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• W2091031576 abstract "Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTP-form of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts. Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTP-form of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts. Skeletal modeling during growth and bone remodeling in the adult skeleton are dependent on bone resorption. Osteoclasts are the cells that are responsible for the degradation of the organic and inorganic bone matrix. These multinucleated cells mature from a monocyte/macrophage lineage of precursor cells and are mainly located in the vicinity of the bone surface in bone marrow (1Suda T. Takahashi N. Martin T.J. Endocr. Rev. 1992; 13: 66-80PubMed Google Scholar). When bone resorption is induced, osteoclasts migrate to the site of the resorption, become highly polarized (2Väänänen H.K. Zhao H. Mulari M. Halleen J.M. J. Cell Sci. 2000; 113: 377-381Crossref PubMed Google Scholar), and form four distinct membrane domains: the ruffled border, sealing zone, functional secretory domain, and basolateral domain (2Väänänen H.K. Zhao H. Mulari M. Halleen J.M. J. Cell Sci. 2000; 113: 377-381Crossref PubMed Google Scholar, 3Salo J. Metsikko K. Palokangas H. Lehenkari P. Väänänen H.K. J. Cell Sci. 1996; 106: 301-307Google Scholar, 4Väänänen H.K. Horton M. J. Cell Sci. 1995; 108: 2729-2732Crossref PubMed Google Scholar). During the process of osteoclast activation, the cells attach to the bone matrix through a ring-like structure called a sealing zone. The membrane-facing bone matrix inside the sealing zone is the ruffled border formed via rapid fusion of intracellular acidic vesicles to the plasma membrane (5Palokangas H. Mulari M. Väänänen H.K. J. Cell Sci. 1997; 110: 1767-1780PubMed Google Scholar, 6Väänänen H.K. Karhukorpi E.K. Sundquist K. Wallmark B. Roininen I. Hentunen T. Tuukkanen J. Lakkakorpi P. J. Cell Biol. 1990; 111: 1305-1311Crossref PubMed Scopus (323) Google Scholar). This results in acidification and release of proteinases into the space between the bone matrix and the ruffled border known as the resorption lacuna. As a result, bone matrix is digested (7Baron R. Neff L. Louvard D. Courtov P.J. J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (598) Google Scholar). The mineral is dissolved by acid and collagen matrix degraded by proteinases, the degradation products are internalized locally, and transcytosed to the functional secretary domain at the top of the polarized osteoclasts for secretion (8Salo J. Lehenkari P. Mulari M. Metsikko K. Väänänen H.K. Science. 1997; 276: 270-273Crossref PubMed Scopus (347) Google Scholar, 9Salo J. Metsikko K. Väänänen H.K. Mol. Biol. Cell. 1994; 5: 431Google Scholar). Thus, the ruffled border, which is assembled by fusion with endosomal membranes, shows not only characteristics of the plasma membrane but also those of the late endosomal/lysosomal membranes (5Palokangas H. Mulari M. Väänänen H.K. J. Cell Sci. 1997; 110: 1767-1780PubMed Google Scholar). Indeed, many proteins present at the ruffled border are also found in endosomal and/or lysosomal membranes, including Rab7, vacuolar H+-ATPase, cathepsin K, and others. We have previously shown that the ruffled border is divided functionally into two subdomains, the fusion zone for secretion and the uptake zone for endocytosis of degraded products (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar). Recent studies have shown that Ras-related small GTPases of the Rab family control the endocytic, secretory, and recycling traffic of intracellular vesicles in mammalian cells (11Zerial M. Stenmark H. Curr. Opin. Cell Biol. 1993; 5: 613-620Crossref PubMed Scopus (343) Google Scholar, 12Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 13Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2730) Google Scholar, 14Cavier P. Gound B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (423) Google Scholar). Approximately 60 Rab proteins have been identified in the human genome. Each Rab protein localizes to a distinct cytoplasmic compartment where it regulates the steps of transport by the recruiting of specific effectors, which connect Rabs to either actin or microtubule-based motor proteins (15Seabra M.C. Coudrier E. Traffic. 