Major myeloid cell functions from adhesion to migration and phagocytosis are mediated by integrin adhesion complexes, also known as adhesome. The presence of a direct integrin binding partner Kindlin-3 is crucial for these functions, and its lack causes severe immunodeficiency in humans. However, how Kindlin-3 is incorporated into the adhesome and how its function is regulated is poorly understood. In this study, using nuclear magnetic resonance spectroscopy, we show that Kindlin-3 directly interacts with paxillin (PXN) and leupaxin (LPXN) via G43/L47 within its F0 domain. Surprisingly, disruption of Kindlin-3–PXN/LPXN interactions in Raw 264.7 macrophages promoted cell spreading and polarization, resulting in upregulation of both general cell motility and directed cell migration, which is in a drastic contrast to the consequences of Kindlin-3 knockout. Moreover, disruption of Kindlin-3–PXN/LPXN binding promoted the transition from mesenchymal to amoeboid mode of movement as well as augmented phagocytosis. Thus, these novel links between Kindlin-3 and key adhesome members PXN/LPXN limit myeloid cell motility and phagocytosis, thereby providing an important immune regulatory mechanism.

This article is featured in In This Issue, p.1699

Cells of myeloid origin including macrophages function in immune surveillance and defense. These functions are critical for a wide range of physiological and pathological processes, ranging from development, metabolism, tissue homeostasis, injury response, and regeneration to human pathologic conditions from cancer to neurodegenerative disorders (1). Their function is dependent on the timely recruitment from the blood stream, a process involving adhesion to endothelium and subsequent transmigration, followed by directed migration to the site of inflammation and engulfment (phagocytosis) of substances ranging from bacteria to necrotic cells (24). Irregularities in the execution of these defensive functions, including either insufficient or excessive performance, result in serious pathological complications arising from immune incompetence to diseases associated with chronic inflammation. All of the processes described above are mediated by integrins, which are cell surface receptors for the extracellular matrix (ECM). Although extracellular domains of integrins bind to their respective ligands either on the surface of bacteria or apoptotic cells or within an ECM, their cytoplasmic domains interact with a regulatory adhesome network of >200 molecules (5, 6). Among these, two direct binding partners of integrins, talin and kindlin, are absolutely required for their function (7). In myeloid cells of both resident (e.g., microglia) and nonresident (i.e., recruited from blood) origin, Kindlin-3 represents the main Kindlin paralog, and a lack of Kindlin-3 in humans leads to severe immunodeficiency, bone problems, and bleeding (8). Kindlin-3 null cells are not recruited to sites of inflammation because of deficiencies in integrin activation-dependent firm adhesion to endothelium, which together with a number of other defects in integrins, result in immune incompetence (8, 9). The defects in Kindlin-3 null cells are expected to prohibit cell migration, which is true for two-dimensional (2D) experiments and in vivo processes that are dependent on integrin activation, such as monocyte recruitment (10). However, myeloid cells exhibit two interchangeable migration modes, namely mesenchymal and amoeboid migration (3, 4). Whereas the mesenchymal migration is dependent on integrins and actin polymerization–driven sheet-like lamellipodia, integrin-independent amoeboid migration relies upon tissue pressure–dependent spherical “blebs” or actin-rich finger-like filopodia and is considered to be faster (1114). The lack of adhesion, increase in cortical contractility, and cell confinement promote the formation of cell blebs and mesenchymal to amoeboid transition (1518). Indeed, neither the lack of integrins, talin (19), nor Kindlin-3 (10) interferes with amoeboid migration in vivo. At the same time, the mechanisms underlying the transition between these two important migration modes are poorly understood.

All kindlins directly bind to membrane phosphatidylinositol-(4, 5)-bisphosphate via their pleckstrin homology (PH) domain (20, 21) and to the cytoplasmic domain of the β-subunit of integrins via their F3 domain (QW615) (10, 20, 22). Kindlin-2 was also shown to interact with integrin-linker kinase (ILK) via its F2PH domain (23) as well as with F-actin (24) and cytoskeleton protein paxillin (PXN) (25, 26) via its F0 domain (27). Importantly, these Kindlin-2 interactions promote integrin-dependent functions (e.g., cell spreading) (23, 26, 28). For instance, Kindlin-2–PXN interactions positively regulate fibroblast adhesion, spreading, the formation of focal adhesions, and cell migration (2527, 29). The same applies to the interactions between Kindlin-2 and ILK (23) as well as Kindlin-2 and actin (24). To date, no interactions negatively regulating or restricting integrin functions are known for kindlins. In addition, despite the importance of Kindlin-3 in myeloid and other hematopoietic cells, its exact role, as well as the mechanisms and consequences of its interactions with adhesome components, are poorly understood. Because these findings have mainly been based on domain deletion or knockout studies, which affect interactions with dozens of binding partners, they generate significant controversies (25, 26, 29). Accordingly, we have based our studies on the structural analysis of the binding interfaces between Kindlin-3 and PXN and its homolog leupaxin (LPXN) through the generation of point mutations and detailed analysis of integrin-mediated functions in myeloid cells. We show that in contrast to other known kindlins binding partners, interactions between Kindlin-3 and PXN/LPXN negatively regulate integrin-dependent functions of myeloid cells. Whereas Kindlin-3 knockout (K3KO) prevents integrin-mediated responses and Kindlin-3 binding to integrin cytoplasmic domain does not affect the Kindlin-3–PXN/LPXN interaction, disruption of Kindlin-3–PXN/LPXN affects integrin β1 activation. Kindlin-3 mutant lacking interactions with PXN/LPXN show enhanced cell spreading, membrane blebbing, migration, and macrophages phagocytosis. Moreover, the consequences of these interactions on integrin-mediated responses are opposite for Kindlin-3 and Kindlin-2 despite the conserved nature of the interaction site.

Human Kindlin-3 (hK3) open reading frame (ORF) from pGFP-hK3 (8) was subcloned into plvx-Ds-red-monomer-c1 (Takara Bio) and PCMV-HA-N (Takara Bio) vectors. PGFP-hK3 was changed from GFP to Flag tag. Human PXN (PXN-γ) ORF and human LPXN ORF (OriGene Technologies) were subcloned into Pmcv-N-myc vector (Takara Bio). The following constructs were used for protein expression in the bacterial system: hK3 F0 (1–105), human PXN (PXN-γ) LIM4 (541–605), and human LPXN LIM4 (324–386) subcloned into pGST-1 (GST) vectors. Kindlin-3 F0 double mutant (G43K, L47E) and Kindlin-3 integrin binding site mutant (K3KI) were generated by using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies), and Kindlin-3 F0 deletion (lack of 1–106) was generated by PCR.

Raw 264.7 cells, NIH3T3 cells, HEK 293 cells, and 293T phoenix cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies) and 50 μg/ml penicillin/streptomycin (Life Technologies). Thp1 cells, K562 cells, Jurkat cells, MPM cells, and L929 cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Life Technologies) and 50 μg/ml penicillin/streptomycin (Life Technologies). SIM-A9 cells were cultured in DMEM: F12 medium (Life Technologies) supplemented with 5% FBS (Life Technologies), 5% horse serum (Life Technologies), and 50 μg/ml penicillin/streptomycin (Life Technologies). Bone marrow–derived macrophages were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Life Technologies), 50 μg/ml penicillin/streptomycin (Life Technologies), and 30% L929 conditional medium.

GST-tagged protein (Kindlin-3 F0, PXN LIM4, or LPXN LIM4) was expressed in Escherichia coli BL21 (DE3) strain (New England BioLabs). Bacteria were initially grown in 50 ml lysogeny broth medium and then amplified in 1–2 L lysogeny broth at 37°C. The culture was induced by 0.4 mM isopropyl β-D-1-thiogalactopyranoside at room temperature (RT) overnight when it reached an OD600 of 0.7. The pellet was collected and suspended in buffer and then frozen at −80°C. For protein purification, the pellet was lysed, and the lysate was subjected to high-speed centrifugation. The supernatant was collected and incubated with Glutathione Sepharose 4B resin (GST beads) at 4°C for 2 h. GST-tagged protein was eluted by buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 10 mM glutathione. By performing the buffer exchange, the GST-tagged protein was finally stored in buffer that contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). For the protein used in nuclear magnetic resonance spectroscopy (NMR) experiments, the GST tag was removed by TEV protease. After incubation with GST beads, the untagged protein was collected and subjected to gel filtration by using Superdex 75 (16/60) (GE Healthcare), which was pre-equilibrated with the buffer containing 50 mM NaH2PO4/Na2HPO4 (pH 6.8), 50 mM NaCl, and 0.5 mM TCEP. 15N-labeled proteins used in NMR studies were achieved by growing bacteria in minimal medium with 15NH4Cl as the sole nitrogen source. For expression of the 15N-labeled PXN LIM4 or LPXN LIM4 domain, ZnCl2 was added into the minimal medium to reach a concentration of 50 μM during isopropyl β-D-1-thiogalactopyranoside induction. Protein concentration was measured by absorbance at 280 nm.

HEK293 cells were transfected with indicated plasmids by Lipofectamine 3000 (Thermo Fisher Scientific). Forty-eight hours posttransfection, cells were lysed with lysis buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 1% Nonidet P-40, 50 mM NaF, 1.5 mM Na3VO4, protease inhibitor mixture [Roche], 1 mM DTT, and 1 mM PMSF) and then incubated with 10 μg of rGST or GST-fusion proteins, which was preimmobilized to 20 μl of Glutathione Agarose (Pierce Biotechnology) at 4°C for 2 h. GST agaroses were then washed four times with the washing buffer (10 mM Tris [pH 7.5], 300 mM NaCl, 1% Nonidet P-40, and 50 mM NaF) and boiled in SDS sample buffer before analysis by Western blotting.

Western blotting was performed using a standard protocol. Briefly, cells were lysed with 1% SDS lysis buffer, and protein concentrations were determined using the BCA Assay Kit (Thermo Fisher Scientific). Protein samples were resolved on 10% SDS-PAGE (Bio-Rad Laboratories) and then transferred to PVDF membranes (MilliporeSigma). Membranes were blocked with 5% nonfat milk and then incubated sequentially with primary and secondary Abs. After washing with 1× TBST buffer, the membrane was detected by ECL Detection Reagent (Thermo Fisher Scientific). The following Abs were used for Western blotting: GAPDH (D16H11) Rabbit mAb (catalog no. 5174; Cell Signaling Technology), Myc-Tag (9B11) Mouse mAb (catalog no. 2276S; Cell Signaling Technology), HA Tag (C29F4) Rabbit Ab (catalog no. 3724S; Cell Signaling Technology), Flag M2 Ab anti-FLAG produced in rabbit (catalog no. F7425; Sigma), GST Tag Ab (8-326) (catalog no. MA4-004; Thermo Fisher Scientific), Rabbit anti-human PXN (H-114) (catalog no. sc-5574; Santa Cruz Biotechnology), Rabbit Kindlin-3 Ab (catalog no. ab68040; Abcam), Mouse Anti-Kindlin-2 (clone 3A3) (catalog no. MAB2617; MilliporeSigma), and Mouse Anti-Hic5 (Clone 34) Ab (catalog no. 611164; BD Biosciences). The secondary Abs for Western blotting were purchased from Cell Signaling Technology.

Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized by reverse transcription using PrimeScrip RT Master Mix (Takara Bio) and subjected to RT-PCR with SYBR Green (Bio-Rad Laboratories) and gene-specific primers. Relative mRNA levels were calculated by normalization to hypoxanthine phosphoribosyltransferase 1 mRNA using the ΔΔCt method.

2D heteronuclear single quantum coherence (HSQC) experiments were performed on a Bruker 600 MHz NMR spectrometer. Samples containing 100 μM 15N-labeled PXN LIM4 or LPXN LIM4 in the absence or presence of Kindlin-3 F0 were studied. Experiments were performed at 25°C in buffer containing 50 mM NaH2PO4/Na2HPO4 (pH 6.8), 50 mM NaCl, 0.5 mM TCEP, and 5% D2O. For HSQC titration, 100 μM 15N-labeled LIM4 PXN LIM4 or LPXN LIM4 was mixed with increasing concentrations of Kindlin-3 F0 to collect 2D-HSQC spectra. The chemical shift changes of six independent, well-resolved residues were fit to obtain the Kd by using an online analysis tool (http://supramolecular.org/) (30). The chemical shift change (Δδobs [HN, N]) of each residue was calculated with the equation Δδobs [HN,N] = [(ΔδHNWHN)2 + (ΔδNWN)2)]1/2, where Δδ (ppm) = δbound – δfree, and WHN and WN are weighting factors (WHN = 1, WN = 0.154).

For cell sorting of the similar level of Ds-red–positive cells, cells were suspended into sorting buffer that contained 25 mM HEPES (pH 7.0), 1× PBS (Ca2+/Mg2+ free), 1 mM EDTA, and 1% FBS (heat-inactivated) and then sorted through a BD flow cytometer using the same gate.

For Ds-red signal detection, cells were suspended into 5% FBS/PBS. Data were then collected in a BD Biosciences flow cytometer and analyzed by Flow Jo software.

For apoptosis analysis, the indicated Raw 264.7 cells were cultured in the tissue culture plates for 24 h, and control cells were treated with 10 μM camptothecin (Sigma) at 37°C for 1 h to induce apoptosis as the positive control. Then, cells were harvested and stained with the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). The data were collected by a BD flow cytometer and analyzed with Flow Jo software.

To detect C5a receptor (C5aR) surface expression, integrin expression, and activation, the indicated Raw 264.7 cells were cultured in the tissue culture plates for 24 h. Cells were then harvested and stained with APC anti-mouse CD88 (C5aR) Ab (catalog no. 135807; BioLegend), APC Rat IgG2b, κ isotype control Ab (catalog no. 400611; BioLegend), Alexa Fluor 647 Rat Anti-CD11b (αM) (catalog no. 557688; BD Biosciences), and FITC-CD18 (catalog no. 4336690; eBioscience), respectively at 4°C for 30 min and then washed and analyzed by flow cytometry. For integrin β1, cells were harvested and stained with primary Abs rat anti-mouse CD29 (9EG7) (catalog no. 553157; BD Biosciences) and Hamster Anti-Rat CD29 (catalog no. 553837; BD Biosciences) at 4°C for 1 h and then fixed with 2% paraformaldehyde at RT for 15 min, washed, and stained with secondary Abs Alexa Fluor 488 goat anti-rat (catalog no. A11006; Thermo Fisher Scientific) and Alexa Fluor 647 AffiniPure Goat Anti-Syrian Hamster IgG (H+L) (catalog no. 107-606-142; The Jackson Laboratory) at 4°C for 30 min and then washed. Data were collected by BD Biosciences flow cytometry and analyzed with Flow Jo software.

K3KO-expressed Ds-red–hK3 Raw 264.7 cells were labeled with PKH67 (Sigma-Aldrich) and Ds-red–hK3pxn cells were labeled with PKH26 (Sigma-Aldrich) according to the manufacturer’s instructions and then mixed at a 1:1 ratio. Fibrin gel was made by labeled cells, 2 mg/ml fibrinogen (Sigma-Aldrich), 1% FBS, 1% penicillin/streptomycin, and 0.5 U/ml thrombin (Sigma-Aldrich), formed at 37°C for 1 h, and then analyzed using a Leica confocal microscope.

For cell spreading and cell protrusions analysis, Raw 264.7 cells were seeded on fibronectin and 0.1% Poly-l-Lysine Solution (sc-286689; Santa Cruz Biotechnology) precoated coverslips for 1 h and treated with or without 20 ng/ml anaphylatoxin C5a (2150-C5-025; R&D Systems) for 2 h. Then, cells were washed with cold PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked in 3% BSA/10% goat serum/PBS for 30 min, and incubated with anti-PXN Ab (catalog no. sc-5574; Santa Cruz Biotechnology) and Alexa Fluor 647 Phalloidin (catalog no. A22287; Thermo Fisher Scientific) in blocking buffer overnight at 4°C. After washing with PBS, cells were incubated with Goat Anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Ab Alexa Fluor 488 (catalog no. A-11008; Thermo Fisher Scientific) for 1.5 h and then washed and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were collected using a Leica confocal microscope and quantified by ImageJ software. Both smaller and bigger protrusions were counted in all the cells.

For cell blebs analysis, Raw 264.7 cells were labeled with PKH67 (Sigma-Aldrich) according to the manufacturer’s instructions, seeded on the coverslip for 20 min, washed with cold PBS, fixed, and proceeded with general immunofluorescence staining and pictured by Leica confocal microscope.

For cell blebs, cells in media (2D) or in fibrin gels (three-dimensional [3D]) were seeded onto 3.5-μm glass-bottom dishes (catalog no. P35G-1.5-14-C; MatTek) and recorded by differential interference contrast (DIC) and z-stack with a 40× dry objective using the Leica TCS-SP8-AOBS inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) configured with HC-PL FLUOTARL 40×/0.75 dry object and OKO-TOUCH Stage Top Incubator (Okolab, San Bruno, CA). Image analysis was performed using Leica Application Suite X (LAS X) software. The protrusions ratio and the relative lifetime of protrusions were quantified from frame to frame of the videos.

For cell migration, cells were cultured in fibronectin-precoated 12-well plates (Corning). Time-lapse recordings were acquired with Leica DMI6000 inverted microscope and LAS X software (Leica Microsystems) equipped with HC-PL FLUOTARL 20×/0.4 dry object, a Hamamatsu Image EM-CCD camera, and a Hamamatsu Flash4 camera (Hamamatsu Photonics, Shizuoka, Japan), PECON Large Chamber Incubator, heating unit 2000, CO2 controller, and TempControl 37-2 Digital two channel (PeCon) every 5 min for 24 h, and then images were transformed into videos by LAS X software, extracted every five pictures with 25-min intervals, and quantified by Image Pro Plus software. All the cells were counted without distinguishing the morphology. Cell trajectories were drawn by chemotaxis and migration software.

Raw 264.7 cells were serum starved for 12 h. Cells (1 × 105) were seeded into the 6.5-μm pore size inserts and induced by serum and 20 ng/ml C5a for 24 h. Cells were then fixed and stained with 0.5% crystal violet. The bottom sides of the inserts were imaged and quantified by ImageJ software.

Latex beads (catalog no. L4530; Sigma-Aldrich) were opsonized with C3bi by incubating with human IgM (Sigma-Aldrich) and fresh mouse serum at 37°C for 1 h. Raw 264.7 cells were incubated with C3bi-opsonized latex beads with or without 150 nM PMA at 37°C for 2 h and then fixed, permeabilized, stained with anti-RFP Ab (catalog no. ab62341; Abcam), and imaged by confocal microscopy using z-stack. The number of beads phagocytized by Raw 264.7 cells was quantified by ImageJ software.

CRISPR–Cas9 technology was used to generate K3KO cells. Two independent single guide RNAs were designed by the Zhang laboratory CRISPR Design Tool. Annealed oligonucleotides were ligated into the vector LentiCRISPRv2 (Feng Zhang’s laboratory; Addgene) and digested with BsmBI (Fermentas). Lentiviral infection was performed with Lenti-X Packaging Single Shots (Takara Bio) in accordance with a Cleveland Clinic Institutional Biosafety Committee protocol. After 72 h, cells were selected with 2 μg/ml puromycin (Thermo Fisher Scientific) for another 48 h. Live cells were collected and sorted by flow cytometry into 96-well plates. Individual clones were examined by Western blotting or genomic DNA sequencing. The single guide RNA primers sequence for K3KO are as follows: K3KO#1, forward: 5′-CACCGACGGGGGAGTCGCACATTGG-3′; K3KO#1, reverse: 5′-AAACCCAATGTGCGACTCCCCCGTC-3′; K3KO#2, forward: 5′-CACCGACAGACGTGTGCTGCGGCTT-3′; and K3KO#2, reverse: 5′-AAACAAGCCGCAGC ACACGTCTGTC-3′.

The lentivirus infection was performed in accordance with the Cleveland Clinic Institutional Biosafety Committee protocol. For overexpression, Phoenix packaging cells (Commercial) were transfected with plvx-Ds-red-monomer-c1, plvx-Ds-red-monomer-hK3, and plvx-Ds-red-monomer–hK3–G43K/L47E by lipofectamine 3000 (Thermo Fisher Scientific) for 48 h. Then, Raw 264.7 cells were infected by lentivirus from the culture medium with Lenti-X Packaging Single Shots (Takara Bio) according to commercial instructions. Seventy-two hours later, cells were subjected to cell sorting for Ds-red–positive cells (BD Biosciences FACSAria II; Cleveland Clinic), followed by Western blotting and functional experiments.

Data are expressed as mean + SEM. Differences were analyzed using Student t tests. Values <0.05 were considered significant: *p < 0.05, **p < 0.005, and ***p < 0.001.

