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Vol. 17, Issue 6, 2811-2823, June 2006

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The Kinesin KIF1C and Microtubule Plus Ends Regulate Podosome Dynamics in Macrophages

Petra Kopp^*, Reiner Lammers, Martin Aepfelbacher, Günther Woehlke, Thomas Rudel^||, Nikolaus Machuy^||, Walter Steffen^¶, and Stefan Linder^*

*Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universität, 80336 Munich, Germany; Medizinische Klinik IV, Universität Tübingen, 72076 Tübingen, Germany; Institut für Infektionsmedizin, Universitätsklinikum Hamburg Epppendorf, 20246 Hamburg, Germany; Institut für Zellbiologie, Ludwig-Maximilians-Universität, 80336 Munich, Germany; ^||Max-Planck-Institut für Infektionsbiologie, 10117 Berlin, Germany; and ^¶Institut für Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany

Submitted November 3, 2005; Revised February 24, 2006; Accepted March 9, 2006
Monitoring Editor: Paul Matsudaira

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end–enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cross-talk between cytoskeletal systems is essential for processes such as cell motility, wound healing, or cell division. In particular, functional coupling of the actin and microtubule cytoskeletons has been the focus of much interest in recent years (reviewed in Goode et al., 2000; Rodriguez et al., 2003; Schliwa and Woehlke, 2003). Here, we investigate the regulatory relationship between actin-based podosomes and microtubule plus ends as well as of motor proteins that may allow the functional linkage of both structures.

Podosomes are actin-rich adhesions typical for cells of the monocytic lineage, but they are also formed in other cell types (reviewed in Linder and Aepfelbacher, 2003; Buccione et al., 2004). Prominent features that distinguish podosomes (and the related invadopodia) from other adhesions are their two-part architecture—a core of F-actin and associated proteins embedded in a ring structure containing plaque proteins such as paxillin or vinculin—and their ability to concentrate matrix metalloproteases and actively engage in matrix degradation (reviewed in Linder and Kopp, 2005). Podosomes are thought to play important roles in migration and invasion of cells. Podosome-mediated adhesion provides local anchorage and may thus stabilize cellular protrusions and support directional migration. Importantly, podosome-localized release of matrix metalloproteases probably contributes to the invasive abilities of podosome-forming cells in physiological and pathological conditions.

Podosomes are highly dynamic organelles with a lifetime of 2–12 min (Kanehisa et al., 1990; Destaing et al., 2003), and their regulation comprises multiple signal transduction pathways, including Src kinase activity (Marchisio et al., 1988) as well as RhoGTPase signaling and actin-regulatory mechanisms (Linder et al., 1999; Burns et al., 2001). Importantly, microtubules have been shown to influence the dynamics (Evans et al., 2003), positioning (Babb et al., 1997; Destaing et al., 2005), and formation (Linder et al., 2000) of podosomes. However, despite the observed close association of podosomes and microtubules, it remained unclear whether both structures indeed interact in living cells. Moreover, the molecular basis for this functional dependency remained unresolved.

Here, we show that the kinesin kinesin-like-family (KIF)1C, a member of Kinesin-3 family (Linder et al., 2000), is critically involved in the microtubule-dependent regulation of podosome dynamics in macrophages. KIF1C has been implicated in Golgi–endoplasmic reticulum (ER) transport (Dorner et al., 1998) and in mouse macrophage resistance to anthrax toxin (Watters et al., 2001). Knockout experiments in mice, however, suggested that KIF1C activity is dispensable for Golgi–ER transport (Nakajima et al., 2002). So far, no function for KIF1C has been described in human cells.

We now show that KIF1C is enriched at a subset of microtubule plus ends that contact podosomes. Contact by KIF1C is followed by enhanced podosome dynamics, i.e., fission or dissolution. Conversely, expression of mutation or truncation constructs of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. We further identify nonmuscle myosin IIA as an interaction partner of KIF1C. Myosin IIA is enriched in regions of high podosome turnover, and its binding to KIF1C may contribute to the efficient targeting of podosomes by microtubule plus ends.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Isolation and Cell Culture
Human peripheral blood monocytes were isolated and differentiated into macrophages as described previously (Linder et al., 1999).

Microinjection of Proteins
Cells for microinjection experiments were cultured for 5–8 d. Proteins were expressed in Escherichia coli, as described previously (Linder et al., 2000). For microinjection, proteins were dialyzed against microinjection buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM MgCl₂), concentrated in Centricon filters (Millipore, Bedford, MA), shock-frozen, and stored at –80°C. Microinjection was performed using transjector 5246 (Eppendorf, Hamburg, Germany) and a Compic Inject micromanipulator (Cell Biology Trading, Hamburg, Germany). Glutathione S-transferase (GST)-KIF1C-protein tyrosine phosphatase-binding domain (PBD) was injected into the cytoplasm at 1, 2, and 3 µg/µl. (Inhibitory) antibodies m74-2, SUK4 (Cytoskeleton, Denver, CO) and K2.4 (HiSS Diagnostics, Freiburg, Germany) against dynein heavy chain, KIF5B or KIF3B, respectively, were injected at 4 or 5 µg/µl (K2.4). Control experiments were performed with GST or microinjection buffer. Injected cells were identified by labeling coinjected rat IgG (5 mg/ml; Dianova, Hamburg, Germany) with fluorescein isothiocyanate-labeled goat anti-rat IgG antibody (Dianova).

Transfection of Cells
Cells were transiently transfected using a Nucleofector I (amaxa, Köln, Germany) according to the manufacturer's instructions and seeded on coverslips at a density of 5 x 10⁵.