2004; 5: 393-399Crossref PubMed Scopus (160) Google Scholar, 16Olkkonen V. Stenmark H. Int. Rev. Cytol. 1997; 176: 1-85Crossref PubMed Google Scholar, 17Waters M.G. Pfeffer S.R. Curr. Opin. Cell Biol. 1999; 11: 453-459Crossref PubMed Scopus (148) Google Scholar, 18Peranen J. Auvinen P. Virta H. Wepf R. Simons K. J. Cell Biol. 1996; 135: 153-167Crossref PubMed Scopus (217) Google Scholar, 19Echard A. Jollivet F. Martinez O. Lacapere J-J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 379: 580-585Crossref Scopus (414) Google Scholar). Rab proteins, like other small G proteins, control this machinery as molecular switches between GTP-bound (active) and GDP-bound (inactive) states. Their on/off regulatory function is restricted to the membrane compartment where they are localized. In this cycling, guanine nucleotide exchange factors replace GDP with GTP, thus activating Rabs, and act as a trigger to recruit effectors. GTPase-activating proteins, on the other hand, inactivate Rabs by inducing hydrolysis of GTP to GDP, release Rabs from the membrane into the cytosol and cause effectors to dissociate from Rabs (11Zerial M. Stenmark H. Curr. Opin. Cell Biol. 1993; 5: 613-620Crossref PubMed Scopus (343) Google Scholar, 12Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 15Seabra M.C. Coudrier E. Traffic. 2004; 5: 393-399Crossref PubMed Scopus (160) Google Scholar). Accumulating evidence suggests that Rab7 is a key regulator of the late steps of the endocytic pathway and also vacuolar fusion (20Feng Y. Press B. Wandinger-Ness A. J. Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (537) Google Scholar, 21Meresse S. Corvel J.-P. Chavier P. J. Cell Sci. 1995; 108: 3349-3358PubMed Google Scholar, 22Vitelli R. Santillo M. Lattero D. Chiariello M. Bifulco M. Bruni C.B. Bucci C. J. Biol. Chem. 1997; 272: 4391-4397Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In the resorbing osteoclast, Rab7 was shown to be at the ruffled border (5Palokangas H. Mulari M. Väänänen H.K. J. Cell Sci. 1997; 110: 1767-1780PubMed Google Scholar, 10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar), whereas in the non-resorbing osteoclast, Rab7 localized around the nuclei where late endosomes and lysosomes accumulated. Moreover, down-regulation of Rab7 by antisense oligonucleotides prevented the formation of the ruffled border and consequently bone resorption (23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). However, the mechanism of how Rab7 regulates the ruffled border formation has remained obscure. Recently, four Rab7 effectors have been found by using the yeast two-hybrid system. One is Rab-interacting lysosomal protein (RILP) 2The abbreviations used are: RILP, Rab-interacting lysosomal protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; TRITC, tetramethylrhodamine isothiocyanate. (25Cantalupo G. Alifano P. Roberti V. Bruni C.B. Bucci C. EMBO J. 2001; 20: 683-693Crossref PubMed Scopus (390) Google Scholar, 26Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), others are rabring7 (27Mizuno K. Kitamura A. Sasaki T. Mol. Biol. Cell. 2003; 14: 3741-3752Crossref PubMed Scopus (73) Google Scholar), XAPC7 (28Dong J. Chen W. Welford A. Wandinger-Ness A. J. Biol. Chem. 2004; 279: 21334-21342Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and hVPS34/p150 (29Stein M.P. Feng Y. Cooper K.L. Welford A.M. Wandinger-Ness A. Traffic. 2003; 4: 754-771Crossref PubMed Scopus (136) Google Scholar). RILP has been shown to induce the recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes and to conduct these vesicles toward the minus end of microtubules surrounding the nucleus in cells with high expression of RILP (26Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). Moreover, RILP has also been reported as a Rab34 effector involved in the spatial positioning of the lysosomes in high RILP expression cells (30Wang T. Hong W. Mol. Biol. Cell. 2000; 13: 4317-4332Crossref Google Scholar). Based on the fact that Rab7 controls the vesicular transport to the fusion zone of the ruffled border where the actin meshwork is present and underlines the membrane, and because we were unable to detect RILP in the cells, 3Y. Sun, K. G. Büki, O. Ettala, J. P. Vääräniemi, and