The kindlin family includes Kindlin-1, Kindlin-2, and Kindlin-3, which contain a conserved F0 domain at the N terminus, an F1-F2 domain, a conserved PH domain, and an F2-F3 domain (Supplemental Fig. 1A) (31, 32). Kindlin-3 is the only kindlin that is expressed in myeloid cells, including Raw 264.7 macrophage cells (Supplemental Fig. 1B). The PXN family includes PXN, Hic5, and LPXN, with a leucine-rich LD protein–protein interaction motif at the N terminus and four LIM domains at the C terminus (Supplemental Fig. 1A) (3335). Two members of the PXN family are present in Raw 264.7 cells: PXN and LPXN (Supplemental Fig. 1B, 1C). 2D NMR HSQC revealed that the interaction between Kindlin-2 and PXN is mediated by the Kindlin-2 F0 domain and the PXN LIM4 domain (27). Disruption of Kindlin-2–PXN interaction by the key amino acids G42/L46 in F0 domain or F577/L579/K580 mutations in LIM4 domain severely impair focal adhesion formation and directed cell migration (27). Because G43/L47 are conserved in both Kindlin-2 and Kindlin-3 (Supplemental Fig. 1D), we tested whether Kindlin-3 binds to PXN/LPXN in a similar fashion. First, both GST pull-down and 2D NMR HSQC confirmed the Kindlin-3–F0 domain interaction with both PXN LIM4 and LPXN LIM4 (Fig. 1A–C). Interestingly, based on HSQC titration, the binding affinity of Kindlin-3 to PXN LIM4 domain is nearly two times stronger than that of Kindlin-3 to LPXN LIM4 (Fig. 1D, 1E), which is consistent with the results of the GST pull-down assay (Fig. 1C). Further, Kindlin-3–F0 deletion disrupted Kindlin-3–PXN interaction, suggesting only the F0 domain but no other domains mediates Kindlin-3–PXN interaction (Supplemental Fig. 1E). To specifically disrupt this interaction, we mutated the conserved interface residues G43 and L47 of Kindlin-3 (corresponding to G42 and L46 of Kindlin-2) (Supplemental Fig. 1D) into lysine and glutamic acid, respectively. Both GST pull-down and 2D NMR HSQC showed that Kindlin-3–F0-G43K/L47E mutations are able to disrupt its binding to both PXN and LPXN LIM4 domains (Fig. 1F–H, Supplemental Fig. 1F). Similar to Kindlin-2, GST pull-down shows that F577/L579/K580 mutations in PXN LIM4 domain also disrupted Kindlin-3–PXN interaction (Supplemental Fig. 1E). Because NMR is used to assess weak protein–protein interactions (36), we were unable to verify by full-length coimmunoprecipitation. Instead, GST pull-down showed that full-length Flag–Kindlin-3, but not Flag–Kindlin–3-F0-G43K/L47E mutations, binds to PXN and LPXN LIM4 domain (Fig. 1I). Together, these results show that Kindlin-3 interacts with PXN/LPXN interaction via the F0 and LIM4 domains as well as the conserved residues within the F0 domain. Interestingly, disruption of the integrin binding site (Kindlin-3 mutant, K3KI) (10) did not affect Kindlin-3–PXN/LPXN interaction (Fig. 1J, 1K), suggesting that Kindlin-3–PXN/LPXN interactions are integrin independent.

FIGURE 1.

Kindlin-3 F0 interacts with both LIM4 domains of PXN and LPXN. (A) NMR 2D-HSQC shows that the PXN LIM4 domain interacts with the Kindlin-3 F0 domain. The HSQC spectra of 100 μM 15N-labeled PXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0 (red) are shown. (B) NMR 2D-HSQC shows that the LPXN LIM4 domain interacts with the Kindlin-3 F0 domain. The HSQC spectra of 100 μM 15N-labeled LPXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0 (red) are shown. (C) GST pull-down demonstrates the interaction between flag–Kindlin-3 expressed in HEK293 cells with both GST–PXN and GST–LPXN. (D) The affinity of Kindlin-3 F0 binding to the PXN LIM4 domain (Kd: 117.2 μM) measured by HSQC titration. Titration curves of four representative residues are shown. (E) The affinity of Kindlin-3 F0 binding to the LPXN LIM4 domain (Kd: 192.3 μM) measured by HSQC titration. Titration curves of four representative resonance peaks are shown. (F) The HSQC spectra of 100 μM 15N-labeled PXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0-G43K/L47E mutant (red) suggest that G43K/L47E mutations block Kindlin-3 F0 binding to PXN LIM4. (G) The HSQC spectra of 100 μM 15N-labeled LPXN LIM4 domain in the absence (black) and presence of 300 μM Kindlin-3 F0-G43K/L47E mutant (red) suggest that G43K/L47E mutations block Kindlin-3 F0 binding to LPXN–LIM4. Values = mean + SD. (H) GST pull-down assay demonstrates that Myc-PXN expressed in HEK293 cells interacts with GST–Kindlin-3–F0 by conserved Kindlin-3–F0-G43/L47. (I) GST pull-down assay demonstrates that Flag–Kindlin-3, but not Flag–hK3pxn expressed in HEK293 cells, interacts with GST-PXN-LIM4 and GST-LPXN-LIM4. (J) GST pull-down assay demonstrates interactions between both HA–Kindlin-3 and HA–K3KI (Kindlin-3 mutant that does not bind to integrin) with PXN/LPXN LIM4 domain. (K) Quantification of Western blotting shows similar binding of HA–Kindlin-3 and HA–K3KI with PXN/LPXN LIM4 (n = 2). FL, full length.

FIGURE 1.

Kindlin-3 F0 interacts with both LIM4 domains of PXN and LPXN. (A) NMR 2D-HSQC shows that the PXN LIM4 domain interacts with the Kindlin-3 F0 domain. The HSQC spectra of 100 μM 15N-labeled PXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0 (red) are shown. (B) NMR 2D-HSQC shows that the LPXN LIM4 domain interacts with the Kindlin-3 F0 domain. The HSQC spectra of 100 μM 15N-labeled LPXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0 (red) are shown. (C) GST pull-down demonstrates the interaction between flag–Kindlin-3 expressed in HEK293 cells with both GST–PXN and GST–LPXN. (D) The affinity of Kindlin-3 F0 binding to the PXN LIM4 domain (Kd: 117.2 μM) measured by HSQC titration. Titration curves of four representative residues are shown. (E) The affinity of Kindlin-3 F0 binding to the LPXN LIM4 domain (Kd: 192.3 μM) measured by HSQC titration. Titration curves of four representative resonance peaks are shown. (F) The HSQC spectra of 100 μM 15N-labeled PXN LIM4 in the absence (black) and presence of 300 μM Kindlin-3 F0-G43K/L47E mutant (red) suggest that G43K/L47E mutations block Kindlin-3 F0 binding to PXN LIM4. (G) The HSQC spectra of 100 μM 15N-labeled LPXN LIM4 domain in the absence (black) and presence of 300 μM Kindlin-3 F0-G43K/L47E mutant (red) suggest that G43K/L47E mutations block Kindlin-3 F0 binding to LPXN–LIM4. Values = mean + SD. (H) GST pull-down assay demonstrates that Myc-PXN expressed in HEK293 cells interacts with GST–Kindlin-3–F0 by conserved Kindlin-3–F0-G43/L47. (I) GST pull-down assay demonstrates that Flag–Kindlin-3, but not Flag–hK3pxn expressed in HEK293 cells, interacts with GST-PXN-LIM4 and GST-LPXN-LIM4. (J) GST pull-down assay demonstrates interactions between both HA–Kindlin-3 and HA–K3KI (Kindlin-3 mutant that does not bind to integrin) with PXN/LPXN LIM4 domain. (K) Quantification of Western blotting shows similar binding of HA–Kindlin-3 and HA–K3KI with PXN/LPXN LIM4 (n = 2). FL, full length.

Close modal

To understand the role of Kindlin-3–PXN/LPXN interactions in myeloid cells, we generated two independent clones of CRISPR–cas9–based K3KO in Raw 264.7 cells, K3KO#1 and K3KO#2 (Fig. 2A, Supplemental Fig. 2A, 2B), and then re-expressed hK3 and Kindlin-3–G43K/L47E (hK3pxn) mutant in these clones independently by lentiviral infection (a schematic is presented in Fig. 2A). Equal levels of protein re-expression were confirmed by flow cytometry (note that in all experiments, Kindlin-3 was re-expressed as a DS-red fusion protein) (Fig. 2B) and further verified by Western blotting (Fig. 2C). As expected, knockout of K3KO completely prevented cell adhesion, subsequent spreading, cell polarity, and cell protrusions both in the presence and absence of an agonist C5a (Fig. 2D–G). Re-expression of Kindlin-3 (hK3) in K3KO cells restored cell spreading and actin reorganization as shown by phalloidin staining (Fig. 2H–M). In comparison with hK3, expression of hK3pxn mutant augmented cell spreading (Fig. 2H, 2J). The area of hK3pxn-expressing cells was ∼30% higher (Fig. 2J), and the cell polarity index was >65% higher compared with hK3-expressing cells (Fig. 2I, 2K). Upon stimulation with C5a, the situation was similar, and hK3pxn cells exhibited augmented spreading compared with hK3 cells (Fig. 2H, 2J). Activation with C5a resulted in increased cell polarity by >2-fold in hK3 and >1.5-fold in hK3pxn, and the difference between these cell types was no longer apparent (Fig. 2K). Importantly, the number of cellular protrusions in hK3pxn-mutant cells was >4-fold higher than in hK3 cells even in the absence of cell stimulation with agonist (Fig. 2I, quantification shown in Fig. 2L). Moreover, although C5a treatment promoted protrusions in hK3, their numbers did not reach the level observed upon expression of hK3pxn mutant, which was >85% higher compared with hK3-expressing cells (Fig. 2I, 2L). Whereas there was no difference in the length of protrusions in native conditions, the length of protrusions increased after C5a treatment in hK3pxn mutant cells compared with hK3 cells (Fig. 2M).

FIGURE 2.

Disruption of Kindlin-3–PXN/LPXN interactions promote cell spreading and cell polarity and an increase in the number of cell protrusions. (A) A representative schematic of the generating process of K3KO and their rescued cells. K3KO cells were generated by CRISPR–cas9 and then re-expressed plasmids (Plvx–Ds-red–mono vector), hK3 (Plvx–Ds-red–mono-hK3), or hK3 (hK3pxn) (Plvx–Ds-red–mono-hK3-G43K/L47E), respectively, by lentiviral infection. (B) Flow cytometry analysis demonstrates equal levels of hK3 and hK3pxn expression in K3KO cell lines based on the Ds-red signal. K3KO cells with hK3 and hK3pxn were analyzed by flow cytometry. Wild-type cells were used as mock (negative control). (C) Western blot showing equal levels of hK3 and hK3pxn expression in K3KO Raw264.7 cells. The protein levels of Kindlin-3 were detected using an Ab for mouse Kindlin-3 (mK3) and hK3 (Ds-red–hK3), and GAPDH was used as a loading control. Both mK3 and Ds-red–hK3 fusion are indicated. (D) Confocal images of control cells and K3KO cells seeded on fibronectin-precoated coverslips for 2 h with or without 20 ng/ml C5a treatment. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. Scale bar, 20 μm. (E and F) Quantification of cell area and cell polarity in control and K3KO cells. Control (without C5a, n = 35, and with C5a, n = 34) and K3KO (without C5a, n = 34, and with C5a n = 27). Values = means + SEM. *p < 0.05, ***p < 0.001 based on Student t test. (G) Quantification of protrusions from (D). Control (without C5a, n = 35, and with C5a, n = 25) and K3KO (without C5a, n = 35, and with C5a, n = 36). Values = means + SEM. ***p < 0.001 based on Student t test. (H) Confocal images of K3KO cells expressing wild-type (WT) Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn) seeded on fibronectin-precoated coverslips for 2 h. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. Ds-red indicates the presence of hK3 or hK3pxn. Scale bar, 20 μm. (I) Confocal images showing actin-rich protrusions of hK3 or hK3pxn cells seeded on fibronectin for 2 h with or without 20 ng/ml C5a. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. White arrows indicate cell protrusions. Scale bar, 10 μm. (J) Quantification of cell area in K3KO cells expressing either WT Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn). K3KO with hK3 (without C5a, n = 55, and with C5a, n = 88) and re-hK3pxn mutant (without C5a, n = 78, and with C5a, n = 113). Values = means + SEM. **p < 0.005, ***p < 0.001 based on Student t test. (K) Quantification of cell area in K3KO cells expressing either WT Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn). K3KO with hK3 (without C5a, n = 69, and with C5a, n = 85) and with hK3pxn (without C5a, n = 86, and with C5a, n = 96). Values = means + SEM. ***p < 0.001 based on Student t test. (L) Quantification of the number of protrusions from (I). K3KO with hK3 (without C5a, n = 38, with C5a, n = 35) and with hK3pxn (without C5a, n = 38, and with C5a, n = 35). Values = means + SEM. **p < 0.005, ***p < 0.001 based on Student t test. (M) Quantification of the length of protrusions from (I). K3KO with hK3 (without C5a, n = 132, and with C5a, n = 354) and with hK3pxn (without C5a, n = 509, and with C5a, n = 648). Values = means + SEM. *p < 0. 05, **p < 0.005, ***p < 0.001 based on Student t test.