Immunofluorescence and Microscopy
Cells were fixed for 10 min in 3.7% formaldehyde solution and permeabilized for 1 min in ice-cold acetone. F-actin was stained with Alexa 568-labeled phalloidin (Molecular Probes, Leiden, The Netherlands), KIF1C was stained with specific primary antibody (Ab) (Cytoskeleton), mitochondria were stained with OxPhos-Ab against cytochrome-c oxidase subunit I (Molecular Probes), myosin IIA was stained with specific primary polyclonal Ab (DakoCytomation Denmark, Glostrup, Denmark), and tubulin was stained with specific primary polyclonal Ab (Cytoskeleton; Note: This antibody also works sufficiently with formaldehyde fixation, which was used when costaining of other proteins in tubulin-specific fixations such as glutaraldehyde/PHEM was not feasible.) Secondary antibodies were Alexa 488- or Alexa 568-labeled goat anti-mouse, goat anti-rabbit, or goat anti-sheep (Molecular Probes). Coverslips were mounted in Mowiol (Calbiochem, Darmstadt, Germany) containing p-phenylendiamine (Sigma-Aldrich, St. Louis, MO) as antifading reagent and sealed with nail polish.

Microscopy was performed as described previously (Linder et al., 2000). Immunofluorescence-stained preparations were observed with a Leica DM RBE microscope using a 100x plan fluotar objective. Images were captured with a Spot-camera (Leica, Wetzlar, Germany). Confocal microscopy was performed with a Leica DM IRB confocal scanning microscope and a 100x plan apo objective.

Live Cell Imaging
Confocal time-lapse microscopy of transiently transfected cells was performed using an UltraView LCI live cell imaging system with UltraView, version 5.5.0.2 [EC] (PerkinElmer Life and Analytical Sciences, Boston, MA), images were captured using a scan interline digital charge-coupled device camera, model Orca ER (Hamamatsu, Herrsching, Germany) with a resolution of 1344 x 1024 pixels and converted to 12-bit-images. Filter settings were 488-nm excitation, 525/50-nm emission, 568-nm excitation, and 697/45-nm emission. Cells were seeded on glass-bottomed dishes (MatTek, Ashland, MA) at a density of 4–8 x 10⁵ and incubated 20 h before the start of the experiment. During the experiment, cells were incubated in a chamber connected to an incubator controller (EMBL, Heidelberg, Germany) at 37°C and 5% CO₂.

To be scored as a contact between podosomes (labeled with monomeric red fluorescent protein [mRFP]-actin) and microtubule plus ends (labeled with green fluorescent protein [GFP]-cytoplasmic linker protein [CLIP]170 or KIF1C-GFP), the respective mRFP and GFP signals had to be directly adjacent or overlapping, without intermediate black pixels. Contact in most cases included 1) apparently directed, not random, movement of plus ends toward podosomes, 2) a momentary stop of movement at podosomes, and 3) a subsequent change or reversal of direction. A correlation between microtubule contact and podosome fate was scored if an alteration of podosome behavior (fission or dissolution) was discernible within 1–2 min after contact by a microtubule plus end (most alterations were observed within shorter periods).

Plasmid Construction and Mutagenesis
For cloning of GST-KIF1C-P, part of the coding sequence of human KIF1C was amplified and cloned into pGEX-1T, resulting in a construct coding for aa 713-811. For cloning and expression of GST-KIF1C-U, the coding sequence of KIF1C was amplified and cloned into pGEX5X-3, resulting in a construct coding for aa 441-623. For cloning and expression of wild-type GFP-KIF1C, the 5' half of the coding sequence up to nucleotide 1847 was amplified from pRKS-KIF1C generating a 5'XhoI and a 3'EcoRI restriction site. The 3' half of the KIF1C coding sequence was amplified generating a 5'EcoRI and a 3'BamHI restriction site. The two regions were cloned successively into vector pEGFP-N1 (Clontech, Mountain View, CA). GFP-KIF1C-K103A was generated by removing the newly generated EcoRI restriction site and by introducing a K103A mutation using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).

Vector encoding GFP-KIF3BNT was a kind gift from V. Gelfand (University of Illinois, Urbana, IL); MBP-kinesin-associated protein (KAP)3 was a kind gift from Y. Takai (Osaka University, Osaka, Japan); GFP-KAP3-wild type (wt) and GFP-KAP3Arm5 were kind gifts from T. Akiyama (University of Tokyo, Tokyo, Japan); KIF4 was a kind gift from Y. Lee (Ajou University, Suwon, South Korea); Myc-KHC wt, Myc-KHC H582, HA-kinesin light chain (KLC) wt, HA-KLC-TPR6, and HA-KLC-L176 were kind gifts from B. Schnapp (Harvard Medical School, Boston, MA); FLAG-KLC2 was a kind gift from T. Ichimura (Tokyo Metropolitan University, Tokyo, Japan); DsRed-EB1 was a kind gift from A. Barth (Stanford University, Stanford, CA); and GFP-CLIP170 was a kind gift from N. Galjart (Erasmus Medical Center, Rotterdam, The Netherlands).

GST-Pull Down Assay
GST-pull downs were prepared as described previously (Linder et al., 1999). Briefly, 12 x 10⁶ cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. After centrifugation at 3500 x g for 10 min at 4°C, the lysate was added to glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 µg of GST-fusion proteins or GST at comparable molar ratios. Beads were incubated with lysate for 1 h at 4°C, washed three times with lysis buffer, pelleted, and mixed with 100 µl of SDS-sample buffer.