H. K. Väänänen, unpublished data. we hypothesized that other unknown effectors of Rab7 might exist in these specific cells. Thus, we chose a new strategy, the bacterial two-hybrid system, using a GTPase-deficient mutant Rab7Q67L as bait to screen a rat trabecular bone and bone marrow cDNA library. We identified a new Rab7-interacting protein, P21 Ras-related C3 botulinum toxin substrate 1 (Rac1), another small GTPase protein. Here we demonstrate further that Rac1 binds Rab7, and they colocalize in resorbing osteoclasts at the fusion zone of the ruffled border. Antibodies and Reagents—All antibiotics were purchased from Sigma. All cell culture media were from Invitrogen. One of the rabbit polyclonal anti Rab7 antibodies was a gift from Dr. S. R. Pfeffer (Stanford University), and the other was from Santa Cruz. Monoclonal anti-histidine antibody was purchased from Qiagen (Santa Clara, CA). The mouse monoclonal antibody against Rac1 was purchased from BD Biosciences. Osteoclast Culture—As described before (3Salo J. Metsikko K. Palokangas H. Lehenkari P. Väänänen H.K. J. Cell Sci. 1996; 106: 301-307Google Scholar, 4Väänänen H.K. Horton M. J. Cell Sci. 1995; 108: 2729-2732Crossref PubMed Google Scholar), osteoclasts were scraped from longitudinally cut long bones of 1–2-day-old rat pups and collected into α-essential medium with 20 mm Hepes, 10% heat-inactive fetal bovine serum, 100 IU/ml penicillin, and 100 μm/ml streptomycin. Subsequently, they were plated on bovine bone slices or on plastic coverslips. After a 30-min incubation at 37 °C in 5% CO2 and 95% air atmosphere for attachment, non-adherent cells were washed away, and attached cells were cultured further for 48 h in the same conditions. Construction of a Rat Trabecular Bone Marrow cDNA Library—Trabecular bone with marrow cells was taken from long bones of 1–2-day-old rat pups into α-essential medium. The mRNA from the sedimented cells was isolated using the Quickprep micro mRNA purification kit (Amersham Biosciences). A cDNA library was constructed with the BacterioMatch two-hybrid system XR cDNA library construction kit, according to the protocols provided by the manufacturer (Stratagene). Briefly, the first strand cDNA was primed with oligo(dT) and synthesized with reverse transcriptase. Double-stranded cDNAs were cloned into pTRG target plasmid opened with EcoRI and XhoI and transformed into XL1-blue MRF′ Kan library pack competent cells (Stratagene). The library plasmids were isolated using the plasmid maxi kit (Qiagen). Plasmid Constructs—The coding region of rat Rab7, Rab9, Rac1, Rac2, Cdc42, and C-terminal Rac1 were obtained by PCR amplification with specific primers designed according to the sequences from the gene data bank from trabecular bone marrow cDNA. The reading frame of Rab7 (GenBank™ accession number AF 286535) and Rab9 (GenBank™ accession number AF325692) were subcloned into SalI/NotI sites in pGEX-4T-1 plasmid (Amersham Biosciences). The complete reading frame of Rac1 and C-terminal Rac1 (GenBank™ accession number AY 491395), Rac2 (GenBank™ accession number XM_345854), and Cdc42 (GenBank™ accession number NM_171994) reading frame were subcloned into SalI/XhoI sites in pQE 80L series vectors (Qiagen). The constitutively active GTP-bound form of Rab7, Rab7Q67L, and the GDP-bound form of Rab7, Rab7T22N, were made from Rab7 in pGEX-4T-1 construct using the Stratagene QuikChange site-directed mutagenesis kit. Rab7Q67L was further subcloned into NotI/XhoI sites in the pBT bait plasmid in which a flexible linker was inserted in front of the start codon of Rab7. All constructs were confirmed by sequencing. Bacterial Two-hybrid Screening—The bacterial two-hybrid system was used to clone the trabecular bone marrow proteins that interact with the full-length constantly GTP-bound form of Rab7. Rat Rab7 was inserted in a frame into pBT bait vector containing full-length bacterial phage λ repressor protein (λCI) with the N-terminal DNA-binding domain. A rat trabecular bone marrow-derived cDNA library in the pTRG target plasmid fused to the N-terminal domain of the α-subunit of RNA polymerase was screened by using Rab7Q67L as a bait protein in BacterioMatch two-hybrid reporter strain. The interactions were evaluated based on