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Originally published In Press as doi:10.1074/jbc.M701501200 on May 11, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19565-19574, July 6, 2007
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Activation of p61Hck Triggers WASp- and Arp2/3-dependent Actin-comet Tail Biogenesis and Accelerates Lysosomes*

Claire Vincent{ddagger}, Isabelle Maridonneau-Parini{ddagger}1, Christophe Le Clainche§, Pierre Gounon, and Arnaud Labrousse{ddagger}

From the {ddagger}Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, 31077 Toulouse Cedex 04, France, §Dynamique du Cytosquelette et Motilité, Laboratoire d'Enzymologie et de Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France, and Centre Commun de Microscopie, Faculté des Sciences, 06108 Nice Cedex 2, France

Received for publication, February 20, 2007 , and in revised form, May 4, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretory lysosomes exist in few cell types, but various mechanisms are involved to ensure their mobilization within the cytoplasm. In phagocytes, lysosome exocytosis is a regulated phenomenon at least in part under the control of the phagocyte-specific and lysosome-associated Src-kinase p61Hck (hematopoietic cell kinase). We show here that p61Hck activation triggered polymerization of actin at the membrane of lysosomes, which resulted in F-actin structures similar to comet tails observed on endocytic vesicles. We correlated this actin-comet biogenesis to a 35% acceleration of p61Hck-lysosomes in cells, which was dependent on actin polymerization and required an intact microtubular network. It was possible to initiate the formation of actin tails on p61Hck-positive lysosomes and on p61Hck-associated latex beads incubated in human phagocyte cytosolic extracts. The in vitro reconstitution on beads indicated that other lysosomal proteins were dispensable in this mechanism. The de novo actin polymerization process was functionally dependent on the kinase activity of Hck, WASp, the Arp2/3 complex, and Cdc42 but not Rac or Rho. Thus, we identified p61Hck as the first lysosomal protein able to recruit the molecular machinery responsible for actin tail formation. Altogether, our results suggest a new mechanism for lysosome motility involving p61Hck, actin-comet tail biogenesis, and the microtubule network.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It becomes increasingly clear that lysosomes in eukaryotic cells can function as secretory organelles and might no longer be considered just as the end point of the endocytic pathway to which proteins and extracellular particles are delivered for degradation. In fact, in several cell types subpopulations of secretory lysosomes are mobilized to achieve various cell functions such as plasma membrane repair during migration (1), destruction of the target cell when lytic granules fuse at the immunological synapse in cytotoxic T lymphocytes (2), protection against ultraviolet irradiation damages when lysosome-related melanosomes are transferred to keratinocytes (3), and lysis of pathogens when lysosomes and lysosome-related granules fuse to phagosomes in phagocytes (4, 5). From one cell type to another the transport of lysosomes to their target membrane relies on different mechanisms involving the actin cytoskeleton and/or the microtubule network either simultaneously or sequentially. In these contexts, molecular motors are involved, and microtubules usually serve as tracks for long distance movements of lysosomes toward the cell periphery (6, 7), whereas filamentous actin (F-actin)2 is used either as tracks for short distance movements (6), anchors for docking of the vesicles before fusion with the plasma membrane (8), or breaks in the directional transport of vesicles along microtubules (9). An actin-dependent motility process, which is not dependent on molecular motors, has been described for late endocytic compartments. This process relies on de novo polymerization of actin at the membrane of the organelle, resulting in the formation of an actin-comet tail and the rocketing of the vesicle (10–16). Such an actin-based motility had been initially described for pathogens (e.g. Listeria monocytogenes, Shigella flexneri) (17) (18) to propel them within the host cytoplasm using the Arp2/3 complex to initiate actin assembly. Although most bacterial proteins that recruit the host actin polymerization machinery have been identified, the molecules responsible for the recruitment of the actin-assembly machinery at the membranes of endocytic vesicles remain largely unknown. We propose here that Hck is a molecular link able to trigger the actin polymerization process at the membrane of lysosomes.

Hck is a protein-tyrosine kinase of the Src (Rous sarcoma virus protein) family that is specifically expressed in phagocytes. In neutrophils, activation of Hck at the lysosomal membrane has been correlated to lysosome (azurophil granule) fusion with phagosomes, which takes place few seconds after the bacteria/cell contact (19). In macrophages, phagolysosome biogenesis takes dozens of minutes to occur. In these cells, Hck activation precedes the translocation of p61Hck-lysosomes toward the phagosomal membrane (20, 21). Hck is expressed as two isoforms, p59Hck and p61Hck, located at the plasma membrane and the membrane of lysosomes, respectively (22). We have previously shown that the activation of both isoforms of Hck is associated with actin remodeling. p59Hck activation triggers the actin-dependent biogenesis of membrane protrusions similar to those involved in the engulfment of particles or cell migration (23). In cells that are not involved in the phagocytic process, p61Hck activation triggers the biogenesis of actin-rich podosomes in a lysosome-dependent manner via a mechanism that requires intact microfilaments and microtubules (24). Similarly, Src has been shown to be associated with endocytic vesicles (25). Its activation occurs during the actin-dependent translocation of the vesicles toward the plasma membrane (16) and triggers their alignment along F-actin cables (16, 26). While constitutively active v-Src is able to reorganize the actin cytoskeleton (27) in a N-WASp (Neural-Wiskott-Aldrich-Syndrome protein)- and Hsp90 (heat-shock protein)-dependent manner (28), Src has not been demonstrated to directly control the movement of vesicles. Hck phosphorylates WASp, and a phospho-mimicking mutation of the phosphorylated tyrosine residue enhances the actin nucleation activity of the molecule (29). We show here that p61Hck is able to promote actin-comet tail biogenesis in a WASp-, Arp2/3-, and Cdc42-dependent process that we correlated to an increased motility of p61Hck-positive lysosomes in cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Lanolin, Vaseline, paraffin, leupeptin, aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin, imidazole, 1,4-diazabicyclo[2.2.2]octane, methylcellulose, nocodazole, and cytochalasin D were purchased from Sigma (Saint Quentin Fallavier, France). Isopropyl β-D-1-thiogalactopyranoside was purchased from Q-Biogene (MP Biomedicals, Illkirch, France). Dithiothreitol was purchased from Bio-Rad (Marnes la Coquette, France). 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) was purchased from TEBU-Bio (Le Perray en Yvelines, France). Rabbit muscle actin (unlabeled, rhodamine- or pyrene-labeled) was purchased from Cytoskeleton Inc. (TEBU-Bio) or kindly given by Dr. L. Blanchoin (Commissariat à l'Energie Atomique, Grenoble, France). Recombinant human N17Cdc42 and N17Rac1 were purchased from Cytoskeleton Inc. (TEBU-Bio). Active and inactive C3 exoenzymes were kindly given by Dr. E. Lemichez (INSERM, Nice). Nicotinamide adenine adenylate-32P dinucleotide was purchased from Amersham Biosciences.

