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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Departments of Surgery and Cell and Developmental Biology, Vanderbilt University School of Medicine, Vanderbilt-Ingram Cancer Center and the Nashville Veterans Affairs Medical Center, Nashville, Tennessee; and 2Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Submitted 22 February 2007 ; accepted in final form 22 June 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Rab11a; immunoglobulin A; trafficking; apical recycling; GP135; early endosome; EEA1; Eps15 homology domain
Rab11a interacts with and is regulated by specific interacting proteins. The exit of cargo from the recycling endosome is dependent on interaction of Rab11a with the actin motor protein myosin Vb (16), as well as with a group of proteins, the Rab11 family interacting proteins (Rab11-FIPs). The growing family of Rab11-FIPs currently include four Rab11-FIP1 proteins (10, 14, 18): Rab11-FIP2, Rab11-FIP3 (10), Rab11-FIP4 (34), and Rab11-FIP5 (pp75/Rip11) (28, 29). The Rab11-FIP proteins each interact with Rab11 family members (Rab11a, Rab11b, and Rab25) at their carboxy termini through predicted coiled-coil regions containing an amphipathic 
-helical Rab binding domain (10, 27). The diversity of multiple Rab11-FIP proteins, all of which bind to Rab11 with similar helices, suggests that each Rab11-FIP may be important in distinct trafficking processes.
In particular, Rab11-FIP2 (abbreviated here as FIP2) appears to form a ternary complex with both Rab11a and myosin Vb (11). In addition, FIP2 and Rab11a were redistributed when the microtubule architecture was modified with either taxol or nocodazole, demonstrating a link between the microtubule network and the recycling system (10). FIP2 has an amino-terminal C2 domain as well as a carboxyl-terminal Rab11 binding domain (10). A truncation of FIP2 lacking its amino-terminal C2 domain [Rab11-FIP2(129-512) or FIP2(
C2)] strongly inhibits plasma membrane recycling (11). This mutant has been used by us and others to assess proteins utilizing the Rab11a recycling pathway (8, 11, 19, 20). Similarly, a mutant myosin Vb lacking the motor domain, myosin Vb-tail, also acts as a dominant-negative mutant and has proven useful in the assessment of trafficking through the Rab11a trafficking pathway (4, 26, 32, 33). However, we recently have reported that FIP2 also has a role in the establishment of cell polarity (7). In this study, we sought to confirm our initial hypothesis that FIP2 is involved in the recycling pathway (10) utilizing a newly identified dominant-negative mutant of FIP2. Unexpectedly, this mutant has differentiable effects from the previously characterized FIP2 mutant FIP2(C2). The availability of multiple mutants for manipulation of the Rab11a pathway allows a greater appreciation of the complexity of the steps required for trafficking through the apical recycling system.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Site-directed mutagenesis. All site-directed mutagenesis of Rab11-FIP2 was performed with Pfu Turbo polymerase according to the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) with a 16-min extension time. Primers were synthesized (Invitrogen) with one nucleotide change per oligonucleotide sequence. All site-directed mutants were created in pEGFP-C2 (Clontech) and subsequently recloned into pET-30a (Novagen) with EcoRI and SalI restriction sites.
Cell culture. Parental T23 Madin-Darby canine kidney (MDCK) cells (2) and the stably transfected cell lines were grown in DMEM supplemented with 10% FBS (GIBCO), penicillin-streptomycin, 2 mM L-glutamine, and 0.1 mM MEM nonessential amino acids (GIBCO-BRL). Media for cell lines also contained 0.5 mg/ml G418 sulfate (Cellgro) and 0.25 ng/ml hygromycin (Invitrogen). In the stable cell lines, expression of the EGFP chimeras was inhibited with doxycycline (20 ng/ml; Calbiochem). To examine EGFP protein expression, cells were grown on 0.4-µm Transwell filters (Costar) without doxycycline in tetracycline-screened FBS (HyClone) medium for 2–4 days.
GFP constructs and transfections.