the promoter activation of transcription of two reporter genes, an ampicillin-resistant gene and a β-galactosidase gene in the BacterioMatch two-hybrid system reporter strain. In the first round of selection, surviving colonies were selected on plates with carbenicillin, kanamycin, chloramphenicol, and tetracycline. The second round was β-galactosidase gene expression assay using X-gal substrate. Finally, the interactions were confirmed by individual recotransformation with the bait and rescued target plasmids and evaluating reporter gene expression. In Vitro Binding Histidine6-tagged Proteins with GST-tagged Rabs— His6-tagged Rac1, either full-length or the C-terminal 149 amino acid residues, Rac2, CDC42, and RhoB were purified from bacterial lysates on nickel-nitrilotriacetic acid-agarose columns (Qiagen). Eluates, 3–5 mg/ml protein, were used without further purification. GST-fused Rab7, Rab7Q67L, Rab7T22N and Rab9 were bound onto glutathione-Sepharose beads directly from bacterial lysates in binding buffer consisting of 50 mm Tris-HCl, 150 mm NaCl, 5 mm MgCl2, and 0.1% Triton X-100, pH 7.4. The beads were washed free of unbound protein. One μg of bound Rab was used in one experimental point in binding buffer containing 100 μm GTP (or GDP for Rab7T22N). His-tagged protein was added in excess, and the beads were incubated for 3 h at 4 °C. After three washings with binding buffer, the bound proteins were separated on SDS-PAGE. Bound His-tagged proteins were detected by monoclonal antibody against His6, and visualized bands were detected by horseradish peroxidase-conjugated anti-mouse secondary antibody with the ECL system (Amersham Biosciences). Indirect Immunofluorescence Microscopy—Osteoclasts were grown on bone slices or on coverslips, washed with PBS, and permeabilized with 0.01% saponin in 80 mm Pipes, pH 6.8, 5 mm EGTA, 1 mm MgCl2 for 5 min at room temperature. The cells were then fixed in 3% paraformaldehyde in PBS for 20 min and quenched with 50 mm NH4Cl for 10 min. The cells were then washed in 2% bovine serum albumin with 0.01% saponin in PBS for 5 min to block nonspecific binding. Primary antibodies in 0.2% bovine serum albumin with 0.01% saponin in PBS buffer were incubated with the permeabilized cells for 1 h at room temperature. After washing three times with the same buffer, the cells were incubated with Alexa Fluor 488-, TRITC- and Alexa Fluor 647-conjugated secondary antibodies in 0.2% bovine serum albumin with 0.01% saponin in PBS buffer 1 h at room temperature. Nuclei were stained with Hoechst 33258; cells were washed and then mounted with 80% glycerol in PBS. Confocal laser scanning microscopy was performed to obtain and analyze the immunofluorescence images with a Leica TCS-SP unit equipped with argon-krypton lasers (Leica Microsystems Heidelberg GmbH) and a ×63 oil immersion lens. Construction of a cDNA Library Derived from Trabecular Bone and Bone Marrow—A cDNA library derived from rat trabecular bone and bone marrow was constructed using commercial library construction kit according to the bacterial two-hybrid approach established by Dove and Joung (31Dove S.L. Joung J.K. Nature. 1997; 386: 627Crossref PubMed Scopus (228) Google Scholar). We used the trabecular bone and bone marrow of 1–2-day-old rat pups because those contain a relatively high number of osteoclasts. About 5 μg of mRNA was collected and used for the library construction. After second strand synthesis, we used a cDNA size fractionation column (Invitrogen) to eliminate cDNA shorter than 500 bp. Gel electrophoresis of the cDNA showed a smear ranging from 500 to 10,000 bp, the densest part being between 1,000 and 3,000 bp. The library represents 2×106 colonies/ml. One-hundred colonies were analyzed randomly by isolating plasmids followed by digestion with the EcoRI and XhoI enzymes. Ninety-five colonies showed inserts, all longer than 500 bp. Identification of the New Rab7-interacting Proteins Using the Bacterial Two-hybrid System—To identify proteins important for the Rab7-regulated vesicle traffic, we constructed a constitutively active, GTPase-deficient mutant, Rab7Q67L as a molecular bait to screen the rat trabecular bone marrow-derived cDNA library in pTRG plasmids via the bacterial two-hybrid