Antibodies—Rabbit polyclonal anti-human WASp (sc-8353) antibodies were purchased from Santa Cruz (TEBU-Bio, France) and were used at dilution 1:500 for Western blot analysis. Mouse monoclonal anti-His-tag antibodies (70796-3) were purchased from Novagen (VWR International, Strasbourg, France). Mouse monoclonal anti-human Cdc42 antibodies (clone 44), rabbit polyclonal anti-human RhoA antibodies, and mouse monoclonal anti-human Rac1 antibodies (clone 23A8) were purchased from BD Biosciences, Santa-Cruz (TEBU-Bio), and Upstate (Millipore, Billerica, MA), respectively. Anti-Cdc42, -RhoA, and -Rac1 antibodies were, respectively, used at dilution 1:250, 1:200, and 1:500 for Western blot analysis.

Plasmids—The coding sequence of wild-type, Y512F constitutively active (ca) or K290E kinase-dead (kd) mutants p61Hck were amplified by PCR using Pfu (Stratagene, Amsterdam, The Netherlands). Oligonucleotide 1 (5'-cgagatccatatgggggggcgctcaagc-3') as a forward primer and oligonucleotide 2 (5'-gcgcctcgagctacggctgctgttggtactgg-3') or 3 (5'-gcgcctcgagctacggctgctgttggaactgg-3') as reverse primers were used for p61Hckwt or p61Hckca, respectively, and the plasmid encoding p61Hck-GFP as template (described in Carreno et al. (22)). The oligonucleotides 1 and 3 were used to amplify the cDNA encoding p61Hckkd using p61Hckkd-GFP as a template (24). The 1.6-kilobase PCR product was cloned into the pPCR-Script vector (Stratagene) before being subcloned into the pET28a vector using the NdeI and XhoI restriction enzymes (Ozyme, St. Quentin-en-Yvelines, France). These pET28a-p61Hck plasmids were checked by sequencing. For expression in NIH3T3 fibroblasts of full-length p61Hck wild type, kinase dead, or constitutively active and of p61Hck-Unique domain alone in fusion with GFP, corresponding plasmids were described before (22).

Recombinant Proteins Purification—The pET28a-p61Hck proteins were produced in Escherichia coli BL21(DE3)pLysS. Briefly, induction of protein expression was carried out at A600 nm = 1.0 in the presence of 0.5 mM isopropyl-β-D-1-thio-galactopyranoside during4hat16 °C. Cells were then collected by centrifugation, washed in 50 mM NaCl, 20 mM Tris-HCl, pH 8, and resuspended in sonication buffer (500 mM NaCl, 10 mM imidazole, 150 mM Tris-HCl, pH 9.5, and protease inhibitor mixture (pepstatin, leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin). Recombinant His6-p61Hck proteins were purified by pseudo-affinity using a 1-ml HiTrap Nickel column (Amersham Biosciences) according to the manufacturer's instructions. Imidazole elution followed by dialysis was performed, and aliquots of p61Hck containing 10% glycerol were stored at -80 °C. p61Hck was identified in the fractions by SDS-PAGE followed by Western blot and mass spectrometry. The pGex-VCA (Verprolin-Cofilin-acidic domain of N-WASp) plasmid was kindly provided by Philippe Chavrier (Curie Institute, Paris). The recombinant GST-VCA protein was produced in E. coli BL21(DE3)pLysS. After a 3-h induction at 37 °C with 1 mM isopropyl-β-D-1-thiogalactopyranoside, the recombinant protein was purified with glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's recommendations. The recombinant human His6-tagged N-WASp was expressed in Sf9 cells using the baculovirus system and purified as described (30).

Cell Culture and Transfection—NIH3T3 fibroblasts were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 1% L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The day before transfection cells were seeded in Lab-TekII chambered cover glass (VWR International) at 3.5 x 104 cells/ml for video-microscopy experiments or in 9-cm Petri dishes at 5 x 104 cells/ml for p61Hck-lysosome-containing post-nuclear supernatant preparation and transfected as previously described (23). U937 cells were cultured in RPMI medium (Invitrogen) supplemented as above at 37 °C in a 5% CO2 atmosphere as previously described (31). The Tet-Off MEF3T3 stable cell line in which p61Hckca-GFP can be expressed was cultured as described (24). Induction of p61Hckca-GFP expression was obtained by doxycycline depletion for 2 weeks as previously described (32). Induced cells were seeded and grown on sterile glass coverslips at a final density of 4 x 104 cells/well. Cells were then fixed in 3.7% paraformaldehyde, and F-actin was labeled with Texas Red phalloidin (Molecular Probes) as described (23).

