In epithelial cells, endocytosed transferrin and its receptor, which cycle basolaterally, have been shown to transit through recycling endosomes which can also be accessed by markers internalized from the apical surface. In this work, we have used an in vitro assay to follow transfer of an endocytosed marker from apical or basolateral early endosomes to recycling endosomes labeled with transferrin. We show that calmodulin (CaM) function is necessary for transfer and identified myr4, a member of the unconventional myosin superfamily known to use CaM as a light chain, as a possible target protein for CaM. Since myr4 is believed to act as an actin-based mechanoenzyme, we tested the role of polymerized actin in the assay. Our data show that conditions which either prevent actin polymerization or induce the breakdown of existing filaments strongly inhibit interactions between recycling endosomes and either set of early endosomes. Altogether, our data indicate that trafficking at early steps of the endocytic pathway in Madin–Darby Canine Kidney cells depends on the actin-based mechanoenzyme myr4, its light chain CaM, and polymerized actin.
The surface of epithelial cells is separated into apical and basolateral plasma membrane domains, which differ in lipid and protein composition. Endocytosis can occur from both domains[1–3], and studies in the epithelial Madin–Darby Canine Kidney (MDCK) cell line have shown that a specific set of early endosomes is associated with each plasma membrane domain[4–6]. In contrast, late endosomes (LE) and lysosomes are common to both pathways. Previous studies indicate that communications exist between apical and basolateral early endosomes along the routes followed by basolaterally recycling transferrin receptors (TfRs) and transcytosing polymeric IgA receptors (pIgR)[3,7,8]. Indeed, both receptors, as well as membrane-bound or fluid-phase markers internalized from the apical surface, have been observed within a network of thin tubules clustered in the apical region of the cell[9–11], which closely resembles the recycling endosome as described in unpolarized cells[12,13]. In these cells, internalized TfR molecules first appear in early sorting endosomes that contain all endocytosed markers, and are then rapidly transferred to recycling endosomes, typically devoid of ligands destined for the lysosomes.
Major progress has been made in the characterization of molecules controlling membrane traffic, in particular vesicle formation and docking/fusion and recent studies are revealing the existence of complex networks of protein–protein, protein–lipid interactions and membrane–cytoskeleton interactions in the regulation of membrane dynamics. Studies on microtubules and associated motor proteins have shown that these interactions contribute to the organization and topology of organelles, and facilitate vesicle movement over long distances along both the endocytic and the secretory pathways. Recent data suggest that actin filaments are also involved in regulating membrane dynamics along the endocytic pathway[15–23]. A large number of actin-based mechanoenzymes, related to muscle myosin and to brush border myosin I, have been identified. This new superfamily encompasses 14 groups of so-called unconventional myosins[24,25], but the precise function of most of these proteins is unknown.
Several reports have suggested that calmodulin (CaM), a general regulator of key cellular functions, is also involved in the endocytic pathway. CaM may act indirectly by binding and modulating essential components of the endosomal transport machinery. For instance, the interaction of pIgR with CaM was shown by direct binding assays and by affinity chromatography. MDCK cells expressing the pIgR were treated with CaM inhibitors and it has been shown that transcytosis of IgA was inhibited, the recycling was concomitantly increased and there was no modification on IgA internalization. EEA1, which has recently been shown to be a central component in the endosome docking and fusion machinery, exhibits a CaM binding IQ motif, suggesting a possible regulation by CaM. Emans and Verkman used a sensitive real-time fluorescence assay to study fusion in endosomes derived from baby hamster kidney cells (BHK-21). They could show that the addition of purified brain CaM to the reaction significantly inhibited endosome fusion. In vacuole fusion assays, CaM was identified as the first protein of the post-docking phase[30,63]. Colombo et al. have demonstrated that Ca2+ stimulated endosome fusion in an in vitro assay derived from a macrophage cell line, and that the addition of CaM stimulated fusion beyond that produced by Ca2+ alone.