Doxycycline-inhibitable expression vectors were generated by excising the FIP2 wild-type and mutant sequences from pEGFP (EGFP is enhanced green fluorescent protein) vectors with NheI and SmaI and ligating into a pTRE2hyg vector (Clontech) cut with NheI and EcoRV. Transfection was performed with Effectene (Qiagen), following the manufacturer's protocol. One microgram of vector was transfected onto a 60-mm plate of T23 MDCK cells in normal medium. The following day, the cells were trypsinized and replated in serial dilutions, including 0.25 ng/ml hygromycin for selection and 20 ng/ml doxycycline for suppression of EGFP expression. Multiple colonies were selected, expanded for 10 days, and then screened for EGFP expression in medium with tetracycline-screened serum. Multiple clones were initially characterized, all of which showed similar expression patterns and levels of the EGFP construct. One clone was selected for each construct to use as the tetracycline-repressible stable cell line [expressing FIP2 wild type, FIP2(C2), FIP2(S229A), FIP2(R413G), or Rab11-FIP2(S229A/R413G) or EGFP-Rab11-FIP2(SARG)].
Electron microscopy procedures. Cells were plated on Costar clear Transwell filters and allowed to polarize for 4–5 days. The cells were washed twice in PBS before they were fixed in 4% glutaraldehyde, 0.1 M cacodylate buffer, and PBS for 1 h on ice. After fixation, the cells were washed twice in 0.1 M cacodylate buffer and 1x PBS. After cells were washed, the filters with the cells were excised from the Transwell and rolled into a cylindrical tube and tied with a strand of hair to prevent unraveling. Once rolled into tubes, the filters were transferred to 1.5-ml Eppendorf tubes and fixed for 2 h on ice in 1% osmium tetraoxide and 0.1 M cacodylate buffer. Next, the cells were washed once with 0.1 M cacodylate buffer followed by an ethanol dehydration series with 10-min incubations on ice. After the final 100% ethanol dehydration, the samples were rocked and incubated in equal volumes of 100% ethanol and Spurrs resin for at least 8 h and then incubated in a 1:2 ratio of 100% ethanol and Spurrs resin for another 8–16 h before undergoing two 8-h pure resin incubations. Transwell filters were then transferred to molds with fresh resin and allowed to polymerize for 24–48 h at 65°C. Sections (50–100 nm thick) were then taken of each filter of cells and collected on 200-nm copper grids. Grids of sections were then stained for 12 min in 1% depleted uranyl acetate and washed for 1 min. After the grids air dried, they were counterstained with saturated lead citrate for 10 min, followed by a 2-min water wash. The sections were imaged with a Philips CM12 electron microscope.
Fluorescent polymeric IgA (pIgA) trafficking in MDCK cells. Fluorescent labeling of pIgA and trafficking experiments were done as previously described (11) except that we used the cell culture medium described above. Cells were loaded from the basolateral side and fixed at 0 and 40 min after a 15-min loading incubation.
Fluorescent transferrin trafficking in MDCK cells. To assess internalization and trafficking of human transferrin, MDCK cells grown on Transwell filters were serum starved for 1 h. Cells were then allowed to internalize 10 µg/ml Alexa 633 human transferrin (Molecular Probes) for 1 h at 37°C and fixed as above. Transferrin localization and trafficking were then imaged with a Zeiss 510 confocal microscope.
Analysis of 125I-labeled IgA postendocytotic fate. 125I-labeled IgA was iodinated by the ICl method to a specific activity of 1.0–2.0 x 107 cpm/µg (3). The postendocytotic fate of a preinternalized cohort of 125I-IgA (at 5–10 µg/ml) that was transcytosed was analyzed as described previously (1). In brief, filter-grown MDCK cells expressing the various Rab11-FIP2 constructs and wild-type polymeric immunoglobin receptor (pIgR) were cultured in the presence or absence of doxycycline, and 125I-IgA was internalized from the basolateral cell surface of the cells for 10 min at 37°C. The basal surface of the cells was rapidly washed three times, and the apical and basolateral media were aspirated and replaced with fresh medium. The cells were then incubated for 3 min at 37°C. This wash procedure takes 5 min at 37°C. Fresh medium was added to the cells, and they were chased for up to 2 h at 37°C. At the designated time points, the apical and basolateral media (0.5 ml) were collected and replaced with fresh media. After the final time point, filters were cut out of the insert and the amount of 125I-IgA was quantified with a gamma counter.