Video-microscopy and Image Analysis—36 h after transfection cells were observed with an inverted Leica microscope equipped with a heated stage adjusted to 37 °C and a HCX PLAPO 63x/1.4-0.6 objective. Fluorescent pictures were taken through a I2 filter-set (Leica Microsystems, Rueil Malmaison, France) using a Cool-Snap HQ CCD camera (Photometrics, Roper Scientific, Evry, France) driven by Metamorph (Roper Scientifics). Pictures were taken every 5 s as stacks. Observations were performed within 60 min. p61Hck-positive vesicles were followed through the stacks, and distances were measured for 10–20 lysosomes per cell. Five to ten cells per transfection conditions were analyzed, and at least three independent experiments were carried out for each condition. For cytochalasin D, latrunculin A, and nocodazole treatments, drugs were added to cells at 0.5, 0.5, and 5 µM, respectively, 25 min before lysosome movements were being monitored. Velocity distributions were fitted to Gaussian curves with the equation y = amplitude x e-0.5((X-mean)/S.D.)2, where y is the number of lysosomes within a velocity range (X), and S.D. stands for Standard Deviation.

Subcellular Fractionation and Cytosol Treatments—The lysosome-containing fraction was prepared after having confirmed that p61Hck-positive vesicles could be loaded with rhodamine-dextran, a characteristic of compartments of the endocytic pathway (22). For p61Hck-lysosome-containing post-nuclear supernatant preparation, NIH3T3 fibroblasts were washed, resuspended in 2 volumes of lysis buffer (0.25 M sucrose, 20 mM Hepes, pH 7.7) with protease inhibitors (1 µg/ml aprotinin, 1 µg/ml pepstatin, 3 µg/ml leupeptin, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride), and lysed by passage through a 27-gauge needle (15–20 times; cell breakage was monitored by phase microscopy). Centrifugation for 10 min at 300 x g provided the p61Hck-lysosome-containing post-nuclear supernatant. For cytosolic extracts preparation, U937 cells were collected and washed twice in phosphate-buffered saline (Invitrogen). Two milliliters of cell suspension (1 x 108 cells/ml) in Relax buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 20 mM Trizma (Tris base), 6 mM EGTA, pH 7.3) supplemented with protease inhibitors (as above) were broken by cavitation as described for neutrophils (19). Lysates were spun at 250 x g and a 15,000-g post-nuclear supernatant was further spun at 100,000 x g for 45 min at 4 °C. The cytosolic fraction was then separated into aliquots and stored at -80 °C. Protein concentration was measured by the Bradford method and varied from 4 to 12 mg/ml. Xenopus oocyte cytosolic extracts were prepared from non-fertilized oocytes as described (33) with the following modifications; cytochalasin D and silicone oil were omitted throughout the protocol, and oocytes were activated by three 2-s 60-V DC electrical pulses. Protein concentrations varied from 20 to 25 mg/ml. To remove the Arp2/3 complex from the U937 cytosolic extracts, 26 µl of glutathione-Sepharose 4B beads (Amersham Biosciences) loaded with 0.3 nmol of recombinant glutathione S-transferase-VCA were incubated with 100 µl of the corresponding cytosols for 1 h at 4°C. Beads were removed by a 1300 x g centrifugation, and the supernatant was directly used in the actin-comet tail assay described below. For GDP loading of the cytosolic extracts, cytosols were supplemented with 100 µM GDP and 5 mM EDTA before being incubated for 10 min at 30 °C and then supplemented with 10 mM MgCl2 before being used in the actin polymerization assay described below. For experiments carried out with a dominant negative mutant of the Rho-GTPases Cdc42 or Rac1, cytosolic extracts were supplemented with an optimized concentration of 10 µM N17Cdc42 or N17Rac1, respectively, and incubated for 30 min at 4 °C before being used in the actin-comet tail assay described below. For experiments carried out with anti-human WASp blocking antibodies, cytosolic extracts were supplemented with rabbit polyclonal anti-human WASp (sc-8353) antibodies at 1:25 dilution before being used in the actin-comet tail assay described below. For experiments carried out in the presence of anti-human Cdc42 antibodies, cytosolic extracts were supplemented with an optimized concentration of 1 µg of antibodies per 150 µg of total cytosolic proteins and incubated for 30 min at 4 °C before being used in the actin-comet tail assay described below. To functionally inhibit Rho, cytosolic extracts were treated with C3 exoenzyme (1 µg of enzyme per 1 mg of total proteins) for 10 min at 37 °C before being used in the actin-comet tail assay described below. To assess the level of Rho inhibition in the cell extracts treated with active or inactive C3 exoenzyme, the extracts were further incubated with an excess of active C3 exoenzyme (100 µg of enzyme per 1 mg of total proteins) for in vitro ADP-ribosylation, which was performed for 30 min at 37 °C in the presence of 50 µCi of adenine adenylate-32P dinucleotide per 1 mg of total proteins (34). Under these conditions, 83% of endogenous Rho were inhibited.

In Vitro Polymerization of Actin on Agarose Beads—Agarose-Ni2+ beads (His-Select nickel affinity gel, Sigma) were washed 3 times in 20 mM NaCl, 50 mM Tris-HCl, pH 8, before being incubated overnight at 4 °C in the presence of recombinant His6-tagged p61Hck. Beads were then washed twice in Relax buffer, then incubated in 20 mM NaCl, 10 mg/ml BSA, 50 mM Tris-HCl, pH 8, for 15 min at 4 °C before being washed in Relax buffer. Beads were then incubated for 1 h at 25°C in a 100-µl aliquot of cytosol. An 8-µl aliquot of the reaction was transferred into a tube containing 2 µl of 10 µM rhodamine-phalloidin (Sigma) before being mounted for fluorescence microscopy observations. Snapshots were taken with a constant exposure time of 200 ms. To allow direct visual comparisons, experiments and the corresponding controls were displayed with the same dynamics of gray levels. For further Western blot analysis, beads were washed in Relax buffer as above, and proteins recruited on beads were then separated by SDS-PAGE before immunoblotting with the corresponding antibodies.