Immunoblotting
Immunolabeling was performed by standard procedure, using the above-mentioned primary antibodies. Secondary antibodies were horseradish peroxidase-coupled anti-mouse or anti-rabbit IgG (Dianova). Protein bands were visualized by using Super Signal kit (Pierce Chemical, Rockford, IL) and X-Omat AR film (Kodak, Stuttgart, Germany).

Validation of Small Interfering RNA by Quantitative Real-Time PCR
Small interfering RNAs (siRNAs) were validated by quantitative real-time PCR (q-PCR) using lysates of transfected HeLa cells, as described previously (Machuy et al., 2005). Briefly, 0.1–0.25 µg of siRNA (final concentration, 80–200 nM) directed against KIF1C, KIF3A, KAP3A, KIF5B, or Luciferase as control and 2 µl Transmessenger reagent (QIAGEN, Hilden, Germany) were added to 10 x 10⁴ cells seeded in 96-well plates. RNA was isolated 48 h later using the RNeasy 96 BioRobot 8000 system (QIAGEN). The relative amount of target mRNA was determined by q-PCR using Quantitect SYBR Green reverse transcriptase-PCR kit following manufacturer's instructions (QIAGEN).

siRNA/Short Hairpin RNA Knockdown Experiments
Duplex siRNA (650 ng) was transfected transiently in primary human macrophages. For visualization of transfected cells, 1.2 µg of pEGFP-N1-vector was cotransfected, and cells were cultured for 24–72 h. For vector-encoded short hairpin RNA (shRNA), different target regions in the KIF1C coding sequence were chosen and cloned into psiSTRIKE U6 (Promega, Madison, WI). Cells were transfected and cultured for additional 24–72 h. Target sequence corresponds to the nucleotides 426–442 in KIF1C. As a control, a scrambled sequence was used.

Reverse Transcriptase Reaction
Cells (6 x 10⁶) were cultured for 7 d, and mRNA was isolated using QuickPrep Micro-mRNA purification kit (GE Healthcare). DNA was removed by DNAse digestion (Novagen, Madison, WI). For cDNA-synthesis, 1 µg of random primer (Promega) was annealed to 2 µg of RNA for 5 min at 70°C, and second strand synthesis was performed using Moloney murine leukemia virus reverse transcriptase (Promega) using an oligonucleotide primer pair corresponding to nucleotides 3030–3059 and 3369–3397 of the KIF1C coding sequence, respectively. As a control for quantitative removal of residual DNA, oligonucleotide primers specific for an exon in the human -actin gene were used, corresponding to nucleotides 1161–1142 and 716–735, respectively.

Podosome Reformation
Podosomes were disrupted by addition of tyrosine kinase inhibitor PP2 (Sigma-Aldrich) at 25 µM for 1 h with subsequent washout. Podosome disruption/reformation does not interfere with microtubule integrity or distribution (Linder et al., 2000). Macrophages normally display 50–150 podosomes per cell. The threshold value for a macrophage containing podosomes was set to 10. This allowed an immediate and clear distinction between cells "containing" or "not containing" podosomes.

Immunoprecipitation
Immunoprecipitations were performed with the µMACS-protein isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Seven- to 14-d-old macrophages were lysed in lysis buffer A (50 mM Tris-HCl, pH 7.5, 100 mM KCl, and 0.5% Triton X-100) or B (20 mM HEPES, pH 7.5, 150 mM NaCl, 250 mM sucrose, 1 mM Na-molybdate, and 1% Igepal), both with Complete protease inhibitors (Roche Diagnostics). Two micrograms of antibody was added to 50 µl of protein A-coupled magnetic beads, incubated with the cleared lysate for 1 h, and applied to the µMACS separation column. After washing four times with washing buffer (20 mM HEPES, 150 mM NaCl, 2 mM peroxovanadate, 0,1% Triton X-100, and 10% glycerol), the immunoprecipitate was eluted with 1x Laemmli buffer. Immunoprecipitations of GFP fusion proteins were performed using the GFP-µMACS epitope tag Protein isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. The cells were lysed in (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 1% Igepal), with Complete Mini protease inhibitors.

Mass Spectrometry
Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel bands were washed twice with water and then covered with a 1:1 mixture of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate and washed twice with water. Clear gel pieces were then treated twice with 50% acetonitrile for 5 min. Trypsin (sequencing grade modified; Promega, Mannheim, Germany) was added, and proteins were digested overnight in 40 mM NH₄HCO₃ buffer, pH 8.0, at 37°C and 650 rpm. For protein identification, probes were used for matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) experiments. Ten microliters of each sample was first purified and concentrated on a C18 reverse phase pipette tip (ZipTip; Millipore, Schwalbach, Germany). Peptides were eluted with 1 µl of -cyano-4-hydroxycinnamic acid (Sigma-Aldrich) and directly spotted on a MALDI sample plate (Applied Biosystems, Foster City, CA). MALDI-TOF measurements were performed on a Voyager-DE STR TOF mass spectrometer (Applied Biosystems). The resulting spectra were then analyzed via Mascot software (Matrix Science, London, United Kingdom) using the NCBInr Protein Databank.

