The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages - PubMed (original) (raw)
The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages
Petra Kopp et al. Mol Biol Cell. 2006 Jun.
Abstract
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.
Figures
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-β-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 β-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–β-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.
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
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 β-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-β-tubulin antibody, respectively. (D and E) Primary macrophage stained with specific primary antibodies for KIF1C (D) and γ-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.
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-β-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.
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 × 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 × 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).
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 × 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).
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 × 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.
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