In the polarized epithelial MDCK cell line, work with synthetic CaM inhibitors implicated an involvement of CaM in transcytosis, recycling[27,32] and transport to the lysosome. However, in these in vivo studies, direct and indirect effects of CaM inhibitors cannot be distinguished, hence it remains unclear at which step or target protein CaM is acting and if it has a fusion propagating or inhibitory function. Therefore, we have adapted a well established in vitro transport assay[4,33] in order to study fusion between early endosomes and recycling endosomes in polarized MDCK cells. Using this assay, we observed that membrane transport within the MDCK apical and basolateral early endosomal systems is facilitated by polymerized actin filaments, and depends on myr4, a type I unconventional myosin, as well as on CaM, which is known to bind myr4 as a light chain.
Results and Discussion
The fate of fluid-phase tracers internalized from the medium in polarized MDCK cells has been studied in detail both in vivo, using confocal and electron microscopy, and in vitro, using a transport assay[4–6]. Apically and basolaterally endocytosed tracers were observed within separate populations of early endosomes, and mixing of these tracers was only detected in the common LE. Recent studies, however, have shown that transferrin (Tf), which is internalized from the basolateral surface of the monolayer, co-localizes, at least in part, with proteins endocytosed from the apical surface[10,47]. Indeed, apically and basolaterally derived tracers were shown to meet in apically located tubular endosomes which are highly reminiscent of the recycling endosomes described by Maxfield and collaborators[12,48] in other cell types.
In vitro fusion properties of MDCK II endosomes
First, we compared the interaction of Tf-labeled recycling endosomes with early endosomes labeled with fluid-phase HRP, using a modified version of the in vitro transport assay we have established previously[33,49]. We used MDCKII cells expressing the human TfR. Previous studies have established that, within minutes after internalization, Tf localizes to early recycling endosomes, consistent with the rapid cycle of its receptor [t1/2=5–10 min; for review see[7,8]]. To ensure efficient labeling of recycling endosomes (ETf) in our in vitro transport assay, biotinylated Tf-HRP conjugate (bTf-HRP) was bound to the basolateral plasma membrane on ice and then endocytosed for 10 min at 37°C. In parallel, the content of apical (Eap) or basolateral (Ebl) early sorting endosomes of a separate cell population was labeled with avidin internalized from the medium corresponding to the appropriate side of the monolayer for 10 min at 37°C, as previously established. Post-nuclear supernatants (PNS) were prepared from each cell population, mixed in the assay in the presence of ATP, and incubated for 40 min at 37°C. If membrane fusion occurred, mixing of the content of the two endosome populations resulted in the formation of an avidin–bTf-HRP complex. The complex was then extracted in detergent, imunoprecipitated with anti-avidin antibodies, and the HRP enzymatic activity of the complex was quantified.
In a first series of control experiments, we tested the fusion properties of apical and basolateral early endosomes, which had been labeled after fluid-phase endocytosis of avidin and biotinylated HRP (bHRP). As previously observed individual elements of each endosomal population exhibited homotypic fusion activity ( Fig. 1a, Eap:Eap and Ebl:Ebl), which is typical of early endosomes both in polarized and non-polarized cells[4,33,49]. Moreover, direct fusion of apical and basolateral early endosomes ( Fig. 1a, Eap:Ebl) did not occur to any significant extent in the assay, consistent with our previous observation. We then used this transport assay to investigate the properties of recycling endosomes (ETf) loaded with Tf internalized from the basolateral surface. As shown inFig. 1a, fusion could be observed between recycling endosomes and both, apical (Eap:ETf) and basolateral (Ebl:ETf) early endosomes. Since direct fusion of apical and basolateral early endosomes did not occur in the assay, yet each population exhibited the capacity to interact with recycling endosomes, one may conclude that the functional properties of recycling endosomes and early sorting endosomes are distinct. However, until now the fusion properties of early endosomes and recycling endosomes in vitro have not been discriminated and both appear to exhibit rabaptin5-dependent fusion activity in non-polarized cells[50,51]. Recycling endosomes were also clearly distinct from LE since they were not fusogenic with LE (LE:ETf,Fig. 1a), whereas late endosomal elements exhibited typical homotypic fusion activity [LE:LE; see].