The postendocytic fate of apically internalized 125I-IgA was essentially as described above. However, after ligand internalization, the apical surfaces of the cells were treated three times for 10 min with 100 µg/ml trypsin at 4°C to strip surface-bound ligand. The cells were then treated with 125 µg/ml soybean trypsin inhibitor for 10 min at 4°C (21). The postendocytic fate was determined as described above.
Statistical significance. Two-way ANOVA with a Bonferroni post hoc test was performed with GraphPad Prism version 4 for Macintosh, GraphPad Software (www.graphpad.com; San Diego, CA). The quantified trafficking numbers were assessed for statistically significant differences between each cell line with and without doxycycline in the media for either apical and basolateral medium collection. If a statistically significant difference was found by ANOVA between the two conditions, the differences at each individual time point were assessed with the post hoc test.
Immunofluorescence. Cells grown on Transwell filters were washed three times with PBS and then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were washed twice with PBS and stored at 4°C in PBS until staining. Cells were blocked with extraction buffer (10% normal donkey serum, 150 mM NaCl, 20 mM sodium phosphate, pH 7.4, 0.3% Triton X-100) for 20 min. Primary antibody was added in antibody buffer (10% normal donkey serum, 150 mM NaCl, 20 mM sodium phosphate, pH 7.4, 0.05% Tween 20) for 2 h. Secondary species-specific Cy dye-labeled anti-IgGs were added for 1 h in antibody buffer. After filters were washed with PBS two times, filters were cut out of the Transwells. These filters were washed once more in PBS and mounted with Prolong Antifade solution (Molecular Probes). Cells were imaged on a Zeiss LSM510 confocal microscope using a x100 lens. Z-sections were 0.5 µm.
Manipulations of the cytoskeleton. Manipulations of the microtubule cytoskeleton (5) were performed as previously described. Briefly, for assessment of the microtubule network, polarized cells were incubated at 4°C for 30 min. Nocodazole was added in a final concentration of 33 µM for 30 min at 4°C. The cells were then moved to 37°C for 1 h before fixation. For manipulation of the actin network, cells were incubated with cytochalasin D at a final concentration of 2 µM for 2 h at 37°C.
Yeast two-hybrid assay.
Yeast two-hybrid assay interactions were initially assessed by growth on triple dropout plates as previously described (10, 22). All interactions among Rab11a, myosin Vb, Eps15, and FIP2 were confirmed by assay of 
-galactosidase activity with development of blue reaction product within 3 h, as previously described (10).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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C), a FIP2 construct lacking the C2 domain (11), which localizes as a central subapical ring. After identification of the double mutant, we assessed the effects of each single mutant in stable cell lines. FIP2(S229A) has no discernible effect on the morphology of the recycling system, with EGFP-labeled vesicles scattered throughout the subapical region as we have previously seen with overexpression of wild-type FIP2 (Fig. 1A) (7, 10). In contrast, FIP2(R413G) had a severe phenotype with collection of the EGFP in tubular cisternae. These results suggested that the FIP2(R413G) mutation was responsible for the altered morphology seen in the double mutant. The cells expressing FIP2(R413G) were difficult to maintain in a uniform monolayer in culture on Transwell filters. Therefore, we utilized the more uniform pattern found in the double-mutant FIP2(SARG) cell line for our further studies (Fig. 1A).
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C2) in the z-axis. As previously reported (11), FIP2 wild type was localized to vesicles in the apical domain. The FIP2(SARG) pattern is a long tubular structure that localizes from the apical membrane down toward the nucleus of the cells. In contrast, the FIP2(C2) localizes in a centrally expansive tangle of tubules reminiscent of a donut shape above the nucleus (Fig. 1, B and C). In multiple cloned cell lines expressing Rab11-FIP2(SARG), the size of polarized cells was smaller than in cells expressing Rab11-FIP2(C2). Our group (35) has previously observed a similar enlarged cell phenotype in MDCK cells overexpressing the constitutively GTP-bound form of Rab11a, Rab11a(S20V).