Microtubule Cosedimentation Assay
Cells (1.2 x 10⁷) were lysed in (80 mM PIPES, pH 6.8, 1 mM MgCl₂, and 1 mM EGTA, with protease inhibitors) and centrifuged at 100 000 x g for 1 h. Tubulin in the cytosol was polymerized by addition of (2 mM GTP, 2 mM MgCl₂, and 20 µM taxol, end concentrations), and motor proteins were allowed to bind for 30 min at room temperature in the presence of 1.5 mM adenyl-5'-yl imidodiphosphate (AMP-PNP). Motor–tubulin complexes were pelleted by centrifugation at 165 000 x g for 1 h, and aliquots of resuspended pellets were analyzed with SDS-PAGE and Western blots.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Podosomes Are Contacted by Microtubule Plus Ends
Podosomes are cell–matrix contacts that localize exclusively to the ventral cell side of substrate-attached macrophages. In contrast, microtubules originate mostly from the microtubule-organizing center (MTOC), which is localized in the dome-shaped central part of the cell containing most of the cytoplasm and also the nucleus. To investigate the spatiotemporal relationship between podosomes and microtubules in living cells, primary human macrophages were transfected with constructs encoding mRFP–-actin, to label podosomes, and GFP-fused CLIP170, a microtubule plus end–binding protein (reviewed in Galjart, 2005). Cells were subsequently analyzed using time-lapse confocal microscopy (Figure 1A). Confirming previous results gained with fixed specimens (Linder et al., 2000), microtubules often extended to the podosome-containing ventral cell side. Moreover, podosomes were found to be targeted, often repeatedly, by microtubule plus ends (Figure 1A; for full appreciation of repeated contacts between microtubules and podosomes, see Supplemental Videos 1–4). Further analysis revealed three basic modes of podosome behavior: dissolution, fission, or static behavior (Figure 1A and Supplemental Videos 2–4).

View larger version (75K): [in this window] [in a new window]   Figure 1. Podosome fate is influenced by contact with microtubule plus ends. (A) Confocal laser scanning micrographs of substrate-attached part of human primary macrophage expressing mRFP--actin (red) and GFP-CLIP170 (green). White boxes in overview image indicate corresponding areas enlarged at the right, showing dissolution (A'), static behavior (A''), or fission of podosomes (A'''. Note repeated contact of CLIP170-labeled microtubule plus ends with -actin–labeled podosomes. Images shown were taken every 60 s from start of the experiment (Supplemental Videos 1–4). White arrows indicate single podosomes. Bar, 10 µm. Podosome behavior and concurrent contact by CLIP170-labeled microtubule plus ends was evaluated in a total of 731 podosomes from three independent experiments. Values are given as mean percentage ± SD of total counts. Podosomes contacted by CLIP170-labeled microtubule plus ends: 31 ± 10% for no change, 53 ± 6% for dynamic behavior; podosomes not contacted by CLIP170-labeled microtubule plus ends: 10 ± 3% for no change, 7 ± 3% for dynamic behavior. (C) Analysis of podosome dynamics in a human macrophage. Confocal laser scanning micrograph of primary macrophage expressing mRFP–-actin (image of time lapse series). Podosomes are color coded according to their behavior during the course of the experiment: static (blue), fission (green), or dissolution (red). Note that small, static podosomes cluster in the cell center, whereas in the cell periphery, larger precursor clusters mostly undergo fission, and smaller podosomes mostly dissolve. Bar, 10 µm.

Podosomes Are Influenced by ATP Hydrolysis but Not by KIF5B or Dynein
Contact by microtubules raised the possibility of motor proteins being involved in podosome regulation. To investigate this question, we evaluated podosome reformation under conditions of motor protein inhibition. This assay takes advantage of a microinjection artifact, because injection of macrophages with high pressure leads to disruption of podosomes, which are subsequently reformed within 1 h (with microtubule integrity not being compromised; Linder et al., 2000). Microinjection with the nonhydrolysable ATP analogue AMP-PNP led to a reduction of 60% in podosome reformation (Figure 2A and Table 1), indicating the general influence of ATP hydrolysis-dependent processes. An antibody (Ingold et al., 1988) against conventional kinesin (KIF5B in the unified nomenclature; Miki et al., 2001), which has been shown to inhibit focal adhesion regulation by microtubules in Xenopus fibroblasts (Krylyshkina et al., 2002), did not influence podosome reformation (Figure 2, A–C, and Table 1). However, in an established control assay testing its functionality (Krylyshkina et al., 2002), the antibody effectively inhibited the plus end–directed transport of mitochondria in macrophages (Supplemental Figure S1). Also, microinjection of a potentially inhibiting antibody against heterotrimeric kinesin (Morris and Scholey, 1997) did not interfere with podosome reformation (our unpublished data). Moreover, inhibition of dynein, either by an inhibitory antibody (Steffen et al., 1997) or by the inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA; Schliwa et al., 1984) did not influence podosome reformation (Figure 2A and Table 1), although, in an established functionality assay (Krylyshkina et al., 2002), both substances inhibited minus end–directed lysosome transport in macrophages (Supplemental Figure S2; our unpublished data). These results indicate that neither conventional kinesin nor dynein are important for the reestablishment of regular podosome dynamics in macrophages.

View larger version (47K): [in this window] [in a new window]   Figure 2. Podosome formation is influenced by KIF1C but not by conventional kinesin or dynein. (A) Evaluation of podosome formation after microinjection (0 or 1 h). Values are given as mean percentage ± SD of total counts in Table 1. For differences between control values and values gained with motor protein inhibitors, a p value < 0.01 was considered significant (indicated by asterisk). (B and C) Fluorescence micrographs of macrophages injected with anti-KIF5B antibody, subsequently fixed at 0 or 1 h and stained for F-actin (red) or rat IgG as an

In a next step, we synthesized siRNAs against KIF1C, KIF3A, KAP3A, and KIF5B, which were validated in HeLa cells and found to be highly effective (>84.0% knockdown; see Materials and Methods). Primary macrophages were transfected with the respective siRNA and evaluated for podosome content 24 and 48 h after transfection. Cotransfected pEGFP-N1 (at substoichiometric concentrations; see Materials and Methods) served as a transfection marker. Compared with controls, KIF3A-, KAP3A- and KIF5B-specific siRNA showed a slight effect on podosome numbers. By contrast, KIF1C siRNA-transfected macrophages showed a marked and significant reduction in podosome content to 60% of controls (Figure 2E and Table 1).