In Vitro Polymerization of Pyrene-Actin—Optimized concentrations of recombinant human GST-VCA (270 nM) and His6-tagged p61Hck (15 nM) were added to the following reaction mix: 200 µl of U937 cytosol (between 6 and 12 mg/ml of proteins), 20 µl of KMEI 10x buffer (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH 8.0), 1 mM ATP/Mg, 1 mM MnCl2, 0.5 µM F-actin, 0.75 µM Mg2+-actin, and 0.75 µM Mg2+-pyrene-actin. F-actin was prepared as follows. monomeric actin was incubated at 55 µM with 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, pH 7.3, for 1 h at room temperature before being stored at 4 °C. F-actin stocks were used within 5 days of storage. Pyrene fluorescence (excitation and emission wavelengths were 366 and 407 nm, respectively) was measured with a SAFAS spectrofluorimeter.

Actin-comet Tail Assay—For electron microscopy of actin tail, samples were fixed following the recommendations and protocols previously described (35). Briefly, samples were fixed with 1% glutaraldehyde, 0.2% tannic acid in 50 mM phosphate buffer, pH 6.8, at room temperature. Preparations were then washed in 0.1 M phosphate buffer, pH 6.8, and post-fixed with 1% OsO4 in 0.1 M phosphate buffer, pH 6.2, for 30 min at 4 °C. Preparations were carefully washed with water then stained en bloc overnight with 0.5% uranyl acetate and washed again with water before dehydration with ethanol and final embedding in epoxy resin. Thin sections were made, stained, and observed under standard conditions.

Recombinant human His6-N-WASp or His6-p61Hck were bound to 1-µm-diameter protein-G-carboxylated beads (Polysciences, TEBU-Bio, France) at a final concentration of 0.4 µM for 2 h at 4°C via anti-His6-tag antibodies (Novagen) and then washed 3 times in 1% Xb-BSA or in 1% BSA-phosphate-buffered saline (PBS), respectively, before being resuspended in 0.1% Xb-BSA or 0.1% BSA-PBS, respectively. Xb buffer contained 100 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, and 10 mM Hepes, pH 7.8. 2 µl of functionalized beads or of p61Hck-lysosome-containing post-nuclear supernatant were then incubated in a biomimetic medium: 6 µl of Xenopus oocyte or U937 cell cytosol, 1.6 µM rhodamine-actin, 10 µM F-actin (see above), 0.06 or 2.1 mM 1,4-diazabicyclo[2.2.2]octane for experiments carried out in U937 or Xenopus oocyte, respectively, 0.2 or 14 mM dithiothreitol for experiments carried out in U937 or Xenopus oocyte, respectively, 0.9 mM ATP, 1.7 mM MgCl2, 0.2 mM MnCl2 and 0.2% methylcellulose. Three microliters of the reaction were squashed between a slide and a 22-mm2 coverslip that was sealed with VALAP (a 1:1:1 mix of Vaseline, lanolin, and paraffin). Rhodamine fluorescence was observed directly using a N2.1 filter set on a DM-RE Leica microscope. Pictures were taken with a CoolSnap HQ CCD camera (Photometrics, Roper Scientifics, Evry, France) driven by MetaView (Roper Scientifics) and contrasts and luminosity were optimized for viewing using the Adobe Photoshop Elements software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
p61Hck Triggers Actin Polymerization on Lysosomes—Our first attempt has been to optimize the detection of a potential actin-dependent process taking place at the lysosomal membrane using a constitutively active mutant of p61Hck (p61Hckca). This mutant has been obtained by point mutating the regulatory C-terminal tyrosine into a phenylalanine, which resulted in a non-regulated kinase activity and a constitutively opened conformation, with both adaptor domains available for interactions (23). p61Hckca, previously shown to target the membrane of lysosome (23), has been stably expressed in an inducible Tet-Off fibroblast cell line. We selected cell clones expressing amounts of Hck similar to the endogenous level in phagocytes (24). We observed that p61Hckca-GFP lysosomes were associated with actin clouds that were often polarized (Fig. 1A). These clouds were very similar to actin-comet tails already described in association to endocytic vesicles in cells (16, 36, 37). By confocal microscopy we observed these lysosome-associated actin tails mainly at the basal part of the cells (Fig. 1A, a and b) and also occasionally at the upper levels of the cytoplasm (Fig. 1A, c and d). In this case p61Hckca-GFP vesicles and associated actin-comet tails could be better visualized. Actin-comet tails were mainly detected in cells with clustered podosomes at the cell periphery (Fig. 1A, a and b), whereas they were very occasionally observed in cells with well established podosomal rosettes (Fig. 1Ae). We previously described these rosettes as being a consequence of p61Hck activation and lysosome targeting to the plasma membrane (24). None of these actin structures was ever observed in cells when Hck expression was not induced in p61Hckca-GFP Tet-Off stable cell line (not shown).


Figure 1
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  		FIGURE 1.
p61Hck promotes the formation of actin-comet tails on lysosomes. A, co-localization of F-actin and p61Hck-GFP on vesicles of transfected murine fibroblasts. MEF3T3 fibroblasts stably expressing p61Hckca-GFP (green) were fixed and stained for F-actin with Texas red phalloidin (red). Actin-comet tails were observed either at the ventral parts of the cells (panels a and b) or in upper parts of the cells (panels c, d, and d'). Panel d, focal plan indicated by the dotted line in panel d'. Panel d', z-section corresponding to the dotted line indicated on panel d. Panel e, ventral part of a cell with well established large podosomal rosettes. Arrowheads show podosomes induced by p61Hckca organized as clusters. Arrows show established rosettes of podosomes. Bars, 10 µm. B, polymerization of actin around lysosomes. Post-nuclear supernatants containing lysosomes of NIH3T3 fibroblasts transiently expressing p61Hckca-GFP (panel a), p61Hckwt-GFP (panel b), p61Hckkd-GFP (panel c), and p61Hckunique-GFP (panel d)(green) were incubated in a medium containing rhodamine-actin (red) and phagocytic cell extracts before being observed by fluorescence microscopy. panel e, a similar experiment as in panel b but in the presence of 1 µM PP1. Bars = 3 µm. Cells and lysosomal vesicles representative of at least three independent experiments are shown.