To gain a better understanding of the morphological features of the Rab11-FIP2 mutants, we analyzed the FIP2(C2), FIP2(SARG), and FIP2 wild-type cell lines using transmission electron microscopy (Fig. 2). Electron microscopic images demonstrated that both Rab11-FIP2 mutants induced extensive tubular systems within the apical region of the cells. Although vesicular elements were always observed throughout the apical region of MDCK cells expressing wild-type Rab11-FIP2, no extensive tubular elements were observed.
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FIP2(SARG) and FIP2(R413G) decrease the rate of transcytosis.
In previous studies, a collapsed cisternal phenotype was associated with decreases in transcytosis. We therefore assessed whether the tubular cisternae of FIP2(SARG) affected the efficiency of trafficking. We examined the ability of the two single and the double Rab11-FIP2 mutants to traffic pIgA. Expression of the FIP2(R413G) mutant resulted in a reproducible decrease in transcytosis (Fig. 4). FIP2(SARG) also induced a marked delay in transcytosis through the plasma membrane recycling system (Fig. 4). The effects of the two mutants were similar to the inhibition of transcytosis observed in cells expressing FIP2(C2). In addition, expression of FIP2(C2) also appeared to promote basolateral recycling of pIgA. Expression of FIP2(S229A) had no effect on transcytosis. FIP2(R413G) had a slight, but significant, decrease on apical recycling. The other three FIP2 mutants did not elicit any apparent effects on the apical recycling of pIgA (Fig. 4).
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C2) or FIP2(SARG). Although in cells expressing FIP2(C2) pIgA was present in small vesicles in FIP2(SARG)-expressing cells, pIgA was present in more tubular structures. In contrast, cells expressing FIP2 wild type do not contain pIgA 30 min after chasing. These results suggested an alteration of trafficking into or out of the recycling system. However, neither FIP2 mutant altered trafficking of the basolateral recycling cargo transferrin (Supplemental Fig. 1). (The online version of this article contains supplemental data.) Similarly, endogenous transferrin receptor stained with the H68.4 monoclonal antibody did not accumulate with either of the FIP2 mutants (data not shown).
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C2) to investigate regulators of cargoes within the apical recycling pathway (8, 20). The FIP2(SARG) mutant has effects on the recycling system that are distinctly separate from the FIP2(C2) mutant. We assessed the localization of these mutants in the context of polarity (7). Our group has previously reported that FIP2 phosphorylation plays an important role in the establishment of epithelial cell polarity (7). Here, we found that the FIP2(SARG) cells have a dynamic localization as the cells polarize. One day postplating, the EGFP-tagged FIP2(SARG) mutant localization looked similar to wild-type localization (Fig. 6 compared with Fig. 1A). The tubular structure of FIP2(SARG) did not emerge until the cells were completely polarized. In contrast, EGFP-tagged FIP2(C2) cells maintained the subapical ring localization regardless of the state of polarity (Fig. 6).
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C2) alters the morphology of the early endosomal system marked by the Rab5-interacting protein EEA1. EEA1 does not colocalize with FIP2(C2), but the EEA1 containing early endosomes appears more centrally clustered around the EGFP-labeled tubular cisternae. However, this alteration was not seen in FIP2(SARG) or FIP2 wild-type cells, thus providing additional evidence that FIP2(SARG) is not simply causing a collapse of the entire endosomal pathway.
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C2) or FIP2 wild-type-expressing cells (Fig. 8). These results indicate that FIP2(SARG), but not FIP2(C2), can alter either recycling or de novo trafficking of GP135.
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C2) collapsed recycling system. Previous studies had shown that stabilization of microtubules with taxol causes relocation of the Rab11a-containing recycling endosomes to one subapical corner of polarized MDCK cells (16). However, taxol did not alter localization of either FIP2(SARG) or FIP2(C2) (data not shown). Furthermore, disruption of microtubules with nocodazole treatment did not affect the localization of either FIP2(SARG) or FIP2(C2) (Fig. 9). In contrast, the localization of FIP2 wild type in cells was affected by both treatments as previously reported by our group (Fig. 9) (10).
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C2) uncoupled the complex from microtubule network.