FIGURE 2.

Disruption of Kindlin-3–PXN/LPXN interactions promote cell spreading and cell polarity and an increase in the number of cell protrusions. (A) A representative schematic of the generating process of K3KO and their rescued cells. K3KO cells were generated by CRISPR–cas9 and then re-expressed plasmids (Plvx–Ds-red–mono vector), hK3 (Plvx–Ds-red–mono-hK3), or hK3 (hK3pxn) (Plvx–Ds-red–mono-hK3-G43K/L47E), respectively, by lentiviral infection. (B) Flow cytometry analysis demonstrates equal levels of hK3 and hK3pxn expression in K3KO cell lines based on the Ds-red signal. K3KO cells with hK3 and hK3pxn were analyzed by flow cytometry. Wild-type cells were used as mock (negative control). (C) Western blot showing equal levels of hK3 and hK3pxn expression in K3KO Raw264.7 cells. The protein levels of Kindlin-3 were detected using an Ab for mouse Kindlin-3 (mK3) and hK3 (Ds-red–hK3), and GAPDH was used as a loading control. Both mK3 and Ds-red–hK3 fusion are indicated. (D) Confocal images of control cells and K3KO cells seeded on fibronectin-precoated coverslips for 2 h with or without 20 ng/ml C5a treatment. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. Scale bar, 20 μm. (E and F) Quantification of cell area and cell polarity in control and K3KO cells. Control (without C5a, n = 35, and with C5a, n = 34) and K3KO (without C5a, n = 34, and with C5a n = 27). Values = means + SEM. *p < 0.05, ***p < 0.001 based on Student t test. (G) Quantification of protrusions from (D). Control (without C5a, n = 35, and with C5a, n = 25) and K3KO (without C5a, n = 35, and with C5a, n = 36). Values = means + SEM. ***p < 0.001 based on Student t test. (H) Confocal images of K3KO cells expressing wild-type (WT) Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn) seeded on fibronectin-precoated coverslips for 2 h. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. Ds-red indicates the presence of hK3 or hK3pxn. Scale bar, 20 μm. (I) Confocal images showing actin-rich protrusions of hK3 or hK3pxn cells seeded on fibronectin for 2 h with or without 20 ng/ml C5a. Staining for PXN (green), F-actin (purple, Alexa Fluor 647–Phalloidin), and DAPI are shown. White arrows indicate cell protrusions. Scale bar, 10 μm. (J) Quantification of cell area in K3KO cells expressing either WT Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn). K3KO with hK3 (without C5a, n = 55, and with C5a, n = 88) and re-hK3pxn mutant (without C5a, n = 78, and with C5a, n = 113). Values = means + SEM. **p < 0.005, ***p < 0.001 based on Student t test. (K) Quantification of cell area in K3KO cells expressing either WT Kindlin-3 (hK3) or Kindlin-3–PXN/LPXN mutant (hK3pxn). K3KO with hK3 (without C5a, n = 69, and with C5a, n = 85) and with hK3pxn (without C5a, n = 86, and with C5a, n = 96). Values = means + SEM. ***p < 0.001 based on Student t test. (L) Quantification of the number of protrusions from (I). K3KO with hK3 (without C5a, n = 38, with C5a, n = 35) and with hK3pxn (without C5a, n = 38, and with C5a, n = 35). Values = means + SEM. **p < 0.005, ***p < 0.001 based on Student t test. (M) Quantification of the length of protrusions from (I). K3KO with hK3 (without C5a, n = 132, and with C5a, n = 354) and with hK3pxn (without C5a, n = 509, and with C5a, n = 648). Values = means + SEM. *p < 0. 05, **p < 0.005, ***p < 0.001 based on Student t test.

Close modal

Therefore, in contrast to Kindlin-2, disconnecting Kindlin-3 from PXN/LPXN promoted cell polarization, spreading, and the formation of actin-rich protrusions in a similar manner to C5a agonist stimulation.

At the same time, flow cytometry revealed no difference in C5aR surface expression between hK3 cells and hK3pxn cells (Supplemental Fig. 2C, 2D), suggesting that the increase in cell spreading, cell polarity, and cell protrusions in hK3pxn cells is not because of an altered expression level of C5aR. None of the used cell lines adhered to 0.1% poly-l-lysine (Supplemental Fig. 2E–H), thereby demonstrating that these effects are integrin dependent. Expression of αMβ2 (CD11b/CD18) complex and β1 integrins was generally similar between hK3 and hK3pxn cells (Supplemental Fig. 3A–D). Integrin β1 activation status measured by 9EG7 staining but not its expression was slightly decreased on hK3pxn cells compared with hK3 line (Supplemental Fig. 3E–H); whereas similar to previous reports, no effect of C5a stimulation on β1 integrin status was observed (37).

Kindlins interact with the cell membrane via their PH domain and with the cytoskeleton, F-actin, and PXN/LPXN via F0 domain, thereby linking the cell membrane with the cytoskeleton (21, 2427, 29). Disruptions of similar intracellular links lead to increased and imbalanced cortical contractility, which, in turn, promote cell blebbing and facilitate the transition to the amoeboid mode of migration (17, 38). Thus, Kindlin-3–PXN/LPXN interactions might serve as the main connection to the cytoskeleton, and its disruption might lead to increased blebbing and transition to amoeboid migration. Several models were used for this analysis: time-lapse live imaging of spontaneous migration on the adhesive substrate in 2D, analysis of blebbing during cell spreading by immunofluorescent staining, evaluation of blebbing and lamellipodia formation during initial and late phases of cell spreading by time-lapse live imaging, apoptosis analysis by flow cytometry, directed cell migration toward substrate, and finally, blebs and lamellipodia analysis by time-lapse live imaging under confinement conditions in 3D, known to promote blebbing and trigger amoeboid mode.

Kindlin-3 deficiency prevented spontaneous cell movement on the adhesive substrate in 2D as evidenced by time-lapse live imaging (Fig. 3A, 3B and Supplemental Videos 1, 2). The average cell velocity was reduced by 2-fold in K3KO cells (Fig. 3B), and K3KO cells were not moving along the substrate but merely exhibited the movements of the membrane (cell migration trajectories in Fig. 3A, Supplemental Video 2).

FIGURE 3.

Knockout of Kindlin-3 augments cell velocity and promotes the transition from mesenchymal migration to amoeboid migration. (A) Representative tracks of CRISPR–cas9 control cells (+Vector) and K3KO cells (+Vector) were recorded for 24 h by phase-contrast microscopy time-lapse imaging. The starting point is at the origin, and each point represents the position of a 25-min interval. Control with vector (Vec) (n = 67) and K3KO with Vec (n = 52). (B) Quantification of the average velocity of motile cells from (A). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Representative confocal images of control cells and K3KO cells seeded on fibronectin for 20 min. Membrane was labeled with PKH67 (green), and Alexa Fluor 647–Phalloidin (purple) indicates F-actin. White arrows indicate cell blebs. Scale bar, 20 μm. (D) Quantification of blebs numbers per cell. Control (n = 18), K3KO#1 (n = 18), and K3KO#2 (n = 32). Values = means + SEM. ***p < 0.001 based on Student t test. (E) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in control and K3KO Raw 264.7 cells at the initial adhesion stage (0–30 min after plating) in 2D culture. Images of the focal layer were extracted from z-stack. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (F) Ratios of various types of cell protrusions from (E) in three independent experiments. Control (n = 27), K3KO#1 (n = 37), and K3KO#2 (n = 26). (G) Bright-field time-lapse imaging of cell blebbing of control and K3KO cells at 16–18 h after plating. Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and lamellipodia/filopodia, respectively. Scale bar, 10 μm. (H) The ratio of cell protrusions from (G) in three independent experiments. Control (n = 23), K3KO#1 (n = 43), and K3KO#2 (n = 45). (I) Quantification of the relative lifetime of protrusions of control and K3KO cells at the firm (late) adhesion stage in 2D. The average interval of each video is 27 s. Control (Lamellipodia, n = 21; filopodia, n = 38; and blebs, n = 0), K3KO#1 (Lamellipodia, n = 0; filopodia, n = 121; and blebs, n = 33), and K3KO#2 (Lamellipodia, n = 0; filopodia, n = 69; and blebs, n = 68). Values = means + SEM. ***p < 0.001 based on Student t test.

FIGURE 3.

Knockout of Kindlin-3 augments cell velocity and promotes the transition from mesenchymal migration to amoeboid migration. (A) Representative tracks of CRISPR–cas9 control cells (+Vector) and K3KO cells (+Vector) were recorded for 24 h by phase-contrast microscopy time-lapse imaging. The starting point is at the origin, and each point represents the position of a 25-min interval. Control with vector (Vec) (n = 67) and K3KO with Vec (n = 52). (B) Quantification of the average velocity of motile cells from (A). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Representative confocal images of control cells and K3KO cells seeded on fibronectin for 20 min. Membrane was labeled with PKH67 (green), and Alexa Fluor 647–Phalloidin (purple) indicates F-actin. White arrows indicate cell blebs. Scale bar, 20 μm. (D) Quantification of blebs numbers per cell. Control (n = 18), K3KO#1 (n = 18), and K3KO#2 (n = 32). Values = means + SEM. ***p < 0.001 based on Student t test. (E) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in control and K3KO Raw 264.7 cells at the initial adhesion stage (0–30 min after plating) in 2D culture. Images of the focal layer were extracted from z-stack. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (F) Ratios of various types of cell protrusions from (E) in three independent experiments. Control (n = 27), K3KO#1 (n = 37), and K3KO#2 (n = 26). (G) Bright-field time-lapse imaging of cell blebbing of control and K3KO cells at 16–18 h after plating. Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and lamellipodia/filopodia, respectively. Scale bar, 10 μm. (H) The ratio of cell protrusions from (G) in three independent experiments. Control (n = 23), K3KO#1 (n = 43), and K3KO#2 (n = 45). (I) Quantification of the relative lifetime of protrusions of control and K3KO cells at the firm (late) adhesion stage in 2D. The average interval of each video is 27 s. Control (Lamellipodia, n = 21; filopodia, n = 38; and blebs, n = 0), K3KO#1 (Lamellipodia, n = 0; filopodia, n = 121; and blebs, n = 33), and K3KO#2 (Lamellipodia, n = 0; filopodia, n = 69; and blebs, n = 68). Values = means + SEM. ***p < 0.001 based on Student t test.