selection. The constantly active form of the protein (Rab7Q67L mutant) was chosen, because we anticipated that Rab effectors would bind mainly to this form. Target proteins, which interacted with the bait, were selected by the activation of ampicillin resistance and next by induced β-galactosidase. Approximately 2 × 106 transformants were screened. The first screening was performed in four antibiotic plates, including chloramphenicol, kanamycin, tetracycline, and carbenicillin, which led to isolation of 2,800 colonies that activated the upstream Ampr reporter gene. Here we used carbenicillin instead of ampicillin to reduce the background, because carbenicillin is a more stable analog of ampicillin. Of the above, 104 colonies turned strongly blue on X-gal plates containing also chloramphenicol, kanamycin, and tetracycline, which meant that the bait-target interaction had been strong enough to trigger the downstream reporter gene, too. Finally, the interactions were confirmed by cotransformation of the bait and the rescued target plasmid pairs individually back into the reporter strain. Only the target plasmids that survived on the four antibiotics plates were selected to be studied further. We finally obtained a limited number of colonies for further studies. One of the strong positive colonies contained a 658-bp long insert, consisting of a 445-bp open-reading frame, a stop codon, and a 213-bp 3′-untranslated region, and a poly(A) tail. A search in the Ensembl rat genome data bank produced 100% sequence match on the p11 band of chromosome 12 (ENSRNOG00000001068 and predicted transcript: ENSRNOT00000001417). The gene on this site codes for Rac1, which is a member of the Rho family small GTPase proteins. Thus, we had isolated the C-terminal 149 residues moiety of Rac1 with the two-hybrid method. To get the full reading frame, we designed a primer pair based on the predicted sequence and PCR-amplified the full-length reading frame and the 3′-untranslated region. We then cloned and sequenced it and deposited the sequence in the GenBank™ (accession number AY491395) (Fig. 1). Comparison with the Rac1 transcript from the rat genome data bank of EMBL, the Rac1 cDNA of mouse (GenBank™ accession number BC003828), and human (GenBank™ accession number BC004247) in the gene data bank revealed a more than 30-base-pair completely conserved sequence starting with the translation start codon. Rat and mouse sequences showed 97% similarity, whereas between rat and human it was 90% in the coding region. At the protein level, Rac1 sequences of rat and mouse are identical, only one amino acid is different in man at position 135, which is isoleucine instead of threonine. In Vitro Interaction of Rab7 and Rac1—After we found that Rac1 strongly interacted with Rab7Q67L in the bacterial two-hybrid screening, we confirmed the direct interaction between Rab7 and Rac1 also with in vitro binding assays using recombinant proteins. Although the His-tagged full-length Rac1 and the isolated 445-bp C-terminal of Rac1 both bound to the GST-fused Rab7Q67L and wild type Rab7, only weak binding was detectable with the GST-fused Rab7T22N, the GDP-bound form of Rab7 (Fig. 2A). We then examined the specificity of the binding. We cloned GST-fused Rab9, another Rab protein that is also located in the late endosomal compartment. Rac1 did not bind to Rab9 in the same assay. Next, we studied interactions of Rab7 with Rac2, CDC42, and Rho B, other Rho family proteins. Rac2 is an isoform of Rac1 and shares 93% similarity in protein sequence, and the cellular functions of Rac1, Rac2, and CDC42 are overlapping, although the tissue distribution of them may be different (32Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 33Moll J. Sansig G. Fattori E. van der Putten H. Oncogene. 1991; 6: 863-866PubMed Google Scholar, 34Shirsat N.V. Pignolo R.J. Kreider B.L. Rovera G. Oncogene. 