The simplest interpretation of our observations is that endocytosed Tf was rapidly transferred from early sorting endosomes, which contained the bulk of endocytosed fluid-phase tracers, to specialized recycling elements, as was shown in other cells[12,48,52,53]. In fact, electron microscopy studies have shown that the tubular endosomal elements, characteristic of recycling endosomes, are only poorly accessible to endocytosed HRP in vivo, whereas they are very rapidly labeled with internalized Tf[48,55]. Indeed, basolaterally internalized Tf arrives within 5 min ( Fig. 1b) in a compartment that is fusion competent with the apical sorting endosome, reaching a plateau after 15 min. These data indicate that internalized Tf is rapidly transferred from basolateral sorting endosomes, which are not fusogenic with apical endosomes, to recycling endosomes, which are competent to undergo fusion with apical endosomes.
The relationship between early sorting endosomes and recycling endosome is still poorly understood, but it becomes increasingly clear that they represent separate compartments [for discussion, see]. The mechanisms of Tf trafficking from sorting to recycling endosomes remain to be elucidated. In vivo experiments in non-polarized cells, however, suggest that these endosomes may communicate by vesicular traffic, perhaps via tubular intermediates. In the text, we will use the term ‘transfer’ when referring to interactions between early sorting endosomes and recycling endosomes, to avoid confusion with homotypic fusion events.
Role of CaM in homotypic fusion and transfer
Only few proteins that regulate the recycling pathway have been identified. Although, CaM antagonists may have multiple targets in vivo, their use suggests that the recycling of receptors depends on CaM in polarized cells[27,32,56]. Similarly, transcytosis and transport to lysosomes were also proposed to be CaM-dependent. Therefore, we used confocal laser immunofluorescence microscopy to determine whether CaM localizes to one of the compartments studied in the fusion assay. For this purpose Alexa™ 488-Tf was internalized from the basolateral medium and HRP through the apical medium for 10 min. Because of the large cytoplasmic pool of CaM, it was necessary to cross-link the internalized HRP first by DAB reaction and extract cytoplasmic CaM prior to fixation (see Materials and Methods). Thereby, the membrane-associated fraction of CaM was analyzed together with the internalized endocytic tracers in a triple labeling experiment ( Fig. 2a–e). CaM partially co-localized with Alexa™ 488-Tf in the supranuclear region underneath the apical plasma membrane ( Fig. 2d) and, to a significantly lesser extent, with HRP that labels apical sorting endosomes and has only limited access to the recycling endosomes ( Fig. 2e). These data suggest that a fraction of CaM is membrane-associated and localizes to recycling endosomes containing endocytosed Tf.
These data prompted us to investigate in our cell-free fusion assay the functional involvement of CaM in the transfer from apical (Eap:ETf) or basolateral (Ebl:ETf) early endosomes to the common recycling endosomes. As shown inFig. 3, in vitro transfer from apical (Eap:ETf) or basolateral (Ebl:ETf) early endosomes to the common recycling endosomes was inhibited by the CaM antagonist W13. In contrast, no inhibition was observed when using the analogue W12, which is 5–10-fold less potent than W13 and has been used as a control for non CaM-mediated effects of the drugs in vivo. These data were further supported by the observation that transfer from either early endosome population to recycling endosomes was also inhibited by the addition of a well-characterized anti-CaM monoclonal antibody ( Fig. 3), known to block many CaM-dependent processes in vitro and in vivo[37,46].
Interestingly, both treatments (W13 and anti-CaM monoclonal antibody) had stimulatory, rather than inhibitory, effects on homotypic early sorting endosome fusion events (Eap:Eap and Ebl:Ebl,Fig. 3), pointing to the existence of a distinct CaM-dependent mechanism in early endosome dynamics. Moreover, both W13 and anti-CaM antibodies enabled apical and basolateral early endosomes to fuse with each other (heterotypic fusion,Fig. 3), a process which otherwise does not occur ( Fig. 1a). We demonstrated that these effects were specific to early endosomes, since the treatments did not cause Tf-containing endosomes to fuse with LE (data not shown). The fact that early endosome fusion and transfer reactions were affected in an opposite manner by both antibodies and antagonists further demonstrates that these two processes are different. Whereas further work is needed to understand these differences, the effects of antagonists and antibodies were clearly specific. Confirmation that CaM mediates the effects was obtained by addition of purified exogenous CaM (Ca2+ was present at physiological levels from rat cytosol), which completely reversed both inhibitory and stimulatory effects induced by drugs or antibodies ( Fig. 3, hatched bars), but had no effect on control, untreated reactions (not shown). We also observed that neither transfer reactions, nor the inhibitory effects of W13 and anti-CaM antibodies, were affected by the so-called slow Ca2+ chelator EGTA (data not shown, see also) suggesting a Ca2+-independent regulatory function of CaM. Similar observations by using EGTA have been obtained recently by another group; however, fusion was inhibited when the fast Ca2+ chelator BABTA was applied. BABTA is a membrane-permeant chelator that therefore also reaches internal Ca2+ pools. These observations led the authors to conclude that fusion of early endosomes, participating in recycling of synaptic vesicles, requires local release of Ca2+ from the endosomal interior. The effects of CaM inhibitors in our in vitro assay agree well with in vivo observations that CaM antagonists increase the size of endosomes and cause mixing of apical and basolateral endosomes to occur, but inhibit TfR recycling[27,56].