Close modal

In the absence of Kindlin-3, cells formed more actin-deficient blebs compared with control cells (Fig. 3C, 3D). DIC microscopy revealed that K3KO cells had more spherical blebs during the initial stages of cell adhesion as compared with control cells, which remained relatively smooth (Fig. 3C, 3D, Supplemental Videos 3, 4). Quantification of membrane movements revealed that although control cells primarily formed lamellipodia, both K3KO#1 and K3KO#2 lines exhibited extensive blebbing (Fig. 3D). In most cases, the phenotype of K3KO#2 cells was more apparent because of the specifics of Kindlin-3 excision (Supplemental Fig. 2A). The protrusions turned over quickly with a similar lifetime in control and K3KO cells (Supplemental Fig. 4A). At later stages, when cells firmly adhered to the substrate (14–18 h after seeding), control cells formed lamellipodia primarily in one direction, whereas K3KO cells continued producing numerous spherical blebs covering the nearly entire surface of the cell (Fig. 3G, 3H, Supplemental Videos 5, 6). Thus, whereas the lack of Kindlin-3 significantly impairs cell velocity on the substrate and directed migration (10), it induces the attributes of amoeboid migration mode, namely the switch from the directional lamellipodia to excessive membrane blebbing (16). Whereas control cells primarily formed lamellipodia and filopodia, K3KO cells exhibited blebbing (Fig. 3G, 3H). The protrusions in K3KO cells had a substantially shorter lifetime than in control cells while exhibiting no lamellipodia (Fig. 3I). The relative lifetime of filopodia decreased by >5-fold in K3KO compared with control cells (Fig. 3I). To verify that increased blebbing is not due to apoptosis, we stained these cells for Annexin V (Supplemental Fig. 3I, 3J). The rate of spontaneous apoptosis in K3KO lines was not higher compared with control cells. Moreover, re-expression of hK3 in K3KO cells did not affect the apoptosis rate, demonstrating that increased blebbing in K3KO cells is not due to apoptosis (Supplemental Fig. 3I, 3J). Together, the lack of Kindlin-3 diminishes cell adhesiveness while increasing cell blebbing and promoting membrane protrusions turnover, which together are likely to aid fast amoeboid migration.

Re-expression of hK3 in K3KO cells reverted the membrane movement and migration velocity on the substrate (Fig. 4A, Supplemental Videos 7, 9, 11). In fact, the behavior of cells that re-expressed hK3 (Supplemental Videos 9, 11) was indistinguishable from that of control cells (Supplemental Videos 3, 5). Cells expressing hK3pxn mutant exhibited a unique set of characteristics, namely substantial spreading on the substrate similar to or greater than that of hK3 cells and, at the same time, a high level of membrane blebbing resembling K3KO cells (Supplemental Videos 10, 12). Importantly, the average velocity on the substrate of hK3pxn mutant–expressing cells was ∼1.5-fold higher compared with hK3 cells (Fig. 4A, 4B). Immunofluorescent staining showed that the number of actin-deficient membrane blebs increased in hK3pxn cells compared with hK3 cells by ∼3.5-fold on K3KO#1 background and by ∼9-fold on K3KO#2 background (Fig. 4C, 4D). DIC microscopy time-lapse imaging showed that although hK3 cells formed primarily lamellipodia (more than 50% of the cells), the proportion of lamellipodia was substantially lower (<30%) in hK3pxn mutant cells, and cell blebbing in hK3pxn mutant cells was respectively higher compared with hK3 cells (Fig. 4E, 4F, Supplemental Videos 9, 10). Similarly, the protrusions in both hK3 cells and hK3pxn cells turned over quickly with a similar lifetime during the initial spreading stage (Supplemental Fig. 4B). Consistently, during the firm adhesion stage when membrane blebbing has generally ceased and transitioned to lamellipodia/filopodia in hK3 cells, hK3pxn mutant cells continued with intense blebbing (∼37% blebs in K3KO#1 and ∼20% in K3KO#2) as compared with hK3-expressing cells at ∼4 and 15%, respectively (Fig. 4G, 4H, Supplemental Videos 11, 12). Moreover, certain hK3pxn mutant cells transitioned from filopodia formation to blebbing (Fig. 4G, 4H, Supplemental Video 12). The protrusions in hK3pxn cells turned over much faster than in hK3 cells (Fig. 4I). The relative lifetime of lamellipodia decreased by ∼20% and by ∼4.5-fold in hK3pxn mutant cells compared with hK3 on the background of K3KO#1 and K3KO#2, respectively (Fig. 4I). The relative lifetime of filopodia in hK3pxn versus hK3 cells decreased by 1.4- and 2-fold in K3KO#1 and K3KO#2 lines, respectively (Fig. 4I). Apoptosis analysis by Annexin V staining revealed no significant difference between hK3 and hK3pxn cells (Supplemental Fig. 3I, 3J), demonstrating that high membrane blebbing in hK3pxn cells is not due to apoptosis. Together, these results show that disruption of Kindlin-3–PXN/LPXN interactions augmented cell blebbing and protrusions turnover, indicating the shift toward amoeboid mode.

FIGURE 4.

Disruption of Kindlin-3–PXN/LPXN interactions augments random cell velocity and promotes the transition from mesenchymal migration to amoeboid migration. (A) Representative tracks of K3KO Raw 264.7 cells re-expressing Kindlin-3 wild-type (hK3) or Kindlin-3–PXN (hK3pxn) mutant within 24 h by phase-contrast microscopy time-lapse imaging. The starting point is at the origin, and each point represents the cell position at 25-min intervals. K3KO#1 re-expressed hK3 (n = 31) and hK3pxn (n = 64), and K3KO#2 re-expressed hK3 (n = 71) and hK3pxn (n = 57). (B) Bar graphs show the average velocity of cells from (A). 2.5 × SD was removed from consideration. Values = means + SEM. **p < 0.005 based on Student t test. (C) Representative confocal images of K3KO with hK3 and hK3pxn seeded on fibronectin for 20 min. Membrane was labeled with PKH67 (green), Alexa Fluor 647–Phalloidin (purple) indicates F-actin, and Ds-red indicates hK3 and hK3pxn expression. White arrows indicate cell blebs. Scale bar, 10 μm. (D) Quantification of blebs numbers per cell. K3KO#1 with hK3 (n = 75) and hK3pxn (n = 75) and K3KO#2 with hK3 (n = 46) and hK3pxn (n = 45) were analyzed. Values = means + SEM. ***p < 0.001 based on Student t test. (E) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in K3KO cells with hK3 or hK3pxn expression at the initial adhesion stage (0–30 min) in 2D in three independent experiments. The images were exacted from z-stacks. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (F) Characterization of cell protrusions and blebbing during adhesion. Bars show the ratios of various protrusions from (E) in three independent experiments. K3KO#1 with hK3 (n = 77) and hK3pxn (n = 74) and K3KO#2 with hK3 (n = 52) and hK3pxn (n = 53) were analyzed. (G) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in K3KO cells with hK3 or hK3pxn expression at the firm adhesion stage (16–18 h after seeding). Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and lamellipodia, respectively. (H) Characterization of cell protrusions and blebbing from Fig. 4G in three independent experiments. Ratios of various protrusions types are shown. K3KO#1 with hK3 (n = 45) and hK3pxn (n = 48) and K3KO#2 with hK3 (n = 59) and hK3pxn (n = 60) were analyzed. (I) Quantification of the relative lifetime of protrusions of hK3 and hK3pxn cells at the firm adhesion stage in Fig. 4G. The average interval of each video is 27 s. hK3 (K3KO#1) (Lamellipodia, n = 37; filopodia, n = 81; and blebs, n = 0) and hK3pxn (K3KO#1) (Lamellipodia, n = 25; filopodia, n = 125; and blebs, n = 52). hK3 (K3KO#2) (Lamellipodia, n = 0; filopodia, n = 17; and blebs, n = 78), and hK3pxn (K3KO#2) (Lamellipodia, n = 17; filopodia, n = 145; and blebs, n = 34). Values = means + SEM. Values = means + SEM. ***p < 0.001 based on Student t test.

FIGURE 4.

Disruption of Kindlin-3–PXN/LPXN interactions augments random cell velocity and promotes the transition from mesenchymal migration to amoeboid migration. (A) Representative tracks of K3KO Raw 264.7 cells re-expressing Kindlin-3 wild-type (hK3) or Kindlin-3–PXN (hK3pxn) mutant within 24 h by phase-contrast microscopy time-lapse imaging. The starting point is at the origin, and each point represents the cell position at 25-min intervals. K3KO#1 re-expressed hK3 (n = 31) and hK3pxn (n = 64), and K3KO#2 re-expressed hK3 (n = 71) and hK3pxn (n = 57). (B) Bar graphs show the average velocity of cells from (A). 2.5 × SD was removed from consideration. Values = means + SEM. **p < 0.005 based on Student t test. (C) Representative confocal images of K3KO with hK3 and hK3pxn seeded on fibronectin for 20 min. Membrane was labeled with PKH67 (green), Alexa Fluor 647–Phalloidin (purple) indicates F-actin, and Ds-red indicates hK3 and hK3pxn expression. White arrows indicate cell blebs. Scale bar, 10 μm. (D) Quantification of blebs numbers per cell. K3KO#1 with hK3 (n = 75) and hK3pxn (n = 75) and K3KO#2 with hK3 (n = 46) and hK3pxn (n = 45) were analyzed. Values = means + SEM. ***p < 0.001 based on Student t test. (E) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in K3KO cells with hK3 or hK3pxn expression at the initial adhesion stage (0–30 min) in 2D in three independent experiments. The images were exacted from z-stacks. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (F) Characterization of cell protrusions and blebbing during adhesion. Bars show the ratios of various protrusions from (E) in three independent experiments. K3KO#1 with hK3 (n = 77) and hK3pxn (n = 74) and K3KO#2 with hK3 (n = 52) and hK3pxn (n = 53) were analyzed. (G) DIC microscopy time-lapse imaging showing the dynamics of cell blebbing in K3KO cells with hK3 or hK3pxn expression at the firm adhesion stage (16–18 h after seeding). Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and lamellipodia, respectively. (H) Characterization of cell protrusions and blebbing from Fig. 4G in three independent experiments. Ratios of various protrusions types are shown. K3KO#1 with hK3 (n = 45) and hK3pxn (n = 48) and K3KO#2 with hK3 (n = 59) and hK3pxn (n = 60) were analyzed. (I) Quantification of the relative lifetime of protrusions of hK3 and hK3pxn cells at the firm adhesion stage in Fig. 4G. The average interval of each video is 27 s. hK3 (K3KO#1) (Lamellipodia, n = 37; filopodia, n = 81; and blebs, n = 0) and hK3pxn (K3KO#1) (Lamellipodia, n = 25; filopodia, n = 125; and blebs, n = 52). hK3 (K3KO#2) (Lamellipodia, n = 0; filopodia, n = 17; and blebs, n = 78), and hK3pxn (K3KO#2) (Lamellipodia, n = 17; filopodia, n = 145; and blebs, n = 34). Values = means + SEM. Values = means + SEM. ***p < 0.001 based on Student t test.

Close modal

Next, directed cell migration toward anaphylatoxin C5a, which is a ligand and agonist for myeloid cells (3941), was assessed in Boyden chambers. As shown previously, K3KO nearly completely abolished directed cell migration, whereas re-expression of hK3 completely restored this response (Fig. 5A, 5B). This is consistent with the concept that the expression of Kindlin-3 is critical for the function of integrins (8, 10). A direct comparison between migrating hK3 and hK3pxn cells is shown in Fig. 5A, and the quantification is in Fig. 5B. The number of hK3pxn mutant cells transmigrated toward C5a was ∼37% higher compared with hK3 cells (Fig. 5B), demonstrating that Kindlin-3–PXN/LPXN interactions limit directed macrophage migration.