1990; 5: 769-772PubMed Google Scholar). We found that none of the three types of Rab7 bound to these Rho family members in our in vitro binding experiments (Fig. 2B). The GST protein alone was also included as a control in the assay system and showed no binding to Rac1 or to the C-terminal Rac1. All experiments were performed in the presence of 100 μm GTP or GDP for Rab7T22N. These experiments indicated that Rab7 specifically interacted with Rac1. Rac1 Colocalizes with Rab7 in Rat Osteoclasts and Fibroblast-like Cells—To visualize subcellular localization of endogenous Rab7 and Rac1, we used indirect immunofluorescence staining and laser confocal microscopy. In non-resorbing osteoclasts, we found that Rab7-positive vesicles were distributed around the nuclei. As also in other cells, Rab7 colocalized with the late endosomal/lysosomal marker LAMP2 (data not shown, see Ref. 23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), whereas Rac1 displayed a rather uniform distribution in the cytoplasm. We found that Rab7 colocalized with Rac1 in some but not all vesicles around the nuclei of osteoclasts where late endosomes and lysosomes were located (data not shown). Next, we examined the location of these two proteins in resorbing osteoclasts on bone. As has been shown previously (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar), Rab7 was redistributed to the fusion zone of the ruffled border just inside the sealing zone (actin ring) (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 35Mattson J.P. Skyman C. Palokangas H. Väänänen H.K. Keeling D.J. J. Bone Miner. Res. 1997; 12: 753-760Crossref PubMed Scopus (27) Google Scholar). We also found a large amount of Rac1 present at same area, clearly colocalizing with Rab7 (Fig. 3, A–F). In the same rat osteoclast culture on the bovine bone slice, we also saw Rab7 clearly colocalizing with Rac1 in numerous vesicles of fibroblast like cells (Fig. 3, G–I). Rab7, a member of the small GTPase family, regulates vesicular traffic from early endosomes to late endosomes and from late endosomes to lysosomes in the late endocytic pathway in eukaryotic cells (20Feng Y. Press B. Wandinger-Ness A. J. Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (537) Google Scholar, 21Meresse S. Corvel J.-P. Chavier P. J. Cell Sci. 1995; 108: 3349-3358PubMed Google Scholar, 22Vitelli R. Santillo M. Lattero D. Chiariello M. Bifulco M. Bruni C.B. Bucci C. J. Biol. Chem. 1997; 272: 4391-4397Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In resting osteoclasts, Rab7 is located in numerous intracellular acidic vesicles (5Palokangas H. Mulari M. Väänänen H.K. J. Cell Sci. 1997; 110: 1767-1780PubMed Google Scholar, 10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar). Strikingly, in the resorbing osteoclasts, Rab7 relocates to the ruffled border, especially on the fusion zone, where acidic vesicles are targeted to release their contents into the resorption lacuna (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar, 35Mattson J.P. Skyman C. Palokangas H. Väänänen H.K. Keeling D.J. J. Bone Miner. Res. 1997; 12: 753-760Crossref PubMed Scopus (27) Google Scholar). We have shown earlier that there are two distinct subdomains in the ruffled border of resorbing osteoclasts (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar). The central area of the ruffled border, “the uptake zone” is reserved for endocytosis of the degraded bone matrix, whereas the peripheral part of the ruffled border is for the fusion of acidic vesicles also containing proteolytic enzymes, mainly cathepsin K, named as the “fusion zone.” This part of the ruffled border contains actin filaments but no microtubules (10Mulari M. Zhao H. Lakkakorpi T.P. Väänänen H.K. Traffic. 2003; 4: 113-125Crossref PubMed Scopus (106) Google Scholar, 24Zhao H. Mulari M. Parikka V. Hentunen T. Lakkakorpi T.P. Väänänen H.K. Calcif. Tissue Int. 2000; 52: S63Google Scholar). We have also shown that the down-regulation of the expression of Rab7 with an antisense oligonucleotide impairs membrane delivery and fusion into the periphery of the ruffled border, and thus interferes with the formation of the ruffled border (23Zhao H. Laitala-Leinonen T. Parikka V. Väänänen H.K. J. Biol. Chem. 2001; 276: 39295-39302Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To find out which molecular machinery is involved in the membrane delivery process controlled by Rab7 in resorbing osteoclasts, we sought the downstream effectors of Rab7. Most Rab proteins have been reported to recruit effectors to specific membrane compartments in their GTP-bound form. The Rab proteins recruit the