Myr4 on MDCK endosomes
Our next step was to identify possible target(s) of CaM in our assay. Since CaM is known to bind (as light chain) to unconventional myosins in a Ca2+-independent manner, we reasoned that a member of this superfamily may function as a target for CaM in our experiments.
Fractions containing the small GTPase rab5, a marker of early endosomes, and the TfR (not shown), were prepared by flotation in a sucrose gradient solubilized under mild conditions (0.2% NP-40), and then incubated in the presence or absence of Ca2+ with purified CaM immobilized on Affi-gel 15 agarose. The resin was washed and eluted either with high salt (and EGTA for Ca2+-independent binding) or with EGTA (for Ca2+-dependent binding). A major protein with an apparent mobility of 110 kDa was retained on the column in a Ca2+-independent manner, and was then identified as the unconventional myosin myr4 using specific antibodies ( Fig. 4a). Indeed, myr4 is known to bind CaM as a light chain, and to contain one Ca2+-independent CaM binding site, in addition to a Ca2+-dependent site. We then tested whether other unconventional myosins were also present in the same endosomal fraction obtained from the gradient, and could detect, in addition to myr4 also myr 1, myr2 and myr3, but not myr5 and myr6 ( Fig. 4b). Consistent with these biochemical observations, confocal laser scanning immunofluorescence microscopy showed that myr4 was present on recycling endosomes labeled with Tf-Alexa™ 488 internalized for 10 min, mostly in the supranuclear region underneath the apical plasma membrane ( Fig. 5). However, the co-localization of myr4 seems also to extend towards the basolateral sorting endosomes along the lateral plasma membrane (compare withFig. 2).
Myr4 regulates transfer from early endosomes to recycling endosomes in vitro
Using a panel of anti-myr antibodies ( Table 1), we then tested whether the myr proteins which were enriched in our gradient fraction were involved in the transport reactions we have studied. As shown inFig. 6, the transfer from apical or basolateral early endosomes to recycling endosomes (Eap:ETf and Ebl:ETf) was strongly inhibited by the anti-myr4 antibodies (Tü12 and Tü13, seeTable 1). The peptide these antibodies were raised against corresponds to a specific extension in the myosin I tail homology motif, which is proposed to mediate membrane association (seeTable 1). In contrast, no inhibitory effects were observed in the assay when using antibodies against other regions of myr4, or against other members of the myosin I family (not shown; seeTable 1), including an antibody blocking the actin-activated ATPase activity of myr3, which was also present in our fractions. None of the antibodies, including anti-myr4 antibodies, had any effect on the homotypic fusion processes (Eap:Eap and Ebl:Ebl), demonstrating that the effects of anti-myr4 antibodies on the transfer reactions from sorting to recycling endosomes were specific. This also suggests that the effects of CaM inhibitors on homotypic fusion processes (seeFig. 3) are myr4-independent. These observations indicate that the actin-based mechanoenzyme myr4, which can bind two CaM molecules as light chains, is involved in trafficking from apical and basolateral early endosomes to recycling endosomes in MDCK cells.