FIGURE 5.

Disruption of Kindlin–PXN/LPXN interactions augments directed cell migration in 2D and promotes the transition from mesenchymal migration to amoeboid migration in 3D. (A) The K3KO cells re-expressed Kindlin-3–PXN/LPXN mutant cells (hK3pxn) migrated better than wild-type Kindlin-3 (hK3) in a transwell migration assay. Fibronectin was used as a ligand. (B) Graph shows the number of migration cells per field in three independent experiments, with triplicates in each experiment. Control (n = 15), K3KO (n = 15), hK3 (n = 15), and hK3pxn (n = 15). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Time-lapse imaging by confocal microscopy of K3KO cells with hK3 or hK3pxn expression in the 3D fibrin gel (0–1.5 h). Cells expressing hK3 were labeled with PKH67 membrane dye (green), and hK3pxn mutant were labeled with PKH26 membrane dye (red) and mixed together at 1:1 in 2 mg/ml 3D fibrin gels at 37°C for 30 min. After polymerization, cells in the gel (120–150 μm above the bottom) were imaged by confocal microscopy. Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (D) Bars show the quantification of cell protrusions from (C) in three independent experiments. K3KO cells with hK3 (n = 10) and hK3pxn (n = 12) were analyzed.

FIGURE 5.

Disruption of Kindlin–PXN/LPXN interactions augments directed cell migration in 2D and promotes the transition from mesenchymal migration to amoeboid migration in 3D. (A) The K3KO cells re-expressed Kindlin-3–PXN/LPXN mutant cells (hK3pxn) migrated better than wild-type Kindlin-3 (hK3) in a transwell migration assay. Fibronectin was used as a ligand. (B) Graph shows the number of migration cells per field in three independent experiments, with triplicates in each experiment. Control (n = 15), K3KO (n = 15), hK3 (n = 15), and hK3pxn (n = 15). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Time-lapse imaging by confocal microscopy of K3KO cells with hK3 or hK3pxn expression in the 3D fibrin gel (0–1.5 h). Cells expressing hK3 were labeled with PKH67 membrane dye (green), and hK3pxn mutant were labeled with PKH26 membrane dye (red) and mixed together at 1:1 in 2 mg/ml 3D fibrin gels at 37°C for 30 min. After polymerization, cells in the gel (120–150 μm above the bottom) were imaged by confocal microscopy. Images were extracted from z-stacks. White arrows and arrowheads indicate cell blebs and filopodia, respectively. Scale bar, 10 μm. (D) Bars show the quantification of cell protrusions from (C) in three independent experiments. K3KO cells with hK3 (n = 10) and hK3pxn (n = 12) were analyzed.

Close modal

Because amoeboid migration is generally restricted to 3D conditions, it is expected that the blebbing phenotype of mutant cells will be augmented in 3D, in which pressure from the substrate promotes amoeboid behavior (12, 42). Cells expressing hK3 and hK3pxn mutant were labeled with green and red dyes PKH67 and PKH26, respectively, and were imaged next to each other at different depths within a fibrin gel (Fig. 5C, 5D, Supplemental Video 13) without interference from the fluorescence of the fusion protein (Supplemental Fig. 4C, 4D). This analysis revealed a substantially higher frequency of blebbing in hK3pxn cells compared with hK3 cells (Fig. 5C, Supplemental Video 13). As seen in Supplemental Video 13, the entire surface of hK3pxn-expressing cells (red, on the top of the screen) was “bubbling” with large round blebs, whereas hK3 cells (green, bottom of the screen) primarily formed sharper lamellipodia-like structures, which were substantially smaller and restricted to ∼30% of the surface area of the cell. Quantification of blebbing in Fig. 5D revealed than whereas 30% of hK3 cells did not form any blebs or lamellipodia, all of the hK3pxn cells showed extensive blebbing. The protrusions turned over quickly, and there was no difference in the relative lifetime between hK3 cells and hK3pxn cells in 3D during the initial spreading stage (Supplemental Fig. 4E). Taken together, these results show that Kindlin-3–PXN/LPXN interactions restrict macrophage migration and membrane blebbing, thereby preventing cellular transition from a slower mesenchymal to a faster amoeboid mode.

Phagocytosis, which is the main function of macrophages, aims to remove cell debris and microbial pathogens (43). Phagocytosis of particles covered with C3bi complement component depends on the αMβ2 integrin and its Kindlin-3–dependent activation by an agonist (8). PMA stimulation leads to a 5.6-fold increase in the number of phagocytosed beads per control cell, demonstrating that the process is, indeed, entirely dependent on agonist stimulation (Fig. 6A, 6B). At the same time, heat inactivation of C3bi complement component on the beads completely abolished phagocytosis, showing the specificity of this assay (Fig. 6A, 6B). The lack of Kindlin-3 in K3KO cells abolished phagocytosis of C3bi-covered beads even after PMA treatment (Fig. 6A, 6B), which is consistent with a deficient ligand recognition by K3KO cells. Re-expression of hK3 in both independent K3KO#1 and K3KO#2 CRISPR macrophage lines completely rescued their phagocytic function to an average number of 40 beads and 30 beads per cell, respectively, upon PMA stimulation (Fig. 6C, 6D), which is nearly identical to the number of beads per control cell prior to Kindlin-3 deletion, emphasizing the key role of Kindlin-3 in phagocytosis. The direct comparison between hK3 and hK3pxn mutant in K3KO cells revealed that PMA-stimulated hK3pxn mutant cells phagocytosed ∼45 and 30% more beads per cell compared with hK3 in K3KO#1 and K3KO#2 lines, respectively (Fig. 6C, 6D). These responses were specific and were completely abolished by heat shock treatment (Fig. 6C, 6D). Increased phagocytosis of hK3pxn cells was consistent with their augmented membrane blebbing, which is known to favor phagocytic and endocytic events (44). Together, these results show that Kindlin-3–PXN/LPXN binding restricts macrophage phagocytosis.

FIGURE 6.

Kindlin-3–PXN/LPXN interactions control macrophage phagocytosis. (A) Confocal images of control and K3KO cells phagocytosis of C3bi-opsonized latex beads. Latex beads (carboxylate-modified polystyrene, fluorescent yellow green) were incubated with human IgM and fresh mouse serum with or without heat shock (HS) at 37°C for 1 h to initiate C3bi opsonization. Cells were incubated with C3bi-opsonized latex beads with or without 150 nM PMA at 37°C for 2 h and then fixed, permeabilized, stained with anti-RFP Ab, and imaged by confocal microscopy. HS inactivated was used as a negative control. (B) Bars show numbers of beads per cells in (A) in the phagocytosis assay. Control (HS: n = 64; no treatment: n = 107; and PMA treatment: n = 95), K3KO#1 (HS: n = 82; no treatment: n = 67; and PMA treatment: n = 160), K3KO#2 (HS: n = 83; no treatment: n = 46; and PMA treatment: n = 131). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Representative confocal images showing phagocytosis of C3bi-opsonized latex beads in K3KO with hK3- or hK3pxn-expressing cells. Latex beads (carboxylate-modified polystyrene, fluorescent yellow-green) were incubated with human IgM and fresh mouse serum with or without HS as indicated at 37°C for 1 h to initiate C3bi opsonization. Cells were stimulated with 150 nM PMA at 37°C for 2 h to activate integrins, or unstimulated cells were incubated with C3bi-opsonized beads. Fixed cells were stained with anti-RFP Ab (red) to visualize hK3. Images were extracted from z-stacks by confocal microscopy. HS inactivated was used as a negative control. (D) Bars show numbers of beads per cell from (C). K3KO#1 line with hK3 (HS: n = 40; no treatment: n = 47; and PMA treatment: n = 45) and hK3pxn (HS: n = 53; no treatment: n = 48; and PMA treatment: n = 28) and K3KO#1 line with hK3 (HS: n = 58; no treatment: n = 133; and PMA treatment: n = 30) and hK3pxn (HS: n = 89; no treatment: n = 97; and PMA treatment: n = 57). Values = means + SEM. *p < 0.05 based on Student t test.

FIGURE 6.

Kindlin-3–PXN/LPXN interactions control macrophage phagocytosis. (A) Confocal images of control and K3KO cells phagocytosis of C3bi-opsonized latex beads. Latex beads (carboxylate-modified polystyrene, fluorescent yellow green) were incubated with human IgM and fresh mouse serum with or without heat shock (HS) at 37°C for 1 h to initiate C3bi opsonization. Cells were incubated with C3bi-opsonized latex beads with or without 150 nM PMA at 37°C for 2 h and then fixed, permeabilized, stained with anti-RFP Ab, and imaged by confocal microscopy. HS inactivated was used as a negative control. (B) Bars show numbers of beads per cells in (A) in the phagocytosis assay. Control (HS: n = 64; no treatment: n = 107; and PMA treatment: n = 95), K3KO#1 (HS: n = 82; no treatment: n = 67; and PMA treatment: n = 160), K3KO#2 (HS: n = 83; no treatment: n = 46; and PMA treatment: n = 131). Values = means + SEM. ***p < 0.001 based on Student t test. (C) Representative confocal images showing phagocytosis of C3bi-opsonized latex beads in K3KO with hK3- or hK3pxn-expressing cells. Latex beads (carboxylate-modified polystyrene, fluorescent yellow-green) were incubated with human IgM and fresh mouse serum with or without HS as indicated at 37°C for 1 h to initiate C3bi opsonization. Cells were stimulated with 150 nM PMA at 37°C for 2 h to activate integrins, or unstimulated cells were incubated with C3bi-opsonized beads. Fixed cells were stained with anti-RFP Ab (red) to visualize hK3. Images were extracted from z-stacks by confocal microscopy. HS inactivated was used as a negative control. (D) Bars show numbers of beads per cell from (C). K3KO#1 line with hK3 (HS: n = 40; no treatment: n = 47; and PMA treatment: n = 45) and hK3pxn (HS: n = 53; no treatment: n = 48; and PMA treatment: n = 28) and K3KO#1 line with hK3 (HS: n = 58; no treatment: n = 133; and PMA treatment: n = 30) and hK3pxn (HS: n = 89; no treatment: n = 97; and PMA treatment: n = 57). Values = means + SEM. *p < 0.05 based on Student t test.

