clarify the redundancy and selectivity of motor transport.
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Materials and methods |
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Cap site hunting
Cap site cDNA and forward primers for the cap (mGppp) site region were purchased from Nippon Gene. Reverse primers were synthesized corresponding to the complementary sequences for the first and second nested PCR, which are conserved in mouse and human KIFC3 (Hoang et al., 1998).
Antibodies
Antibodies against gp135 and annexin XIIIb were gifts from Drs. G.K. Ojakian (State University of New York Health Care Center, Brooklyn, NY) and F. Lafont (European Molecular Biology Laboratory, Heidelberg, Germany). Anti-Fyn, anti-rab 11, anti-DIC, and anti-HA antibodies were obtained from Transduction Laboratory, Zymed Laboratories, Chemicon International, Inc., and Advanced ImmunoChemical, Inc., respectively.
Polyclonal antibody against recombinant KIFC3 fragments corresponding to the NH2-terminal half of the rod domain (aa 100250) was raised in rabbits and used after affinity purification.
Western blotting
Tissue specimens were homogenized with RIPA buffer (50 mM Tris-HCl, pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% SDS, and a protease inhibitor cocktail), centrifuged at 20,000 g for 10 min, and boiled. For immunoprecipitation, 1 mg of each tissue homogenate was reacted with 0.6 µg of affinity-purified anti-KIFC3. Immunoblots were detected with alkaline phosphataseconjugated antirabbit IgG antibody (Cappel) or 125I-labeled protein A (ICN).
ATPase and in vitro motility assays
For the ATPase and in vitro motility assays, C3S, a motor domain construct produced by PCR, was cloned into the pET21B expression vector (Novagen). Recombinant protein was expressed and purified as described (Okada and Hirokawa, 1999). MT-activated ATPase activity and single motor motility assay were performed as described previously (Okada and Hirokawa, 1999). In brief, Alexa (Molecular Probes)-labeled C3S protein was observed to move along Bodipy-labeled, polarity-marked MTs prepared according to the method of Hyman and Mitchison (1991). The resultant images were analyzed on a Macintosh computer using NIH Image software with a custom-made macroprogram for semiautomatic centroid analysis.
Cell culture
MDCK II cells were provided by Dr. E. Rodriguez-Boulan (Cornell University Medical College, New York, NY). The cells were maintained in DME (GIBCO BRL) supplemented with 5% FCS. Polarized culture was performed on Transwell (Costar) as described by Bennett et al. (1988), and then harvested for 6 d.
Immunofluorescence microscopy and immuno-EM
Pre- or nonpretreated cells were fixed with a PLP fixative (4% paraformaldehyde, 10 mM NaIO4, 75 mM L-lysine monohydrochloride, and 37.5 mM phosphate buffer) for 15 min, stained by the avidin biotin complex method or according to a standard immunofluorescence protocol (Noda et al., 1995), and observed under a confocal laser-scanning microscope LSM510 (ZEISS).
For immuno-EM, the sample was fixed with the PLP fixative for 2 h and processed according to the method described by Funakoshi et al. (1996).
Quantitative analysis of fluorescence intensity of immunocytochemistry
The apical plane of polarized MDCK cells was judged as upper limit of E-cadherin staining (Takara Biomedicals), or as the level of gp135. The mean density of fluorescence intensity of each cell in that plane was measured using NIH Image. Each set of data was collected from two to four independent experiments. The fluorescence intensity was normalized by the corresponding group data set, and then fitted with a logarithmic normal distribution for statistical comparison by Student's t test. To guarantee the robustness of this statistical analysis, nonparametric method (Wilcoxon's rank-sum test) was also performed.
Flotation assay
Polarized MDCK cells were extracted with 1% Triton X-100 containing TBS buffer (pH 8.0) for 30 min at 4°C. Crude extract was processed and applied at the bottom of the 5/30% sucrose discontinuous gradient as described by Rodgers et al. (1994).
Immunoprecipitation and GST pulldown
Recombinant KIFC3 fragments corresponding to the NH2-terminal tail domain (aa 1100) were expressed in Escherichia coli pGEX vector as GST fusion protein. They were recovered on glutathione Sepharose Fast Flow beads (Amersham Pharmacia Biotech). Polarized MDCK cells were resuspended in sucrose buffer (0.25 M sucrose, 20 mM Tris-HCl, pH 8.0, 2 mM EGTA, 1 mM DTT, 0.1% gelatin, and a protease inhibitor cocktail), homogenized by pipetting through 27-gauge needles, and centrifuged at 1,000 g for 20 min. The recovered supernatant was then incubated with bait-conjugated glutathione Sepharose beads or antiannexin XIIIb antibody-fixed protein A-Sepharose 4 Fast Flow beads (Amersham Pharmacia Biotech) at 4°C overnight. The beads were washed and prepared for SDS-PAGE.
Construction of adenovirus vectors
Full-length KIFC3 and its COOH-terminally truncated form (aa 1515) were inserted into the pAdex apCAW vector. The recombinant virus was purified and amplified according to Terada et al. (1996). Polarized MDCK cells were infected from the basolateral side at 100 moi and observed after 1824 h (Delporte et al., 1997).
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Footnotes |
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Acknowledgments |
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This work was supported by a special Grant-in-Aid for the Center of Excellence (N. Hirokawa) from the Japan Ministry of Education, Science, Sports, Culture and Technology.
Submitted: 8 August 2001
Revised: 29 August 2001
Accepted: 29 August 2001
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References |
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