This was further confirmed by transfection of a construct which allows bicistronic expression of KIF1C-specific shRNA and enhanced green fluorescent protein (EGFP) for unequivocal identification of transfected cells (STRIKE-KIF1C). Test transfection of human umbilical vein endothelial cells (HUVECs) showed efficient reduction of endogenous KIF1C protein expression levels, compared with a construct encoding a scrambled sequence (STRIKE-scrambled; Figure 2F). Primary macrophages transfected with the KIF1C-specific construct showed a (gradual) reduction of podosomes (Supplemental Figure S3A), to levels similar to those of cells directly transfected with siRNA (Figure 2G). In addition, those cells transfected with the KIF1C-specific construct but still containing podosomes showed decreased podosome fission (13.8 ± 7.4% for STRIKE-KIF1C versus 30.6 ± 10.7% for STRIKE-scrambled; Figure 2H). In sum, interference with KIF1C function during podosome reformation either by expression of mutant constructs or through transfection/expression of specific siRNA or shRNA seemed to disrupt regular podosome dynamics and led to increasing numbers of cells devoid of podosomes.

KIF1C Is Enriched at Podosome-contacting Microtubule Plus Ends
To confirm endogenous expression of KIF1C in primary human macrophages, several assays were performed. The presence of KIF1C mRNA in macrophages was confirmed by reverse transcriptase-PCR using specific primers and subsequent sequencing of the PCR product (Figure 3A), whereas Western blots of KIF1C immunoprecipitations, developed with a specific antibody, confirmed expression of the corresponding polypeptide in macrophage lysates (Figure 3B). Moreover, in a microtubule cosedimentation assay, KIF1C was present in the microtubule-containing pellet (Figure 3C), indicating a microtubule-binding ability of the protein. Immunofluorescence analysis of endogenous KIF1C showed an enrichment at the periphery of the ventral cell side (Figure 3, D and F) and also at a juxtanuclear position, which, by staining for -tubulin, was shown to overlap with the centrosome/MTOC (Figure 3, E and F).

View larger version (26K): [in this window] [in a new window]   Figure 3. KIF1C is expressed in primary human macrophages. (A) Reverse transcriptase-PCR using primers specific for KIF1C (left), control reaction using primers for an exon in the -actin gene (right), agarose gel stained with ethidium bromide; size in base pairs on left. (B) Immunoprecipitation of macrophage lysates using anti-KIF1C antibody (left), rabbit IgG was used as control (right); Western Blot probed with anti-KIF1C antibody. Molecular mass in kilodaltons is indicated on the left. (C) Microtubule cosedimentation assay using macrophage lysates. KIF1C is pelleted together with polymerized microtubules stabilized by taxol treatment. Western blot of cytosolic supernatant and pellet, probed with anti-KIF1C or anti--tubulin antibody, respectively. (D and E) Primary macrophage stained with specific primary antibodies for KIF1C (D) and -tubulin (E), overlay shown in (F). Superimposition of each time two confocal laser scanning micrographs with a z-distance of 4 µm. Cell circumference depicted by dashed white line. White bar, 10 µm.

View larger version (70K): [in this window] [in a new window]   Figure 4. KIF1C at microtubule plus ends contacts podosomes. (A) KIF1C-GFP is enriched at sites of high podosome turnover. Confocal micrograph series of substrate-attached part of primary macrophage expressing KIF1C-GFP (green) and mRFP--actin (red; Supplemental Videos 5 and 6). Time since start of experiment is indicated in top right corners. White box indicates enlarged area. Bar, 10 µm. (B) KIF1C-GFP (green) localizes to DsRed-EB1–labeled (red) microtubule plus ends. Confocal laser scanning micrograph of substrate-attached part of cell (Supplemental Videos 7 and 8). White box indicates enlarged area. Bar, 10 µm. (C) Statistical analysis of podosome development in primary macrophage. Podosomes were analyzed for behavior (static, fission, or dissolution) and concurrent contact by KIF1C-GFP–containing punctate structures. For each value, a total of 601 podosomes from three different experiments were evaluated. Values are given as mean percentage ± SD of total counts. Contact by KIF1C-GFP accumulations: 14 ± 4% for no change, 16 ± 4% for fission, 26 ± 8% for dissolution; no contact by KIF1C-GFP accumulations: 23 ± 8% for no change, 7 ± 5% for fission, 15 ± 5% for dissolution.

Expression of a P-Loop Mutant of KIF1C Leads to Podosome Loss
Typical for kinesins, KIF1C features a P-loop sequence (GQTGAGKS; aa 97-104; Dorner et al., 1998), which is supposedly involved in nucleotide binding (Sack et al., 1999). Mutations in the corresponding motif of KIF5B have been shown to abrogate ATP hydrolysis and induce a rigor state (Nakata and Hirokawa, 1995). Therefore, attempting to generate a dominant negative construct of KIF1C, we introduced a K103A mutation (Figure 5A). Mutant KIF1C-K103A-GFP was mostly dislocalized from microtubule plus ends and instead concentrated at a dot-like juxtanuclear position (Figure 5B), which, by costaining for -tubulin, was identified as the centrosome/MTOC (our unpublished data). The expression of KIF1C-K103A-GFP led to a clear reduction in cellular podosome content (Figure 5B), without apparently compromising microtubule number, length or dynamics (our unpublished data). Quantifying this phenomenon in the podosome reformation assay, we found that only 45% of KIF1C-K103A–expressing cells formed podosomes, compared with 75% of control cells (Figure 5C). The inability of KIF1C-K103A–expressing cells to reform podosomes was clearly coupled to an aberrant localization of the mutant protein, because 80% of cells without podosomes showed an accumulation of KIF1C-K103A at the MTOC, in contrast to 35% of the cells that were still able to form podosomes (Figure 5C).