Close modal

Our study has identified PXN and LPXN as new and direct intracellular binding partners for a key regulator of myeloid cells, Kindlin-3, and it has defined its structure–functional significance in myeloid cell biology. NMR and pull-down approaches identified G43/L47 residues, which are conserved among all kindlin paralogs, within the Kindlin-3 F0 domain as a binding site for both PXN and LPXN. Site-directed mutagenesis, equal re-expression of mutants in two independent CRISPR macrophage lines, and a subsequent analysis of integrin-dependent functions in macrophages revealed that this Kindlin-3–PXN/LPXN interaction stands apart from interactions with other known partners for Kindlin-3 as well as from the interaction between Kindlin-2 and PXN. Although other adhesome binding partners of kindlins, including ILK and actin, were shown to promote their integrin-dependent functions (24, 45), this interaction seems to have an inhibitory role for integrin-mediated macrophage responses. Disruption of Kindlin-3 and PXN/LPXN binding augments cell spreading, increases cell polarization and the formation of protrusions on the substrate, increases cell blebbing, promoting protrusions turnover and the transition from mesenchymal to faster amoeboid mode of migration, increases cell velocity, and, finally, augments phagocytosis. The lack of Kindlin-3–PXN/LPXN binding does not substantially interfere with ligand recognition by integrins and therefore does not impact cell adhesion. Kindlin-3 binding to integrin cytoplasmic domain does not affect Kindlin-3–PXN/LPXN interaction; therefore, these sites operate independently. Most importantly, disruption of this link between Kindlin-3 and the cytoskeleton destabilizes the membrane, leading to augmented blebbing and faster protrusions turnover, which in turn permits a high rate of migration and increases phagocytosis, which are two of the main functions of macrophages. Cell blebbing is an important prerequisite for a switch to the faster amoeboid migration, which is generally adhesion independent and does not require functions of integrins or their activators talin (19, 46) and Kindlin-3 (47). It was shown previously that adhesion-free conditions or diminished integrin function or expression are sufficient to force a mesenchymal to amoeboid transition when cells are exposed to confinement (16). This explains the movement of adhesion-deficient Kindlin-3 null cells in 3D in vivo and extensive blebbing of K3KO cells in both 2D and 3D conditions. One of the paradoxical aspects of Kindlin-3–G43K/L47E mutant is the combination of high adhesiveness with extensive blebbing, which is indicative of high amoeboid migration potential. High adhesiveness is expected to slow down both spontaneous and directed cell migration (16); however, this is not the case for the Kindlin-3–G43K/L47E mutant.

Interestingly, whereas Kindlin-3–PXN binding (Kd = 117.2 ± 6.0 μM) is stronger than Kindlin-2–PXN binding (Kd = 200.8 ± 7.2 μm) (27), the Kindlin-2 F2 domain binds much stronger to the integrin-linked kinase (ILK)–PINCH–Parvin complex compared with Kindlin-3 (45). In contrast to Kindlin-3, Kindlin-2 localizes to focal adhesions where other components of adhesome, such as ILK are recruited (45). Thus, mechanistically, Kindlin-3 binding to PXN might compensate for the weak interaction with other cytoskeletal proteins, exemplified by ILK (45), representing the main link to the cytoskeleton. In addition, whereas stable focal adhesions are characteristic for cells expressing Kindlin-2, Kindlin-3–expressing cells do not form stable contacts with the ECM. Leukocytes, circulating in the blood or lymph in a mixture of soluble ligands, such as fibrinogen and fibronectin, are programmed to avoid ECM interactions because integrin–ligand binding results in immediate immobilization of these cells, either within the blood clot or on the surface of the inflamed endothelium (4850). It appears that in contrast to Kindlin-2, which requires an interaction with the cytoskeleton to support integrin-dependent functions, Kindlin-3 binding to the cytoskeletal adaptor PXN serves as an inhibitory connection, restricting cell adhesiveness and preserving membrane stability to limit amoeboid cell migration and excessive phagocytosis. Thus, the consequences of Kindlin-3 anchoring to the cytoskeleton are opposite to that of Kindlin-2, demonstrating one of the first critical distinctions between Kindlin paralogs.

There are a number of discrepancies in the literature between the effects of individual Kindlin or PXN knockouts in various cells. For instance, PXN was shown to generally promote integrin-dependent functions in most Kindlin-2–expressing cells (51); however, in platelets, PXN seems to have an opposite effect (52). These studies highlight the differences between Kindlin-2–expressing cells with their dependence on stable focal adhesions containing numerous binding partners for kindlins and Kindlin-3–expressing hematopoietic cells lacking stable focal adhesions.

Because amoeboid migration is a feature of myeloid precursor cells, whereas a more adhesive and elongated shape is a signature of more mature macrophages (53), it seems that Kindlin-3 interactions with cytoplasmic proteins, including PXN, might be associated with myeloid cell maturation and/or neoplastic transformation. Indeed, the acquisition of the amoeboid migration serves as a key feature of cancer transformation and aggressiveness (54). We have predicted several cancer-associated mutations that might directly interfere with the Kindlin-3–PXN binding interface. Kindlin-3 L46F mutant in human mucinous stomach adenocarcinoma and G44E mutant in human prostate cancer (data from 11K cases and all TCGA tumor types) (5560) (Supplemental Fig. 4F) might drive tumorigenesis by reducing Kindlin-3–PXN/LPXN binding.

Together, we show a new regulatory pathway suppressing myeloid cell adhesion and extensive blebbing associated with amoeboid motility and phagocytosis, thereby affecting not only immune defense but also pathogenesis of inflammatory diseases, degenerative diseases, and cancer.

We acknowledge the Cleveland Clinic Imaging Core and Flow Core equipment and services. We thank Michael McCoy and Greg DeGirolamo for useful discussions and Chris Nelson, Gautam Mahajan, Daniel Nascimento, and Evan Welch for manuscript proofing.

This work was supported by National Institutes of Health Grants HL071625, HL142772, and HL073311.

The online version of this article contains supplemental material.

Abbreviations used in this article:

C5aR

C5a receptor

2D

two-dimensional

3D

three-dimensional

DIC

differential interference contrast

ECM

extracellular matrix

hK3

human Kindlin-3

hK3pxn

Kindlin-3–G43K/L47E

HSQC

heteronuclear single quantum coherence

ILK

integrin-linker kinase

K3KO

Kindlin-3 knockout

LAS X

Leica Application Suite X

LPXN

leupaxin

NMR

nuclear magnetic resonance spectroscopy

ORF

open reading frame

PH

pleckstrin homology

PXN

paxillin

RT

room temperature

TCEP

tris (2-carboxyethyl) phosphine.