FIP2(SARG) does not alter interactions with EHD proteins.
Feig and colleagues (6) have previously noted that Eps15 binds to NPF motifs in the carboxyl-terminal half of FIP2. We have also noted the interaction of FIP2 with Eps15 in yeast two-hybrid library screens (data not shown). We found that FIP2(SARG) and FIP2(R413G) also interacted with Eps15 by yeast-two hybrid assays (Supplemental Table 1). Galperin et al. (9) have suggested that EHD3-containing vesicles may regulate vesicular microtubule-dependent movement. Because previous studies have reported that Rab11-FIP2 interacts with EHD1 and EHD3 (22) and because we observed an uncoupling of FIP2(SARG) from the microtubules, we examined the effect of the FIP2(SARG) mutant on these interactions. We found that FIP2(SARG) is able to associate with endogenous EHD1 by immunoprecipitation (data not shown). We visualized associations with EHD proteins through transfection into FIP2(SARG)- and FIP2(C2)-expressing cells (Fig. 10). EHD1 showed extensive colocalization with the cisternal accumulations in both Rab11-FIP2 mutant cell lines (data not shown). In addition, a portion of the overexpressed EHD3 population accumulated in the FIP2(SARG) and FIP2(C2) tubular structures (Fig. 10).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The inhibitory effects on FIP2 action appear to accrue from the Rab11-FIP2(R413G) mutation. We have utilized the cell lines expressing the dual FIP2(SARG) mutant for a number of our studies on apical recycling system morphology, since the cell lines expressing the single mutation demonstrated significant cell shape alterations over time. Both of these point mutants have a pronounced inhibitory effect on trafficking of pIgA in transcytosis assays, a critical Rab11a-directed pathway (10). Although the FIP2(SARG) double mutant exhibited a somewhat lower level of inhibition than the single R413G mutant, all of the effects on the morphology of the recycling system were similar. This collapsed cisternal structure displayed differentiable characteristics compared with the previously characterized FIP2(C2) mutant cell lines. Although the FIP2(SARG) localization was a tighter, more junctionally located tubular structure, the FIP2(C2) localized in a donut shape near the middle of the subapical region. Electron microscopy confirmed that these structures were tubular tangles and not multivesicular bodies. The altered shapes of these two inhibited recycling systems suggest that the mutants result in different levels of membrane retention within the recycling endosomes.
FIP2(SARG) has effects on the plasma membrane recycling system that are separable from those of FIP2(C2) (11) or myosin Vb tail (16), two previously characterized dominant-negative Rab11a trafficking mutants. In direct comparison with FIP2(C2), we noticed that the stages of the pathway affected appeared morphologically distinct. The ring shape of FIP2(C2) is different from the tight cisternal tubules seen with FIP2(SARG). The stage of the pathway affected by these mutants was also distinguishable by the localization of the EEA1-positive early endosomes. Cells expressing FIP2(C2) showed a redistribution of EEA1-positive early endosomes that was not apparent in FIP2(SARG)-expressing cells, suggesting that these two mutants affect different aspects of the FIP2 pathway. The FIP2(C2) mutant appears to alter a broader range of steps in the Rab11a pathway, whereas the FIP2(SARG) mutant may impact later events in the pathway. Nevertheless, it is important to note that both overexpressed FIP2(C2) and FIP2 mutants carrying the R413G mutation appear to inhibit transcytosis by inhibiting passage of trafficking vesicles through the recycling system. Thus IgA receptor-containing vesicles accumulate in apposition to the tubular system containing Rab11a. This failure of membranes to move into and through the recycling system may reflect the general uncoupling of recycling system membranes from the cytoskeleton. Thus treatment with either nocodazole or cytochalasin had no effect on the morphology of FIP2(SARG) or FIP2(C2)-containing tubular cisternae. The inhibition of trafficking therefore accrues either through blockade of trafficking into Rab11a-containing membranes or a sequestration of Rab11a and Rab11a-dependent effectors with the FIP2 mutants.