View larger version (33K): [in this window] [in a new window]   Figure 5. A KIF1C P-loop mutant is dislocalized and leads to podosome loss. (A) Domain organization of KIF1C and expression constructs used in this study: P-loop sequence (P; aa 97-104), motor domain signature (MD; aa 242-253), Unc104 domain (UD; aa 483-593), PTPD-1–binding domain (PTPD; aa 714-809), unspecified 14-3-3 binding region (14-3-3). Numbers indicate first and last amino acid residues of constructs. (B) Primary macrophage expressing GFP-labeled KIF1C K103A mutant (green), labeled for F-actin, confocal laser scanning micrograph of substrate-attached part of cell (superimposition of 4 images with z-distances of 1 µm). Note MTOC-like localization of GFP signal and scarcity of podosomes. (C) Evaluation of podosome reformation in macrophages expressing the KIF1C K103A mutant construct. Cells were treated with PP2 (0 h), followed by washout (1 h). Values for podosome formation are given as mean percentage ± SD of total counts. For each bar, 3 x 30 cells were evaluated. Control (pEGFP-C1): 0 h, 2.2 ± 1.5%; 1 h, 74.4 ± 1.5% for cells with podosomes; KIF1C-K103A: 1 h, 44.4 ± 7.8% for cells with podosomes, concurrent with 16.7 ± 2.6% of total cells showing a MTOC-like localization of the GFP signal; 55.6 ± 7.8% for cells without podosomes, concurrent with 45.6 ± 14.1% of total cells showing a MTOC-like localization of the GFP signal. For differences between control values and values gained with KIF1C-K103A expression (light gray bars), a p value < 0.01 was considered significant (indicated by asterisk); for a correlation between absence of podosomes (black bar) with an MTOC-like accumulation of GFP-KIF1C-K103A (dark gray bar), a p value < 0.02 was considered significant (indicated by asterisk). (D) Effect of microtubule disruption in cells expressing either KIF1C-GFP wt or KIF1C-K103A-GFP. Podosome formation was evaluated in untreated cells (unlabeled bars), after disruption with 25 µM PP2 and subsequent washout (+PP2), after addition of nocodazole (1 µM; +noc.), or after disruption with PP2 and addition of nocodazole to washout (+PP2+noc.). For each bar, 3 x 30 cells were evaluated. Values are given as mean percentage ± SD of total counts in Table 2, a p value < 0.04 was considered significant (indicated by asterisk).

View larger version (56K): [in this window] [in a new window]   Figure 6. KIF1C interacts with nonmuscle myosin IIA. (A) HUVEC lysates immunoprecipitated with anti-GFP antibody coupled to magnetic beads. Silver-stained PAA gel, left lane: cells transfected with pEGFP-N1 as control; right lane: cells transfected with KIF1C-GFP construct. Arrow indicates band subsequently identified by MALDI as nonmuscle myosin IIA. Molecular mass in kilodaltons is indicated on the left. (B) KIF1C and myosin IIA coprecipitate from macrophage lysates. Immunoprecipitation of macrophage lysates, using rabbit IgG as control, KIF1C-specific antibody or myosin IIA-specific antibody. Western blots developed with antibody indicated at the right. (C) GST-pull down of macrophage lysates. Western blot probed with anti-myosin IIA antibody. Denominations of GST-fused polypeptides used for pull down are given above each lane. (D) Microinjection of macrophages with GST-KIF1C-P disrupts podosomes. Laser scanning confocal micrograph of substrate-attached part of cell, specimen stained for F-actin and rat IgG (inset) as an injection marker. Bar, 10 µm. (E) Evaluation of podosome formation in macrophages microinjected with GST-KIF1C-P polypeptide. Values are given as mean percentage ± SD of total counts. For each value, 3 x 30 cells were evaluated. Cells with podosomes: GST control (0.5 µg/µl), 73.3 ± 7.7%; GST-KIF1C-P (1 µg/µl), 34.4 ± 1.5%; GST-KIF1C-P (2 µg/µl), 30.0 ± 7.3%; GST-KIF1C-P (3 µg/µl), 17.8 ± 1.5%. For differences between control values and values gained with KIF1C-P injections, a p value < 0.03 was considered significant (indicated by asterisk).

Inhibition of Nonmuscle Myosin IIA Leads to Podosome Loss
This is further underscored by the fact that both nonmuscle myosin IIA and KIF1C-GFP localize preferentially to the periphery of the ventral cell side in macrophages (Figure 7A). Moreover, inhibition of nonmuscle myosin II (A and B) via the specific inhibitor blebbistatin (Limouze et al., 2004) resulted in a dose-dependent decrease of podosome-containing cells (Figure 7B). Interestingly, even in cells that still contain podosomes, podosome disruption could be observed in the cell periphery (Figure 7C), where myosin IIA is mostly concentrated (Figure 7A). In this context, it is noteworthy that 1) dynamic podosomes also localize to this region (Figure 1C) and 2) KIF1C-GFP–decorated plus ends preferentially contact podosomes in the cell periphery, whereas GFP-CLIP170–decorated microtubule plus ends contact podosomes irrespective of their subcellular localization (see Supplemental Figure S4). In sum, both KIF1C-decorated microtubule plus ends and myosin IIA preferentially localize to the region of dynamic podosomes, and inhibition of either motor protein disrupts podosome turnover.