Actin dependence of transfer from early to recycling endosomes in vitro
Since myr4 is known to interact with F-actin, our observations raise the possibility that the myr4-dependent transfer from apical or basolateral early endosomes to recycling endosomes also depends on actin filaments. A fluorescence analysis after FITC-phalloidin staining revealed that actin filaments, in contrast to polymerized microtubules, were indeed present under our in vitro conditions (not shown). Formation of filaments in the assay could be prevented by monomer sequestration in a 1:1 complex with DNase1 [not shown; see]. Alternatively, existing filaments could be severed with recombinant gelsolin or with the S1-3 active domain of gelsolin [not shown; see[42,61]]. S1-3 binds two actin molecules in the presence or absence of Ca2+ and it severs and caps filaments like full-length gelsolin although it does not nucleate polymerization. Under both conditions, transfer from either early endosomal population to common recycling endosomes was significantly inhibited ( Fig. 7, Eap:ETf and Ebl:ETf). Therefore, our data suggest that, after disruption of the cellular organization in our assay, dynamic interactions between endosomal elements can be facilitated by the presence of polymerized actin filaments. Similarly, taxol-stabilized microtubules were shown to facilitate in vitro interactions between endosomes at late stages of the pathway[4,45]. Our in vitro findings agree well with the in vivo observations that actin filaments facilitate Tf recycling in a hepatoma cell line and with actin sequestering experiments performed in perforated A431 cells. This role of actin filaments also agrees with their in vivo localization to a sub-cortical region in MDCK cells, which contains early endosomes and recycling endosomes[10,15,27]. We stained MDCKII cells grown on tissue culture filters were for CaM and myr4 using specific antibodies and for F-actin using phalloidin-Alexa™ 568. All three markers co-localized in the supranuclear region underneath the apical plasma membrane (data not shown). Altogether, these observations strongly suggest that actin filaments are involved in the endocytic/recycling pathway.
In conclusion, our data show that, in MDCK cells, recycling endosomes can communicate with both apical and basolateral early endosomes, and that this process differs at the molecular level from the homotypic fusion process within the early endosomal compartments. Transfer reactions depend on actin, CaM and myr4, an actin-based mechanoenzyme, in contrast to the transport between early and LE, which depends on microtubules and other mechanochemical motors[4,45].
It is unclear at present whether myr4 in our system acts as a motor protein and moves endosomal vesicles on actin cables, or whether the protein tethers or anchors endosomal membrane domains on actin filaments and thereby contributes to the dynamics or the functional organization of the early endosomal membrane system. These possible functions of unconventional myosins in membrane dynamics are still debated, and have been discussed in detail by Coudrier et al. and Mooseker and Cheney. Recently, evidence was provided that the vesicle delivery function of Rho3 in budding yeast is mediated by the unconventional myosin Myo2. In addition, molecular genetic analysis of the Dictyostelium unconventional myosin VII reveals that this motor protein plays a specific and significant role in phagocytosis. While this paper was under review, Durrbach et al. reported the involvement of an acto-myosin-driven mechanism in trafficking of basolaterally internalized molecules to the apical plasma membrane. Brush border myosins might also be involved in membrane trafficking occurring between endosomes and lysosomes in unpolarized cells. These observations, together with our results, indicate that unconventional myosins underlie the elusive crosstalk between endosomes and the actin cytoskeleton that is required for membrane trafficking along the recycling pathway in polarized epithelial cells.
Materials and Methods
Cells and reagents
An MDCKII cell line expressing the human TfR (cDNA kindly provided by R. Davies, U. Mass. Med. School Worcester, MA) was generated as outlined. We verified that> 90% of the surface-expressed TfR was basolateral, and that internalization and recycling of human transferrin (Tf) occurred with kinetics similar to those of canine Tf via the endogenous receptor (W. Hunziker, unpublished data). Cells were grown and passaged as described. Synthetic CaM antagonists[27,32] (N-[4-Aminobutyl]-2-naphtalenesulfonamide W12 or W13) were from Sigma-Aldrich, Vienna, Austria (A-3168, A-0666). The anti-CaM monoclonal antibody was described previously. Polyclonal antibodies against myr proteins (see alsoTable 1) were raised against synthetic peptides derived from the tail or head domains of myr proteins and purified as described previously[34,38–41]. Polyclonal antibodies specific for HRP were generated in the laboratory. Lyophilized DNase1 from bovine pancreas (Roche Diagnostics, Vienna, Austria, 1284932) was reconstituted in ddH20, aliquoted and stored at −20°C. Human plasma gelsolin and gelsolin domain S1-3 were expressed in Escherichia coli, purified and dialyzed overnight (10 mM Tris, pH 8.0, 0.2 mM EDTA, 1 mM DTT, 100 mM NaCl). Purified bovine brain CaM (40 000 units/mg,> 98% purity as judged by SDS-PAGE) was obtained from Calbiochem/Novabiochem, Darmstadt, Germany (208694-B). Alexa™ 488-labeled secondary antibodies and Phalloidin-Alexa™ 568 were obtained from Molecular Probes. Cy3™- and Cy5™-labeled secondary antibodies were from Jackson ImmunoResearch Laboratories.