 
Hck being specifically expressed in phagocytes, post-nuclear supernatants containing vesicular compartments from p61Hck-expressing fibroblasts were incubated in cytosol from a human phagocytic cell line (U937) supplemented as described under "Experimental Procedures" to optimize the actin polymerization process. We observed that p61Hckca-positive lysosomes were surrounded by polarized actin-rich structures, resulting from a de novo polymerization activity at the membrane of lysosomes (Fig. 1Ba). Such actin tails were never observed around lysosomes prepared from cells expressing either p61Hckkd (kinase-dead), a Hck mutant that has both its adaptor domains constitutively available for interactions with partner proteins, and its kinase activity impaired (Fig. 1Bc) or the p61Hck N-terminal unique domain (Fig. 1Bd) used as a negative control to target GFP to lysosomes as previously described (22).


Figure 2
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  		FIGURE 2.
p61Hck induces actin polymerization to generate actin-comet tails in a kinase-dependent manner. A, p61Hck promotes the formation of actin-comet tails in vitro. One-micrometer diameter latex beads bearing recombinant His6-p61Hck or His6-N-WASp were incubated in cytosols from U937 cells or Xenopus oocytes and added to a rhodamine-actin-containing medium before being observed by fluorescence microscopy. Arrows indicate the position of the bead. Bars = 5 µm. B, transmission electron microscopy observations of p61Hck- or N-WASp-induced actin-comet tails. Same as in A using U937 cell extract-containing medium and observed by electron microscopy. Insets show latex beads surrounded by a ring of F-actin. Bars = 0.5 µm. C, activated p61Hck induces de novo polymerization of actin. Recombinant p61Hckca (triangles), GST-VCA (black circles), or buffer alone (white circles) were added to a pyrene-actin-containing reaction medium in the presence of U937 cytosol. D, p61Hck ability to induce actin-comet tails is dependent on the kinase activity of Hck. Latex beads bearing recombinant N-WASp, p61Hckwt (in the absence or in the presence of 1 µM PP1), p61Hckca, or p61Hckkd were incubated in U937 cytosol-containing medium as in A. The percentage of beads associated to an actin-comet tail was quantified after a 90-min incubation. A total of 345 (n = 7), 360 (n = 8), 292 (n = 3), 391 (n = 8), and 106 (n = 2) beads was counted for each condition.

 
In a second attempt we ensured that the actin polymerization process described above was not due to the constitutively active state of p61Hckca. To do so similar experiments were performed using wild-type (wt) p61Hck. As shown in Fig. 1Bb, p61Hckwt-positive lysosomes were also associated to actin tails in a kinase-dependent manner, since in the presence of PP1, a pharmacological inhibitor of the Src kinases (38), F-actin tails were not formed (Fig. 1Be). Altogether these data demonstrate the ability of p61Hck to trigger the polymerization of actin on p61Hck-positive lysosomes in a kinase-dependent manner.

p61Hck Promotes the Genesis of Actin-comet Tails in Vitro in a Kinase-, WASp-, Arp2/3-, and Cdc42-dependent Manner—We then investigated whether p61Hck required partners at the lysosomal membrane to generate such polymerized actin structures. To this aim we developed an assay using recombinant His6-p61Hck bound to 1-µm-diameter latex beads and incubated in a cell-free assay containing cytosolic extracts from either U937 cells or Xenopus oocytes. In these experiments recombinant His6-human N-WASp bound to the latex beads was used as a positive control. We showed that p61Hck was able to generate actin tails that were morphologically similar to those induced by N-WASp; beads were surrounded by a ring of actin, and the tails were of comparable lengths in a U937 cytosol-containing reaction mix (Fig. 2A), showing that p61Hck is able to initiate polymerization of actin in the presence of soluble factors only, independently of any other lysosome-associated proteins. Similar results were obtained with NIH3T3 fibroblasts cytosolic extracts (not shown). Xenopus oocytes have been described as a source of cytosolic extracts able to facilitate the formation of actin-comet tails (12, 39). When incubated in such cell extracts, p61Hck-functionalized beads were also associated to an actin-comet tail that could be 10s of micrometers long for both N-WASp and p61Hck (Fig. 2A). The electron microscopy observations confirmed that N-WASp- and p61Hck-induced actin rings and actin tails had similar morphologies (Fig. 2B). p61Hck was also able to trigger de novo actin polymerization in a pyrene-actin assay in which actin filament elongation was monitored by fluorescence (40). As shown in Fig. 2C, p61Hckca induced a remarkable increase in the fluorescence when added to a pyrene-actin-supplemented U937 cytosol as compared with buffer alone.

We then wanted to better characterize the process by which p61Hck controlled actin-comet tail biogenesis in vitro. As shown in the quantification plot, p61Hckwt was slightly less efficient than p61Hckca in generating actin tails with 44 ± 5% (n = 8) and 63 ± 4% (n = 8) of the beads associated with such actin structures, respectively (Fig. 2D). This result indicates that, although less able than the constitutively active mutant, p61Hckwt can be activated and generate actin-comet in this in vitro assay. The formation of actin-comet tails followed different kinetics; N-WASp-induced tails appeared within the first 10 min, whereas those dependent on p61Hckca and p61Hckwt appeared within 20 and 40 min, respectively. The kinase activity of p61Hck was required in this process since only 4.5 ± 0.5% of the beads bound to the kinase-dead mutant of p61Hck were associated with a comet tail (n = 2), and p61Hckwt generated only 2.0 ± 0.1% (n = 3) of actin tails in the presence of PP1 (Fig. 2D).