tethering, docking, and fusion factors and assemble them as a complex to control the membrane docking and fusion events (11Zerial M. Stenmark H. Curr. Opin. Cell Biol. 1993; 5: 613-620Crossref PubMed Scopus (343) Google Scholar, 12Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 13Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2730) Google Scholar). Some effectors of Rab proteins indirectly attach actin or microtubule-related motor proteins, and thus they control vesicular trafficking. Cantalupo et al. (25Cantalupo G. Alifano P. Roberti V. Bruni C.B. Bucci C. EMBO J. 2001; 20: 683-693Crossref PubMed Scopus (390) Google Scholar) and Jordens et al. (26Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar) found an effector of Rab7, RILP, which controls lysosomal transport by inducing the recruitment of dynein-dynactin motors to the minus ends of microtubules. RILP has also been reported as the Rab34 effector involved in the lysosomal spatial positioning in high RILP expression cells (30Wang T. Hong W. Mol. Biol. Cell. 2000; 13: 4317-4332Crossref Google Scholar). However, the interaction between RILP and motor proteins may be indirect. We examined the endogenous RILP in resorbing osteoclasts, but we could not find Rab7 colocalized with RILP (antibody kindly provided by Dr. J. Neefjes). The absence of RILP-Rab7 in osteoclast prompted us to look for more Rab7-interacting proteins/effectors. The two-hybrid system is generally used for this purpose. We chose the relatively new bacterial two-hybrid system for the following reasons. The yeast two-hybrid system has been used by many others, and we judged that its potential for Rab7 may be exhausted. Yeast has its own Vps small GTP-binding proteins that might confound the finding of further effectors. Cantalupo et al. (25Cantalupo G. Alifano P. Roberti V. Bruni C.B. Bucci C. EMBO J. 2001; 20: 683-693Crossref PubMed Scopus (390) Google Scholar) have reported that prenylation of Rab7 in yeast can cause a strong background in yeast, so they deleted the last three (prenylable) amino acid residues from the bait sequence, although those might be important in effector recognition. In bacteria, there is no Rab-like protein remaining; there is no prenylation, and full-length Rab7 can be used as bait. Making a cDNA library for the target constructs from rat osteoclasts would have been a major effort, considering the scarcity of these cells. Therefore, we have chosen newborn rat bone marrow, which is a rich source of osteoclasts. For the bait construct, the full-length Rab7 was chosen with a mutation in its GTP-binding region. The Q67L mutation renders the protein unable to hydrolyze GTP, keeping it in a constantly activated state (25Cantalupo G. Alifano P. Roberti V. Bruni C.B. Bucci C. EMBO J. 2001; 20: 683-693Crossref PubMed Scopus (390) Google Scholar, 26Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). To our satisfaction, we found new Rab7-interacting peptide sequences with the bacterial two-hybrid assay, one of them was Rac1, which is subject of this paper. The direct protein-protein interaction between Rab7 and Rac1 was also proven by GST-fused protein pull-down experiments. Apparently, the interaction is specific for Rac1, because other Rho GTPases such as RhoB, Rac2, or CDC42 have no affinity to Rab7 in these pull-down assays. Rab7 and Rac1 also colocalize in osteoclasts. Fluorescent images show punctate staining of this colocalization, which indicates that they stain the same intracellular vesicles. The overlap is far from 100%; this also corroborates with the notion that Rab7 does not need Rac1 for the full-length of transport. Another, possibly far reaching, observation is that the partial colocalization of Rab7 and Rac1 is not confined to resorbing osteoclasts, it can be seen in other cell types, i.e. the phenomenon is general. In these cells, the costained structures are mostly perinuclear, where the late endosomes usually reside. Functions of the Rho family of GTPases are well known. They organize the actin cytoskeleton for cell shape, polarization, and migration through their effectors near the cell cortex (40Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar), like IQGAP1, PAK, PIP5K, P140sra-1, and Por1 (41Nomura N. Nagase