Internalization and immunofluorescence
MDCK II cells expressing human Tf and pIg receptors were grown on permeable filter supports (Transwell-clear, Cat.-No. 3450, Costar) as described[36,43]. Transepithelial electrical resistance (TER) was determined to confirm the tightness of the epithelial monolayer and was usually around 150 Ωcm2. The cells were washed with HBS (Gibco) followed by a 3-h incubation in serum-free medium containing desferoxamine mesylate as Fe2+ chelator (50 μM, Sigma) at 37°C to enrich ligand-free TfR on the cell surface. After two washes with ice-cold PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2 (PBS++), pre-warmed medium containing 50 μg/ml Tf-Alexa™ 488 (Molecular Probes) was added basolaterally and medium containing 10 mg/ml HRP (Sigma) apically. Internalization was continued for different time periods at 37°C and stopped by placing the filters in ice-cold PBS++ containing 5 mg/ml BSA. Free Tf and HRP was removed by three washes in ice-cold PBS++. Subsequently, cells were fixed with 4% PFA in CB (cytoskeleton buffer: 10 mM PIPES pH 6.8, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl2). Alternatively, internalized material was cross-linked by incubating the cells with 1 mg/ml 3,3′-diaminobenzidine (DAB), 0.03% H2O2 in PBS++ 20 min on ice and extracted with 0.05% saponin in CB for 6 min on ice before fixation. The filter pieces were submerged in blocking buffer (CB containing 0.3% TX-100, 1% gelatin, 5% FCS, 5% goat serum and 50 mM NH4Cl) processed for indirect immunofluorescence and finally mounted in 50% glycerol, 4% n-propyl gallate (Sigma) in CB. Confocal microscopy images were obtained with a Leica TCS NT confocal microscope (Leica, Heidelberg, Germany) and processed using the Imaris and Co-localization software packages (Bitplane AG, Zurich, Switzerland) after deconvolution using measured point-spread functions with the Huygens-software (Scientific Volume Imaging, Hilversum, Netherlands).
Preparation and labeling of endosomes
For isolation of endosomes, cells were seeded at high density in 75 mm diameter, 0.4 μm pore size Transwell filters as described previously[36,43]. Apical (Eap) and basolateral early endosomes (Ebl) were labeled by internalization of 3 mg/ml avidin or 1.9 mg/ml biotinylated horseradish peroxidase (bHRP) for 10 min at 37°C from the apical or basolateral side of separate monolayers[4,33]. To label LE, avidin was internalized for 20 min at 37°C, followed by a 30-min chase in marker-free medium[4,33]. To label recycling endosomes (ETf), filters were incubated from the basolateral side for 30 min on ice with biotinylated peroxidase-conjugated human Tf (bTf-HRP), and rinsed three times in ice-cold PBS++ containing 5 mg/ml BSA. Then, pre-bound bTf-HRP was internalized for 10 min at 37°C. Alternatively; bTF-HRP was internalized continuously from the basolateral side for varying amounts of time. PNSs were prepared as described.