Figure 3
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  		FIGURE 3.
p61Hck-induced actin tail biogenesis is dependent on WASp, the Arp2/3 complex, and Cdc42. A, p61Hck recruits WASp and requires both WASp and the Arp2/3 complex to polymerize actin. Panel a, agarose beads bearing (lane 1) or not (lane 3) recombinant His6-p61Hckwt were incubated in U937 cytosol before being subjected to a Western blot analysis with anti-human WASp antibodies (Abs, arrow). Lane 2, molecular weight markers. Panels b–b'', agarose beads bearing (panels b and b'') or not (panel b') recombinant His6-p61Hckwt were incubated in U937 cytosol containing (panel b'') or not (panels b and b') anti-human WASp blocking antibodies before being labeled with rhodamine-phalloidin and observed by fluorescence microscopy. Panel c, latex beads bearing p61Hckwt, p61Hckca, or N-WASp were incubated in U937 cytosol-containing medium (as in Fig. 2) in the presence of anti-WASp antibodies (n = 3) or after depletion of the Arp2/3 complex (n = 2). The number of beads associated to an actin-comet tail was determined after a 90-min incubation, and results were expressed as a percentage of inhibition compared with the corresponding control with neither anti-WASp antibodies nor Arp2/3-depletion. B, p61Hck promotes actin polymerization in a Cdc42-dependent manner. Panels a–a'', agarose beads bearing (panels a and a'') or not (panel a') recombinant His6-p61Hckwt were incubated in U937 cytosol that was pre-loaded with GDP (panel a'') or not (panels a and a') before being labeled with rhodamine-phalloidin and observed by fluorescence microscopy. Panel b, agarose beads bearing (lane 2) or not (lane 1) recombinant His6-p61Hckwt were incubated in U937 cytosol before being subjected to a Western blot analysis with anti-Cdc42, -RhoA, or -Rac1 antibodies. Lane 3 was loaded with the U937 cytosolic extracts used in this experiment. Panel c, latex beads bearing N-WASp, p61Hckwt, or p61Hckca were subjected to the actin-comet tail biogenesis assay in the presence of 10 µM N17Cdc42 (n = 2), 10 µM N17Rac1 (n = 2), or 1 µg of C3 exoenzyme/mg total proteins (n = 2). The number of beads associated to an actin tail was determined after a 90-min incubation At least 150 beads were counted in each condition. Results were expressed as a percentage of inhibition compared with the corresponding control in the absence of recombinant N17Cdc42, N17Rac1, or C3 exoenzyme.

 
We then set out to identify the proteins involved in the Hck-to-actin signaling pathway. To do so we used a candidate protein approach. We hypothesized that the WASp-Arp2/3 pathway might be involved in the actin polymerization signaling of Hck because (i) WASp has been demonstrated to control actin nucleation in the actin-comet tail biogenesis process (41), (ii) WASp is a known partner and substrate of Hck (29, 42), and (iii) N-WASp- and p61Hck-induced actin tails had similar morphology (Fig. 2B). To show that WASp was specifically recruited to beads bearing recombinant p61Hck, we used p61Hckwt-functionalized agarose beads of large diameter (70–90 µm) to optimize the detection of recruited proteins. The Western blot analysis on Fig. 3Aa shows that WASp was indeed present on p61Hck-beads after incubation in U937 cytosolic extracts. When the experiments were carried out in the presence of blocking anti-WASp antibodies, fluorescent-phalloidin staining revealed no F-actin on the beads, confirming the functional involvement of WASp in the process (Fig. 3A, b–b''). Moreover, the anti-WASp antibodies also inhibited the formation of actin-comet tails around latex beads by 93.4 ± 2.0% (n = 3) and 89.9 ± 1.1% (n = 3) for p61Hckwt and p61Hckca, respectively (Fig. 3Ac). The involvement of the WASp effector Arp2/3 complex in this p61Hck-controlled process was established using a depleted U937 cytosol with which actin tail biogenesis was inhibited by 95.3 ± 0.6% (n = 2) and 89.6 ± 2.0% (n = 2) for p61Hckwt and p61Hckca, respectively, inhibition effects comparable with what was obtained with the N-WASp control. These data showed that both WASp and the Arp2/3 complex were downstream effectors of p61Hck in the actin-assembly signaling pathway. Another Src kinase has been shown to control actin polymerization; v-Src (the viral constitutively active mutant of Src) can phosphorylate and activate WASp in a Hsp90-dependent manner (28). In our experimental conditions, Hsp90 did not seem to be involved in the Hck-induced actin polymerization process since no effect could be measured when we carried out the assay in the presence of up to 500 nM geldanamycin, a known inhibitor of Hsp90 (not shown).

Among other candidate proteins potentially involved in this process, we also tested the role of GTPases of the Rho family because they have been shown to belong to the signaling pathway downstream to Hck (23, 24). We checked that p61Hck required GTPase activities to polymerize actin by using a phagocyte GDP-loaded cytosol in which GTPases were blocked in their inactive GDP-bound conformation. Under these experimental conditions, no polymerized actin could be detected on p61Hck-agarose beads (Fig. 3B, a–a''). We then wanted to identify the Rho-GTPase(s) involved in this process. We observed that all three proteins, RhoA, Rac1, and Cdc42, were recruited on p61Hck-agarose beads when incubated in U937 cells cytosol (Fig. 3Bb) but that only Cdc42 was functionally involved in the p61Hck-induced actin tail biogenesis (Fig. 3Bc). In fact, the presence of Cdc42dn strongly inhibited actin tail formation for p61Hckwt, p61Hckca, and N-WASp by 68.5 ± 2.4, 70.2 ± 4.6, and 53.1 ± 1.2% (n = 2), respectively (Fig. 3Bc). In a parallel experiment performed with an optimized concentration of anti-Cdc42 antibodies, we achieved similar levels of significant inhibition of actin tail biogenesis (70.0 ± 1.2, 75.0 ± 1.8, and 72.6 ± 2.8 for p61Hckwt, p61Hckca, and N-WASp respectively; n = 2). In contrast, the use of cytosolic extracts containing a dominant-negative mutant of Rac1 or extracts treated with a specific inhibitor of Rho (C3 exoenzyme) had no effect on the biogenesis of either N-WASp- or p61Hck-induced actin tails (Fig. 3Bc). Therefore, although recruited on p61Hck-agarose beads, RhoA and Rac1 are not functionally involved in the process of actin tail formation. Taken together, these data indicate that among Rho-GTPases, only Cdc42 plays a functional role in the in vitro p61Hck-induced actin tail biogenesis, which is consistent with the well established function of Cdc42 as a major co-activator of WASp (43).