1
Wynn
,
T. A.
,
A.
Chawla
,
J. W.
Pollard
.
2013
.
Macrophage biology in development, homeostasis and disease.
Nature
496
:
445
455
.
2
Kim
,
K. W.
,
N.
Zhang
,
K.
Choi
,
G. J.
Randolph
.
2016
.
Homegrown macrophages.
Immunity
45
:
468
470
.
3
Renkawitz
,
J.
,
M.
Sixt
.
2010
.
Mechanisms of force generation and force transmission during interstitial leukocyte migration.
EMBO Rep.
11
:
744
750
.
4
Friedl
,
P.
,
B.
Weigelin
.
2008
.
Interstitial leukocyte migration and immune function.
Nat. Immunol.
9
:
960
969
.
5
Zaidel-Bar
,
R.
,
S.
Itzkovitz
,
A.
Ma’ayan
,
R.
Iyengar
,
B.
Geiger
.
2007
.
Functional atlas of the integrin adhesome.
Nat. Cell Biol.
9
:
858
867
.
6
Horton
,
E. R.
,
J. D.
Humphries
,
J.
James
,
M. C.
Jones
,
J. A.
Askari
,
M. J.
Humphries
.
2016
.
The integrin adhesome network at a glance.
J. Cell Sci.
129
:
4159
4163
.
7
Plow
,
E. F.
,
J.
Meller
,
T. V.
Byzova
.
2014
.
Integrin function in vascular biology: a view from 2013.
Curr. Opin. Hematol.
21
:
241
247
.
8
Malinin
,
N. L.
,
L.
Zhang
,
J.
Choi
,
A.
Ciocea
,
O.
Razorenova
,
Y. Q.
Ma
,
E. A.
Podrez
,
M.
Tosi
,
D. P.
Lennon
,
A. I.
Caplan
, et al
.
2009
.
A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans.
Nat. Med.
15
:
313
318
.
9
Stadtmann
,
A.
,
A.
Zarbock
.
2017
.
The role of kindlin in neutrophil recruitment to inflammatory sites.
Curr. Opin. Hematol.
24
:
38
45
.
10
Meller
,
J.
,
Z.
Chen
,
T.
Dudiki
,
R. M.
Cull
,
R.
Murtazina
,
S. K.
Bal
,
E.
Pluskota
,
S.
Stefl
,
E. F.
Plow
,
B. D.
Trapp
,
T. V.
Byzova
.
2017
.
Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages.
JCI Insight
2
:
93002
.
11
Friedl
,
P.
,
K.
Wolf
.
2010
.
Plasticity of cell migration: a multiscale tuning model.
J. Cell Biol.
188
:
11
19
.
12
Petrie
,
R. J.
,
K. M.
Yamada
.
2016
.
Multiple mechanisms of 3D migration: the origins of plasticity.
Curr. Opin. Cell Biol.
42
:
7
12
.
13
Small
,
J. V.
,
T.
Stradal
,
E.
Vignal
,
K.
Rottner
.
2002
.
The lamellipodium: where motility begins.
Trends Cell Biol.
12
:
112
120
.
14
Chhabra
,
E. S.
,
H. N.
Higgs
.
2007
.
The many faces of actin: matching assembly factors with cellular structures.
Nat. Cell Biol.
9
:
1110
1121
.
15
Bergert
,
M.
,
S. D.
Chandradoss
,
R. A.
Desai
,
E.
Paluch
.
2012
.
Cell mechanics control rapid transitions between blebs and lamellipodia during migration.
Proc. Natl. Acad. Sci. USA
109
:
14434
14439
.
16
Liu
,
Y. J.
,
M.
Le Berre
,
F.
Lautenschlaeger
,
P.
Maiuri
,
A.
Callan-Jones
,
M.
Heuzé
,
T.
Takaki
,
R.
Voituriez
,
M.
Piel
.
2015
.
Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.
Cell
160
:
659
672
.
17
Ruprecht
,
V.
,
S.
Wieser
,
A.
Callan-Jones
,
M.
Smutny
,
H.
Morita
,
K.
Sako
,
V.
Barone
,
M.
Ritsch-Marte
,
M.
Sixt
,
R.
Voituriez
,
C. P.
Heisenberg
.
2015
.
Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.
Cell
160
:
673
685
.
18
Chikina
,
A. S.
,
T. M.
Svitkina
,
A. Y.
Alexandrova
.
2019
.
Time-resolved ultrastructure of the cortical actin cytoskeleton in dynamic membrane blebs.
J. Cell Biol.
218
:
445
454
.
19
Lämmermann
,
T.
,
B. L.
Bader
,
S. J.
Monkley
,
T.
Worbs
,
R.
Wedlich-Söldner
,
K.
Hirsch
,
M.
Keller
,
R.
Förster
,
D. R.
Critchley
,
R.
Fässler
,
M.
Sixt
.
2008
.
Rapid leukocyte migration by integrin-independent flowing and squeezing.
Nature
453
:
51
55
.
20
Li
,
H.
,
Y.
Deng
,
K.
Sun
,
H.
Yang
,
J.
Liu
,
M.
Wang
,
Z.
Zhang
,
J.
Lin
,
C.
Wu
,
Z.
Wei
,
C.
Yu
.
2017
.
Structural basis of kindlin-mediated integrin recognition and activation.
Proc. Natl. Acad. Sci. USA
114
:
9349
9354
.
21
Liu
,
J.
,
K.
Fukuda
,
Z.
Xu
,
Y. Q.
Ma
,
J.
Hirbawi
,
X.
Mao
,
C.
Wu
,
E. F.
Plow
,
J.
Qin
.
2011
.
Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.
J. Biol. Chem.
286
:
43334
43342
.
22
Shi
,
X.
,
Y. Q.
Ma
,
Y.
Tu
,
K.
Chen
,
S.
Wu
,
K.
Fukuda
,
J.
Qin
,
E. F.
Plow
,
C.
Wu
.
2007
.
The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility.
J. Biol. Chem.
282
:
20455
20466
.
23
Kadry
,
Y. A.
,
C.
Huet-Calderwood
,
B.
Simon
,
D. A.
Calderwood
.
2018
.
Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading.
J. Cell Sci.
131
:
jcs221184
.
24
Bledzka
,
K.
,
K.
Bialkowska
,
K.
Sossey-Alaoui
,
J.
Vaynberg
,
E.
Pluskota
,
J.
Qin
,
E. F.
Plow
.
2016
.
Kindlin-2 directly binds actin and regulates integrin outside-in signaling.
J. Cell Biol.
213
:
97
108
.
25
Böttcher
,
R. T.
,
M.
Veelders
,
P.
Rombaut
,
J.
Faix
,
M.
Theodosiou
,
T. E.
Stradal
,
K.
Rottner
,
R.
Zent
,
F.
Herzog
,
R.
Fässler
.
2017
.
Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.
J. Cell Biol.
216
:
3785
3798
.
26
Theodosiou
,
M.
,
M.
Widmaier
,
R. T.
Böttcher
,
E.
Rognoni
,
M.
Veelders
,
M.
Bharadwaj
,
A.
Lambacher
,
K.
Austen
,
D. J.
Müller
,
R.
Zent
,
R.
Fässler
.
2016
.
Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin.
Elife
5
:
e10130
.
27
Zhu
,
L.
,
H.
Liu
,
F.
Lu
,
J.
Yang
,
T. V.
Byzova
,
J.
Qin
.
2019
.
Structural basis of paxillin recruitment by kindlin-2 in regulating cell adhesion.
Structure
27
:
1686
1697.e5
.
28
Liu
,
Y.
,
Y.
Zhu
,
S.
Ye
,
R.
Zhang
.
2012
.
Crystal structure of kindlin-2 PH domain reveals a conformational transition for its membrane anchoring and regulation of integrin activation.
Protein Cell
3
:
434
440
.
29
Gao
,
J.
,
M.
Huang
,
J.
Lai
,
K.
Mao
,
P.
Sun
,
Z.
Cao
,
Y.
Hu
,
Y.
Zhang
,
M. L.
Schulte
,
C.
Jin
, et al
.
2017
.
Kindlin supports platelet integrin αIIbβ3 activation by interacting with paxillin.
J. Cell Sci.
130
:
3764
3775
.
30
Brynn Hibbert
,
D.
,
P.
Thordarson
.
2016
.
The death of the Job plot, transparency, open science and online tools, uncertainty estimation methods and other developments in supramolecular chemistry data analysis.
Chem. Commun. (Camb.)
52
:
12792
12805
.
31
Rognoni
,
E.
,
R.
Ruppert
,
R.
Fässler
.
2016
.
The kindlin family: functions, signaling properties and implications for human disease.
J. Cell Sci.
129
:
17
27
.
32
Yates
,
L. A.
,
A. K.
Füzéry
,
R.
Bonet
,
I. D.
Campbell
,
R. J.
Gilbert
.
2012
.
Biophysical analysis of Kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal β-subunit NPXY motif.
J. Biol. Chem.
287
:
37715
37731
.
33
Schaller
,
M. D.
2001
.
Paxillin: a focal adhesion-associated adaptor protein.
Oncogene
20
:
6459
6472
.
34
Brown
,
M. C.
,
C. E.
Turner
.
2004
.
Paxillin: adapting to change.
Physiol. Rev.
84
:
1315
1339
.
35
Tumbarello
,
D. A.
,
M. C.
Brown
,
S. E.
Hetey
,
C. E.
Turner
.
2005
.
Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin delta.
J. Cell Sci.
118
:
4849
4863
.
36
Vaynberg
,
J.
,
J.
Qin
.
2006
.
Weak protein-protein interactions as probed by NMR spectroscopy.
Trends Biotechnol.
24
:
22
27
.
37
Weber
,
C.
,
J.
Kitayama
,
T. A.
Springer
.
1996
.
Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.
Proc. Natl. Acad. Sci. USA
93
:
10939
10944
.
38
Diz-Muñoz
,
A.
,
M.
Krieg
,
M.
Bergert
,
I.
Ibarlucea-Benitez
,
D. J.
Muller
,
E.
Paluch
,
C. P.
Heisenberg
.
2010
.
Control of directed cell migration in vivo by membrane-to-cortex attachment.
PLoS Biol.
8
:
e1000544
.
39
Chenoweth
,
D. E.
,
S. W.
Cooper
,
T. E.
Hugli
,
R. W.
Stewart
,
E. H.
Blackstone
,
J. W.
Kirklin
.
1981
.
Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins.
N. Engl. J. Med.
304
:
497
503
.
40
Guo
,
R. F.
,
P. A.
Ward
.
2005
.
Role of C5a in inflammatory responses.
Annu. Rev. Immunol.
23
:
821
852
.
41
Ward
,
P. A.
2004
.
The dark side of C5a in sepsis.
Nat. Rev. Immunol.
4
:
133
142
.
42
Keren
,
K.
2011
.
Cell motility: the integrating role of the plasma membrane.
Eur. Biophys. J.
40
:
1013
1027
.
43
Rougerie
,
P.
,
V.
Miskolci
,
D.
Cox
.
2013
.
Generation of membrane structures during phagocytosis and chemotaxis of macrophages: role and regulation of the actin cytoskeleton.
Immunol. Rev.
256
:
222
239
.
44
Diz-Muñoz
,
A.
,
D. A.
Fletcher
,
O. D.
Weiner
.
2013
.
Use the force: membrane tension as an organizer of cell shape and motility.
Trends Cell Biol.
23
:
47
53
.
45
Huet-Calderwood
,
C.
,
N. N.
Brahme
,
N.
Kumar
,
A. L.
Stiegler
,
S.
Raghavan
,
T. J.
Boggon
,
D. A.
Calderwood
.
2014
.
Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.
J. Cell Sci.
127
:
4308
4321
.
46
Lämmermann
,
T.
,
M.
Sixt
.
2009
.
Mechanical modes of ‘amoeboid’ cell migration.
Curr. Opin. Cell Biol.
21
:
636
644
.
47
Meller
,
J.
,
I. B.
Rogozin
,
E.
Poliakov
,
N.
Meller
,
M.
Bedanov-Pack
,
E. F.
Plow
,
J.
Qin
,
E. A.
Podrez
,
T. V.
Byzova
.
2015
.
Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness.
Mol. Biol. Cell
26
:
786
796
.
48
Obenauf
,
A. C.
,
J.
Massagué
.
2015
.
Surviving at a distance: organ-specific metastasis.
Trends Cancer
1
:
76
91
.
49
Schwartz
,
M. A.
,
D.
Vestweber
,
M.
Simons
.
2018
.
A unifying concept in vascular health and disease.
Science
360
:
270
271
.
50
Yurdagul
,
A.
 Jr.
,
A. W.
Orr
.
2016
.
Blood brothers: hemodynamics and cell-matrix interactions in endothelial function.
Antioxid. Redox Signal.
25
:
415
434
.
51
Deakin
,
N. O.
,
C. E.
Turner
.
2008
.
Paxillin comes of age.
J. Cell Sci.
121
:
2435
2444
.
52
Sakata
,
A.
,
T.
Ohmori
,
S.
Nishimura
,
H.
Suzuki
,
S.
Madoiwa
,
J.
Mimuro
,
K.
Kario
,
Y.
Sakata
.
2014
.
Paxillin is an intrinsic negative regulator of platelet activation in mice.
Thromb. J.
12
:
1
.
53
McNally
,
A. K.
,
J. M.
Anderson
.
2002
.
β1 and β2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation.
Am. J. Pathol.
160
:
621
630
.
54
Krakhmal
,
N. V.
,
M. V.
Zavyalova
,
E. V.
Denisov
,
S. V.
Vtorushin
,
V. M.
Perelmuter
.
2015
.
Cancer invasion: patterns and mechanisms.
Acta Naturae
7
:
17
28
.
55
Ellrott
,
K.
,
M. H.
Bailey
,
G.
Saksena
,
K. R.
Covington
,
C.
Kandoth
,
C.
Stewart
,
J.
Hess
,
S.
Ma
,
K. E.
Chiotti
,
M.
McLellan
, et al
MC3 Working Group
; 
Cancer Genome Atlas Research Network
.
2018
.
Scalable open science approach for mutation calling of tumor exomes using multiple genomic pipelines.
Cell Syst.
6
:
271
281.e7
.
56
Gao
,
Q.
,
W. W.
Liang
,
S. M.
Foltz
,
G.
Mutharasu
,
R. G.
Jayasinghe
,
S.
Cao
,
W. W.
Liao
,
S. M.
Reynolds
,
M. A.
Wyczalkowski
,
L.
Yao
, et al
Fusion Analysis Working Group
; 
Cancer Genome Atlas Research Network
.
2018
.
Driver fusions and their implications in the development and treatment of human cancers.
Cell Rep.
23
:
227
238.e3
.
57
Hoadley
,
K. A.
,
C.
Yau
,
T.
Hinoue
,
D. M.
Wolf
,
A. J.
Lazar
,
E.
Drill
,
R.
Shen
,
A. M.
Taylor
,
A. D.
Cherniack
,
V.
Thorsson
, et al
Cancer Genome Atlas Network
.
2018
.
Cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer.
Cell
173
:
291
304.e6
.
58
Liu
,
J.
,
T.
Lichtenberg
,
K. A.
Hoadley
,
L. M.
Poisson
,
A. J.
Lazar
,
A. D.
Cherniack
,
A. J.
Kovatich
,
C. C.
Benz
,
D. A.
Levine
,
A. V.
Lee
, et al
Cancer Genome Atlas Research Network
.
2018
.
An integrated TCGA pan-cancer clinical data resource to drive high-quality survival outcome analytics.
Cell
173
:
400
416.e11
.
59
Sanchez-Vega
,
F.
,
M.
Mina
,
J.
Armenia
,
W. K.
Chatila
,
A.
Luna
,
K. C.
La
,
S.
Dimitriadoy
,
D. L.
Liu
,
H. S.
Kantheti
,
S.
Saghafinia
, et al
Cancer Genome Atlas Research Network
.
2018
.
Oncogenic signaling pathways in the cancer genome atlas.
Cell
173
:
321
337.e10
.
60
Taylor
,
A. M.
,
J.
Shih
,
G.
Ha
,
G. F.
Gao
,
X.
Zhang
,
A. C.
Berger
,
S. E.
Schumacher
,
C.
Wang
,
H.
Hu
,
J.
Liu
, et al
Cancer Genome Atlas Research Network
.
2018
.
Genomic and functional approaches to understanding cancer aneuploidy.
Cancer Cell
33
:
676
689.e3
.

The authors have no financial conflicts of interest.