Reconstitution of fusion in vitro was essentially as described[4,33]. Equal amounts of each PNS (50 μl of a 20 mg/ml PNS) with internalized avidin, bHRP or bTf-HRP were used. Latency of internalized bHRP was 77±5% (mean±SD, n=5). Rat liver cytosol (24 mg/ml) was prepared as described[44,45]. Briefly, avidin, bHRP or bTf-HRP containing endosome fractions were combined at 4°C in the presence of 5 mg/ml rat liver cytosol (containing endogenous amounts of calcium), ATP and salts. Synthetic CaM antagonists[27,32] (N-[4-Aminobutyl]-2-naphtalenesulfonamide W12 or W13), an inhibitory anti-CaM monoclonal antibody[37,46], or CaM purified from bovine brain were added to the fractions prior to the fusion reaction and incubated for 30 min on ice. Final concentrations were 60 μM for W12 and W13, 0.5 nM for the antibody or 30 nM for exogenous CaM. Affinity purified antibodies against each myr protein ( Table 1) were added in equal amounts to the fractions prior to the assay, and the mixture was preincubated for 30 min on ice (antibody at 5.8 μg/ml in the assay). DNase1 (1, 5, 10 μg), gelsolin (15 μM), the active S1-3 domain of gelsolin (15 μM) and dialysis buffer were also added prior to assay and incubated for 30 min on ice. Under these conditions, no polymerized actin could be observed after incubating the fusion competent fractions with FITC-phalloidin (data not shown). S1-3 binds two actin molecules in the presence or absence of Ca2+ and it severs and caps filaments like full-length gelsolin but does not nucleate polymerization. The fusion and transfer reaction was carried out 40 min at 37°C in the presence or absence of ATP.
CaM-agarose affinity chromatography
To assay the in vitro interaction of endosomal myr4 with CaM, 0.5 ml of settled CaM-agarose (1 mg/ml CaM coupled to Affi-gel 15 agarose [Calbiochem/Novabiochem] linked via a secondary amine to a 15 atom hydrophilic spacer arm) was equilibrated in buffer A (TBS, pH 7.3, 0.2% NP-40, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml antipain). Endosomal fractions (220 μg) from four Transwell filters (100 mm diameter, 0.4μm pore size) were solubilized in Buffer A and sonicated for 1 min on ice. The sample was then split into two equal aliquots and either supplemented with 2 mM EGTA (buffer B, Ca2+-independent binding) or with 100 μM CaCl2 (buffer C, Ca2+-dependent binding). Samples were then separately incubated with the resin in the corresponding buffer for 2 h at 4°C with moderate agitation on a rotating platform. The resin was poured into a 2 ml syringe. After collecting the flow-through, the column was eluted as follows: i) for Ca2+-independent binding, 500 μl fractions were eluted in buffer A containing 0.5 M or 1 M NaCl, then the column was washed with buffer D (TBS, pH 7.3, 0.2% NP-40, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml antipain, 2 M NaCl) and re-equilibrated in buffer A; ii) Ca2+-dependent binding, 500 μl fractions were eluted in buffer A containing 1 or 2 mM EGTA, and the remaining protein was eluted in 500 μl of buffer A containing 1 M NaCl. All samples were concentrated by acetone precipitation and analyzed by SDS-PAGE and western blotting.
Western blotting of endosomal fractions
Endosomal fractions were prepared by flotation in a sucrose gradient as described. Myr1-6 proteins were mapped in the fractions (E), total homogenate (H) and PNS (P). Samples containing equal amounts of protein (20 μg) were separated on a 10% SDS-PAGE, transferred onto nitrocellulose and probed with specific polyclonal antisera against each myr protein. Briefly, filters were prewashed in PBS, 4% milk, 0.2% Tween 20 and 0.1% sodium azide (blocking buffer) for 2 h at room temperature and incubated for 1 h with primary antibodies diluted in blocking buffer. After extensive washing, filters were incubated with a HRP labeled secondary antibody (Biorad, Richmond, USA) for 1 h, washed and developed using an enhanced chemoluminescence method (ECL, Amersham Pharmacia, Freiburg, Germany).
The authors thank Dr Matt Cotten for carefully reading the manuscript and for helpful discussions. We wish to acknowledge Marie-Hélène Beuchat for expert technical assistance. This work was supported by the I.M.P. and by grants from the Austrian Science Foundation (FWF, P13577-GEN, the Johnson & Johnson Focused Giving Program to L.A.H. and J.G. as well as by the Swiss National Science Foundation (31-37296.93, to J.G.) and the National Institutes of Health (CA75205 to D.B.S.).