Figure 4
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  		FIGURE 4.
Activated p61Hck accelerates lysosomes in an actin-dependent manner. Average speed of p61Hck-positive lysosomes was measured in transiently transfected murine fibroblasts either in the absence (black bars) or in the presence of 0.5 µM cytochalasin D (white bars). A, results are expressed as mean velocity ± S.D. (n ≥ 6) as a function of the expressed p61Hck construct. B, velocity distributions of lysosomes were determined, and a Gaussian curve was fitted to each distribution. n indicates the number of lysosomes measured in each experimental condition. The vertical dotted line indicates the average reference speed of the p61Hckunique-associated lysosomes.

 
Activation of p61Hck Increases the Motility of Lysosomes in Cells in an Actin- and Kinase-dependent Manner—We then examined whether the biogenesis of actin-comet tails at the membrane of p61Hck-lysosomes could modulate the motility of lysosomes. Average velocities of lysosomes in NIH3T3 fibroblasts expressing the wild-type, the constitutively active, or the kinase-dead mutants of p61Hck in fusion with GFP were compared. As shown in Fig. 4, p61Hckca significantly accelerated lysosomes from 300 nm/s up to 403 nm/s. The kinase activity of p61Hck was clearly necessary for this 35% acceleration since the constitutively opened but kinase-dead mutant of p61Hck had no effect on the motility of lysosomes (Fig. 4A). As a control, the unique N-terminal domain of p61Hck was used to target GFP to lysosomes and measure their motility independently of the p61Hck kinase activity and its adaptor domains. These lysosomes moved at the same average speed as those harboring p61Hckwt or p61Hckkd (Fig. 4A). Indeed, Fig. 4B shows that the velocity distribution of lysosomes harboring p61Hckca was clearly shifted to higher values compared with that of lysosomes harboring the other constructs. As a matter of fact, 53% of p61Hckca-lysosomes had an average speed higher than 380 nm/s, whereas only 19% of p61Hckwt-, 18% of p61Hckkd-, and 16% of p61Hck-unique lysosomes did. These velocity values were comparable to those measured for endocytic vesicles by others in living cells (9, 26).

We then investigated whether p61Hckca-induced acceleration of lysosomes was dependent on the actin cytoskeleton. To address this issue, the same video-microscopy experiments were carried out in the presence of actin-depolymerization drugs. As shown in Fig. 4, when p61Hckca-expressing fibroblasts were treated with 0.5 µM cytochalasin D for 25 min, lysosomes speed was reduced to the control values. When full-length Hck was present at the lysosomal membrane but not constitutively activated, lysosome average speed was not affected by the drug treatment (see Fig. 4, p61Hck wt and kd). The same results were obtained with a treatment of 0.5 µM latrunculin A for 25 min (not shown). The microtubule network was not affected after either drug treatments (not shown). These results indicated that the p61Hckca-induced acceleration of lysosomes required an intact actin dynamics.

We also studied whether microtubules could be involved in p61Hck-lysosome movements. A 5-µM nocodazole treatment of the cells completely abolished the movement of p61Hck-positive lysosomes whichever mutant of Hck was used (not shown). This result indicated that an intact microtubule cytoskeleton was required for p61Hck-lysosome motility. Under our experimental conditions, microtubules were fully depolymerized, and the actin network remained intact (not shown). Taken together these results showed that the acceleration of the microtubule-dependent movement of p61Hck-lysosomes required p61Hck activation and de novo actin polymerization.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous work showed that Hck-positive secretory lysosomes constitute a subpopulation of lysosomes the exocytosis of which is regulated by and correlated to the activation of Hck (19–21, 24). We demonstrate here that Hck is able to trigger actin polymerization on p61Hck-lysosomes to control at least in part the motility of these vesicles.

In fibroblasts expressing the constitutively active mutant of the kinase, we observed polarized F-actin tails associated with p61Hck-lysosomes mainly at the basal part of the cells. We also occasionally observed p61Hck-lysosome-associated comet tails in the volume of the cells as assessed by confocal microscopy. Although we showed that p61Hckwt was able to generate actin-comet tails on lysosomes and on latex beads in vitro within phagocytic cell extracts, we have not been able to detect such lysosome-associated F-actin structures when wild-type p61Hck was expressed in fibroblasts. This was probably due to the fact that actin polymerization around p61Hck-lysosomes can be triggered when p61Hck gets activated, which may not be the case in resting fibroblasts. Moreover, only p61Hckca-lysosomes were accelerated in these cells, whereas those harboring p61Hckwt moved at the same speed as those associated to the Hck negative control p61Hck-unique. Taken together, these data indicated that there was a strong correlation between the presence of the comet tails on p61Hck-lysosomes and the enhancement of vesicle motility. They were also consistent with the fact that actin depolymerization drugs such as cytochalasin D and latrunculin A prevented this p61Hckca-induced acceleration.

In a cell-free assay, where the cytosol was supplemented to optimize actin polymerization, lysosomes harboring p61Hckwt were associated to comet-like F-actin structures similar to those induced by p61Hckca. The p61Hckkd mutant, which has both its adaptor domains constitutively available for interactions with partner proteins and its kinase activity impaired, did not generate F-actin clouds around lysosomes. The same results were obtained when the assay was performed with p61Hckwt in the presence of the pharmacological inhibitor PP1. This indicated that the presence of available Src Homology domain 2 (SH2) and SH3 adaptor domains was not sufficient in this mechanism, which clearly required the Hck kinase activity.