View larger version (45K): [in this window] [in a new window]   Figure 7. Inhibition of nonmuscle myosin II leads to podosome loss. (A) Both myosin IIA and KIF1C-GFP localize preferentially to the cell periphery. Laser scanning confocal micrograph of substrate-attached part of a cell expressing KIF1C-GFP (green), specimen stained for myosin IIA (red), small black-and-white images show localization of KIF1C-GFP (top) or of myosin IIA (bottom). Bar, 10 µm. (B and C) Myosin II inhibitor blebbistatin impairs podosome formation. (B) Statistical evaluation of cells containing podosomes 30 min after addition of respective reagent. Values for podosome formation are given as mean percentage ± SD of total counts. For each bar, 3 x 30 cells were evaluated. Control (medium + 1% dimethyl sulfoxide), 80.0 ± 9.2%; blebbistatin, 10 µM, 65.6 ± 3.9%; 25 µM, 51.1 ± 7.8%, 30 µM, 31.1 ± 3.9%; 40 µM, 22.2 ± 5.7%. For differences between control values and values gained with blebbistatin, a p value < 0.03 was considered significant (indicated by asterisk). (C) Primary macrophage 30 min after addition of 30 µM blebbistatin. Confocal laser scanning micrograph of substrate-attached cell side, specimen stained for F-actin. Note loss of podosomes in the cell periphery. Bar, 10 µm. (D) Model of KIF1C-dependent podosome regulation. Individual podosomes are connected by actin cables. KIF1C at microtubule plus ends binds (directly or indirectly) to myosin IIA, thereby coupling the actin and tubulin cytoskeletons. This may enable an actomyosin/kinesin-based "homing mechanism" for microtubules to podosomes (a). Alternatively, KIF1C could bind podosome-localized myosin IIA, which may temporarily stabilize the contact between both structures (b). Both possibilities are not mutually exclusive.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our data now show that macrophage podosomes are targeted, sometimes repeatedly, by microtubule plus ends. Interestingly, contacted podosomes, and especially those located in the cell periphery, show a certain tendency toward dynamic behavior, i.e., fission or dissolution. This correlation is not very stringent, but together with previous data showing the overall importance of microtubules for human macrophage podosomes (Linder et al., 2000), it seems to imply that contact by microtubules may be necessary for podosome regulation, whereas not all contacts are equally productive. Indeed, experiments using nocodazole showed the absolute necessity of microtubules for podosome reformation after disruption, whereas in cells already containing podosomes, microtubules have only an impact of 30–40% on the upkeep of these structures. The latter observation may indeed reflect microtubule-dependent fission of podosome precursors leading to the formation of new podosomes.

Overall, these results are reminiscent of work on focal adhesions, which shows that microtubules repeatedly target these structures, thereby inducing their dissolution (Kaverina et al., 1999; Krylyshkina et al., 2003). In human macrophages, however, microtubule plus ends influence both dissolution and fission of podosomes, i.e., not only the breakdown but also the generation of podosomes. Ultimately, both events might be based on the same phenomenon, namely, the localized or overall dissolution of the podosome structure. For fission, for example, it could be argued that a dissolution process would have to affect only the connecting structure between future daughter podosomes. However, the factors inducing or even fine-tuning such disruption processes are unknown at the moment.

Surprisingly, and in contrast to previous results showing roughly equal rates of podosome fission and fusion in IC-21 mouse macrophages (Evans et al., 2003), we could only rarely detect fusion events (<1% of all observed podosomes) in our system. It is not clear why this difference occurs; possible explanations are the use of different species (mouse versus human), the use of a cell line versus primary cells, or the observation of migrating versus quiescent cells. Regardless, this demonstrates that podosome behavior is complex, and specifics may differ in various cell systems.

Contact with microtubules suggested the involvement of microtubule-based motor proteins in podosome regulation—a possibility unexplored so far. Initial experiments using microinjection of inhibitory antibodies or inhibitors against conventional kinesin or dynein showed no effect on podosome reformation. This is in clear contrast to the regulation of focal adhesions, where microtubule-dependent disassembly has been shown to involve conventional kinesin (Krylyshkina et al., 2002). However, we cannot rule out a possible long-term role for dynein in podosome dynamics.

A broader approach, combining overexpression of diverse kinesin constructs and siRNA transfection, led to the identification of KIF1C, a member of the Kinesin-3 family, as a motor protein potentially involved in podosome regulation. The involvement of other kinesins such as heterotrimeric kinesin in this process is possible and even likely. However, in all assays, interference with KIF1C expression or function gave the most consistent and significant results. This evaluation was further confirmed by time-lapse confocal microscopy, which revealed KIF1C accumulations being localized at microtubule plus ends and contacting regions of high podosome turnover. A comparison with CLIP170-labeled plus ends showed that KIF1C was only present at a subset of microtubules, and contact of podosomes with these plus ends was coupled with high percentages of podosomes showing fission or dissolution. Conversely, transfection of KIF1C-specific shRNA or expression of a mutant carrying a point mutation in the P-loop, which has been shown to inactive ATPase activity (Honegger et al., 1987), led to decreased podosome dynamics and podosome deficiency. Together, these data argue for KIF1C being a microtubule plus end–localized regulator of podosome dynamics.