T. Miyajima N. Sazuka T. Tanaka A. DNA Res. 1994; 1: 223-229Crossref PubMed Scopus (133) Google Scholar, 42Weissbach L. Settleman J. Kalady M.F. Snijders A.J. Murthy A.E. J. Biol. Chem. 1994; 269: 20517-20521Abstract Full Text PDF PubMed Google Scholar, 43Knaus U.G. Wang Y. Reilly A.M. Warnock D. Jackson J.H. J. Biol. Chem. 1998; 273: 21512-21518Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 44Weernink P.A.O. Meletiadis K. Hommeltenberg S. Hinz M. Ishihara H. Schmidt M. Jakobs K.H. J. Biol. Chem. 2004; 279: 7840-7849Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Recent studies have shown that one of the Rac1/Cdc42 effectors, IQGAP1 (45Fukata M. Watanabe T. Noritake J. Nakagawa M. Yamaga M. Kuroka S. Matsuura Y. Iwamatsu A. Perez F. Kaibuch. K. Cell. 2002; 109: 873-885Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar), also an actin-binding protein (46Fukata M. Kuroka S. Fujii K. Nakamura T. Shoji I. J. Biol. Chem. 1997; 272: 29579-29583Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 47Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), directly interacts with CLIP-170 (45Fukata M. Watanabe T. Noritake J. Nakagawa M. Yamaga M. Kuroka S. Matsuura Y. Iwamatsu A. Perez F. Kaibuch. K. Cell. 2002; 109: 873-885Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar) that specifically accumulates at the plus ends of growing microtubules and tethers the endosomes to the microtubules (48Perez F. Diamantopoulous G.S. Stalder R. Kreis T.E. Cell. 1999; 96: 517-527Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 49Pierre P. Scheel J. Kreis T.E. Cell. 1992; 70: 887-900Abstract Full Text PDF PubMed Scopus (313) Google Scholar). Thus, these three molecules (Rac1/IQGAP1/CLIP-170) make a complex that bridges the plus ends of MT and the cortical actin meshwork (45Fukata M. Watanabe T. Noritake J. Nakagawa M. Yamaga M. Kuroka S. Matsuura Y. Iwamatsu A. Perez F. Kaibuch. K. Cell. 2002; 109: 873-885Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). On the other hand, so far only tubulin-tracking motor effector proteins, dynein and dynactin, were found for Rab7 (25Cantalupo G. Alifano P. Roberti V. Bruni C.B. Bucci C. EMBO J. 2001; 20: 683-693Crossref PubMed Scopus (390) Google Scholar, 26Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). The connection of actin and tubulin skeleton by Rac1 and its effectors would be a simple explanation to the puzzling observation that although no microtubules reach the peripheral ruffled border, Rab7-directed acidic vesicles do actually arrive there (Fig. 4). The knocked out Rac1 gene is lethal (50Sugihara K. Nakamura K. Nakao K. Hashimoto R. Otani H. Sakagami H. Kondo H. Nozawa S. Aiba A. Katsuki M. Oncogene. 1998; 17: 3427-3433Crossref PubMed Scopus (278) Google Scholar). Conditionally knocked out Rac1 was done by using the cre-LoxP system with either interferon-inducible (51Wells C.M. Walmsley M. Ooi S. Tybulewicz V. Ridley J.A. J. Cell Sci. 2004; 117: 1259-1268Crossref PubMed Scopus (156) Google Scholar) or with cell line-specific cre expression (with lysozyme promoter-cre) (52Glogauer M. Marchal C.C. Zhu F. Worku A. Clausen E.B. Foerster I. Marks P. Downey P.G. Dinauer M. Kwiatkowski J.D. J. Immunol. 2003; 170: 5652-5657Crossref PubMed Scopus (255) Google Scholar). The analysis of macrophages and leukocytes in these papers showed anomalies in cell-dependent immunity. It may well have been that some of these defects were caused by the absence of functional Rab7·Rac1 complex that was needed for normal endocytosis. In a Chlamydia-induced arthritis model, it was found that the clearance of pathogen in Rac1-deficient animal was defective (53Zhang X. Glogauer M. Zhu F. Kim T.H. Chiu B. Inman R.D. Arthritis Rheum. 2005; 52: 1297-1304Crossref PubMed Scopus (54) Google Scholar). All these findings point in the direction of Rac1 being an integral component of the endocytic/recycling pathway. We thank Dr. Suzanne R. Pfeffer (Stanford University) for kindly providing polyclonal rabbit anti-Rab7 antibody." @default.
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• W2091031576 title "Possible Role of Direct Rac1-Rab7 Interaction in Ruffled Border Formation of Osteoclasts" @default.
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