We have been able to reconstitute the p61Hck-induced actin-comet tail formation in vitro using latex beads functionalized with recombinant human p61Hck. It is the first time that a Src protein-tyrosine kinase is shown to directly trigger the formation of actin-comets. Moreover, these results showed that p61Hck did not require other lysosome-associated proteins in this signaling pathway but needed its kinase activity, WASp, the Arp2/3 complex, and the GTPase Cdc42. The functional involvement of Cdc42, and not Rho or Rac, in the p61Hck-induced actin tail biogenesis is consistent with the molecular mechanisms described for WASp activation (43, 44). In this process we hypothesize that Cdc42 and Hck act together to fully activate WASp. Moreover, previous work in the laboratory has established the involvement of Cdc42, Rho, and Rac in the p61Hck-signaling pathway resulting in the formation of podosomal structures (24). We now suggest that Rho and Rac act downstream of the lysosome recruitment step in the process of podosome formation (24). Hck has been able to generate actin-comet tails in NIH3T3 fibroblast and Xenopus oocyte extracts that contain N-WASp and in phagocytic cells extracts that contain WASp. This result showed that Hck can recruit both proteins, but because Hck is specific to the phagocytic cell lineage, its physiological downstream effector must be WASp and not N-WASp. The involvement of these factors in the genesis of the p61Hck-induced actin-comet tails is consistent with previous studies establishing WASp as a partner and substrate for Hck (29, 42). In addition, both WASp and the Arp2/3 complex have already been involved in the formation of comet tails induced by pathogens (17) and at the membrane of other intracellular vesicles (12, 14, 15).

In some cases, actin-comet tails have been shown to be associated with endocytic vesicles. On the one hand, actin clouds have been shown to form at the membrane of Src-positive endosomes (16). On the other hand, Src was activated during the actin-dependent translocation of these vesicles toward the cell periphery. However, it was not determined whether Src activation triggers the formation of actin tails on endosomes, which consequently should propel the vesicles through the cytosol (16). In mouse bone marrow-derived macrophages, some endosomes and some latex bead-containing phagosomes bear actin-comet tails, but neither the molecular mechanism involved nor the destination of the moving vesicles have been described (45). The presence of actin-comet tails has also been observed occasionally on late endosomes in a cell-free assay of fusion with phagosomes (46). However, whether these actin-tail-associated vesicles were targeted to the purified phagosomes was not determined. We have previously shown that p61Hck activation triggers the formation of phagolysosomes (19–21) and the biogenesis of podosome rosettes in a lysosome-dependent manner (24). Podosomes are actin-rich structures involved in cell adherence and extracellular matrix degradation (47, 48). Thus, we hypothesize that actin-comet tails play a role in the targeting of p61Hck-lysosomes either to the phagosomal or to the plasma membrane. In this context the biogenesis of actin-comet tails on lysosomes should precede the formation of rosettes of podosomes and could be a pre-requisite for the Hck-positive lysosomes to be targeted and then fuse with their target membrane. This could explain why we observed actin tails on lysosomes only in cells displaying no rosettes or forming rosettes. In contrast, in cells where podosomal rosettes were already formed, we rarely observed actin tails associated to p61Hck-lysosomes, probably because either p61Hck-lysosomes were not recruited anymore to the plasma membrane or there was not enough monomeric actin left to generate detectable actin tails.

The diversity of the molecular mechanisms involved in the traffic of lysosomes probably reflects the specific roles they play from one cell type to another. Actually, the delivery of secretory lysosomes in cytotoxic T lymphocytes, melanocytes, and phagocytes clearly occurs at different sites in response to distinct stimuli. Examples of intracellular traffic involving both cytoskeletal tracks and actin-comet tails have been described. Vaccinia virus moves to the cell periphery along microtubule tracks before the Src-controlled switch to an actin-comet tail-dependent movement (49). Yeast mitochondria have been proposed to simultaneously use actin microfilament tracks and actin-comet tails to ensure proper distribution during cell division (50, 51). We propose a new model relying on an original cooperation between the actin and the microtubule cytoskeletons, where the p61Hck-induced polymerization of actin at the lysosomal membrane would add up to the microtubule-dependent forces to make the secretory lysosomes move faster along the microtubular tracks toward their target membrane.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Institut de Pharmacologie et de Biologie Structurale, CNRS-UMR5089, 205 route de Narbonne, 31077 Toulouse Cedex 04, France. Tel.: 33-5-61175458; Fax: 33-5-61175994; E-mail: Isabelle.Maridonneau-Parini{at}ipbs.fr.

2 The abbreviations used are: F-actin, filamentous actin; Arp, actin-related protein; BSA, bovine serum albumin; ca, constitutively active; GFP, green fluorescent protein; Hck, hematopoietic cell kinase; kd, kinase dead; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine; Tet, tetracycline; VCA, Verprolin-Cofilin-acidic domain; WASp, Wiskott-Aldrich syndrome protein; wt, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. C. Leclerc and Dr. M. Moreau (Centre de Biologie du Développement, Toulouse, France) for technical help with the Xenopus oocytes collection, Dr. L. Blanchoin (Commissariat à l'Energie Atomique-CNRS, Grenoble, France) for the generous gift of rabbit actin (unlabeled and pyrene-labeled) as well as technical help with the actin-pyrene assay, Dr. P. Chavrier for the gift of the pGEX-GST-VCA plasmid, Dr. E. Lemichez (INSERM, Nice) for the gift of active and inactive C3 exoenzymes, and Dr. V. Le Cabec (Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse) for technical help with the ADP-ribosylation assay.



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C. Tu, C. F. Ortega-Cava, G. Chen, N. D. Fernandes, D. Cavallo-Medved, B. F. Sloane, V. Band, and H. Band
Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts
Cancer Res., November 15, 2008; 68(22): 9147 - 9156.
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