In addition to microtubule plus ends, KIF1C also localizes to a region surrounding the MTOC. Although endogenous KIF1C can be found at both locations, the KIF1C P-loop mutant localized preferentially around the MTOC, and this was accompanied by a drastic decrease in podosomes. Interestingly, a rigor mutant of the related KIF1B shows a similar localization (Honegger et al., 1987). Rigor mutations may therefore lead to nonprocessive motors that are still able to bind microtubules/minus ends but can no longer move along the filaments to reach the plus ends. In this context, it is also noteworthy that overexpressed KIF1C has been shown to form heterodimers with the endogenous motor (Dorner et al., 1999). In consequence, functional defects in overexpressed forms of KIF1C, such as the P-loop mutant we used here, probably lead to functional impairment of the endogenous form, resulting in defective regulation of podosomes.

In theory, also KIF1C unassociated to microtubules may have cellular effects. However, at least in the confocal plane of podosomes, KIF1C was almost exclusively associated with microtubules or their plus ends. Moreover, expression of the P-loop mutant, which is only defective in ATP hydrolysis, also results in podosome disruption. Therefore, the movement of KIF1C along microtubules (toward their plus ends) seems to be essential for KIF1C to exert its effect on podosomes.

The search for a potential interaction partner of KIF1C in macrophages led to the identification of nonmuscle myosin IIA. Functional coupling of kinesin and myosin is needed for the coordination of cellular processes such as vesicle transport, for example through binding of both motors to the same cargo vesicle (Schliwa and Woehlke, 2003). However, kinesins and myosins can also interact directly, as shown for KhcU and MyoVa (Huang et al., 1999). Here, we show that KIF1C can interact with nonmuscle myosin IIA through its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons. The possible connection between both motors is underscored by the fact that microinjection of the myosin IIA binding domain of KIF1C, thought to compete with the interaction of both proteins, led to a dose-dependent decrease of podosomes.

Interestingly, myosin IIA is enriched in the cell periphery, where also dynamic podosomes localize, and inhibition of myosin II by blebbistatin leads to loss of podosomes preferentially in this region. Actomyosin-generated force has been shown to regulate the formation and size of focal adhesions, and it has been suggested that microtubules might trigger the dissolution of focal adhesions through the local inhibition of myosin II-dependent contractility (Kaverina et al., 2002; Bershadsky et al., 2003). A similar mechanism could be envisaged for podosomes. Alternatively, myosin IIA could also act as a bridging molecule between podosomes and microtubules. Indeed, myosins have been shown to be involved in binding or capturing microtubule plus ends and targeting them to the cell cortex (reviewed in Gundersen et al., 2004).

Considering previous findings of 1) podosomes being interconnected by a fine meshwork of actin filaments (Gavazzi et al., 1989), 2) microtubule growth sometimes occurring along actin bundles (Salmon et al., 2002), and 3) microtubules targeting focal adhesions along established "tracks" (Krylyshkina et al., 2003), one can speculate that KIF1C binding to myosin IIA could couple microtubule plus ends to podosome-connecting actin cables, thus establishing a "homing mechanism" for microtubules to efficiently target podosomes (Figure 7D). A similar model has been proposed for microtubule targeting of focal adhesions (reviewed in Small et al., 2002; Rodriguez et al., 2003). Alternatively, podosome-localized myosin IIA may enable a stabilized, productive contact by microtubules without being directly involved in microtubule targeting per se (Figure 7D). Of course, both alternatives would not be mutually exclusive.

In sum, our data demonstrate that the kinesin KIF1C is a central player in the microtubule-dependent regulation of podosomes and that the KIF1C–myosin IIA interface may play a role in facilitating podosome dynamics in a subcellularly fine-tuned manner. KIF1C is the first kinesin protein to be implicated in podosome regulation, and conversely, regulation of podosome dynamics is the first function of KIF1C described in human cells. It will be interesting to determine whether KIF1C simply acts as a "tether" for microtubule plus ends or whether it also transports regulatory factors to podosomes. Indeed, it is very likely that further molecules other than KIF1C or myosin IIA are involved in (functionally or physically) linking podosomes and microtubule plus ends. The identification of these factors and of the regulatory signals supposedly being delivered by microtubules are interesting challenges to be solved in future experiments.

	ACKNOWLEDGMENTS

 
We thank Tetsu Akiyama, Angela Barth, Niels Galjart, Vladimir Gelfand, Tohru Ichimura, Young Mi Lee, Bruce Schnapp, Kazuya Shimizu, and Yoshimi Takai for various constructs; the EURIT team for help with siRNA validation; Lars Israel and Tilman Schlunck for mass spectrometry analysis; Andreas Schröder for help with live cell imaging; Raphael Kland for help with video processing; Manfred Schliwa for critical reading of the manuscript; Jürgen Heesemann and Peter C. Weber for continuous support; and Barbara Böhlig for expert technical assistance. This work is part of the doctoral thesis of P. K. Work of our laboratories has been supported by grants to S. L. and M. A. from the Deutsche Forschungsgemeinschaft (SFB 413, GRK 438), by Friedrich Baur Stiftung, and by August Lenz Stiftung.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-11-1010) on March 22, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Stefan Linder ( stefan.linder{at}med.uni-muenchen.de)

Abbreviations used: AMP-PNP, adenyl-5'-yl imidodiphosphate; CLIP, cytoplasmic linker protein; EHNA, erythro-9-[3-(2-hydroxynonyl)]adenine; KAP, kinesin-associated protein; KIF, kinesin-like-family; KLC, kinesin light chain; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; mRFP, monomeric red fluorescent protein; MTOC, microtubule-organizing center; PBD, protein tyrosine phosphatase-binding domain; PTPD, protein tyrosine phosphatase; siRNA, small interfering RNA; shRNA, short hairpin RNA

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