Analysis of the data presented here suggests that FIP2 is involved in multiple stages in passage through the Rab11a-associated recycling system. The alteration of the early endosomes with the FIP2(C2) mutant suggests that FIP2 may also be involved in an early hand-off stage. Previous reports have shown that a point mutation in Rab11a, S29F, caused Rab11a to colocalize with EEA1 (25), providing precedent for a role of Rab11a in the transition from the EEA1-positive early endosomes to the recycling endosome. In contrast, the accumulation of GP135 with FIP2(SARG) but not with FIP2(C2) suggests that some recycling cargoes may enter the recycling system at different points within the tubular recycling system. Multiple points of entry into the Rab11a/FIP2 recycling system may be exploited depending on the origin of the protein and possibly its destination. This model supports a dynamic vision of the recycling system trafficking. The R413G mutant of FIP2 had a small but significant effect on apical recycling. Although our group has previously reported that cells overexpressing FIP2(C2) showed a small decrease in apical recycling (11), in the present studies, using more quantitative methods, we were unable to demonstrate a significant effect on apical recycling across the entire time of trafficking. However, it is important to note that the presence of IgA receptor in the apical recycling pathway in MDCK is an ectopic scenario and may not be analogous to all apical recycling pathways for endogenous cargoes. The FIP2(C2) mutant can inhibit apical recycling of aquaporin-2 in collecting duct cells (23). Therefore, processing through apical recycling systems is likely cell and cargo dependent.
The precise mechanism responsible for inhibition of transcytosis by FIP2(R413G) mutants remains unclear. The FIP2(R413G) and FIP2(SARG) mutants retain their ability to bind Rab11a and myosin Vb, as assessed by yeast two-hybrid analysis. EHD1 and EHD3, both previously shown to regulate plasma membrane recycling, can interact with FIP2 (6, 9, 22). However, FIP2 mutants can still alter the distribution of EHD1 and EHD3. We have found that both FIP2(C2) and FIP2(SARG) are no longer dependent on the microtubule cytoskeletal network. Thus disruption of microtubules did not alter the morphology of the collapsed recycling system. Given these findings, it is tempting to speculate that FIP2(R413G) causes stabilization of protein complexes regulating apical recycling system trafficking. Although the 413 residue lies adjacent to the region involved in FIP2 dimerization (13), the R413G mutation did not alter self-interaction of FIP2 in two-hybrid assays. Whereas we could not determine any discrete alteration of interactions with FIP2(R413G), it is also possible that the R413G mutation may lead to stabilization of higher-order trafficking complexes. Similar inhibition of plasma membrane through stabilization of complexes was recently suggested for overexpression of a combination of cytoplasmic Lek1 (cytLEK1) and synaptosomal-associated protein of 25 kDa (SNAP-25) (26). A number of studies including this work have suggested that trafficking through the recycling system may be regulated by multiple hand-offs between regulatory complexes defining discrete subdomains within the plasma membrane recycling system (14). Thus stabilization of regulatory complexes may be a potent mechanism for inhibition of trafficking through the complex tubular recycling system. Future studies will be required to determine the precise mechanisms that may mediate the inhibitory effects of the FIP2(R413G) on transcytosis.
In summary, the studies presented here characterize the first point mutant of Rab11-FIP2 that exerts a dominant-negative influence on recycling system trafficking. Comparison of the FIP2(SARG) mutant with the FIP2(C2) truncation mutant demonstrates a differentiable set of FIP2-regulated trafficking steps along the endocytic pathway from early endosomes to the recycling endosomes. Although these studies support the role of Rab11-FIP2 in the regulation of transcytosis in polarized epithelial cells, they also establish Rab11-FIP2(SARG) as a specific inhibitor of Rab11a-dependent processes. Although previous studies have utilized a myosin Vb tail truncation mutant as a useful construct for examination of general plasma membrane trafficking pathways (16), recent studies indicate that myosin Vb is involved in multiple recycling pathways (30). Thus the FIP2(SARG) mutant may provide a more specific reagent for examination of cargoes trafficking through Rab11a-regulated transcytotic pathways in polarized epithelial cells.
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Confocal images were generated through the use of the VUMC Cell Imaging Shared Resource (supported by National Institutes of Health Grants CA-68485, DK-20593, DK-58404, HD-15052, DK-59637, and EY-08126).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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