Profilin 1 is required for abscission during late cytokinesis of chondrocytes
2009; Springer Nature; Volume: 28; Issue: 8 Linguagem: Inglês
10.1038/emboj.2009.58
ISSN1460-2075
AutoresRalph T. Böttcher, Sebastian Wiesner, Attila Braun, Reiner Wimmer, Alejandro Berna‐Erro, Nadav Elad, Ohad Medalia, Alexander Pfeifer, Attila Aszódi, Mercedes Costell, Reinhard Fässler,
Tópico(s)Tendon Structure and Treatment
ResumoArticle5 March 2009free access Profilin 1 is required for abscission during late cytokinesis of chondrocytes Ralph T Böttcher Ralph T Böttcher Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Sebastian Wiesner Sebastian Wiesner Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Attila Braun Attila Braun Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Reiner Wimmer Reiner Wimmer Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Alejandro Berna Alejandro Berna Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain Search for more papers by this author Nadav Elad Nadav Elad Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel Search for more papers by this author Ohad Medalia Ohad Medalia Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel Search for more papers by this author Alexander Pfeifer Alexander Pfeifer Institute for Pharmacology and Toxicology, University of Bonn, Bonn, Germany Search for more papers by this author Attila Aszódi Attila Aszódi Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Mercedes Costell Mercedes Costell Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain Search for more papers by this author Reinhard Fässler Corresponding Author Reinhard Fässler Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Ralph T Böttcher Ralph T Böttcher Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Sebastian Wiesner Sebastian Wiesner Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Attila Braun Attila Braun Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Reiner Wimmer Reiner Wimmer Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Alejandro Berna Alejandro Berna Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain Search for more papers by this author Nadav Elad Nadav Elad Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel Search for more papers by this author Ohad Medalia Ohad Medalia Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel Search for more papers by this author Alexander Pfeifer Alexander Pfeifer Institute for Pharmacology and Toxicology, University of Bonn, Bonn, Germany Search for more papers by this author Attila Aszódi Attila Aszódi Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Mercedes Costell Mercedes Costell Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain Search for more papers by this author Reinhard Fässler Corresponding Author Reinhard Fässler Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Author Information Ralph T Böttcher1, Sebastian Wiesner1, Attila Braun1, Reiner Wimmer1, Alejandro Berna2, Nadav Elad3, Ohad Medalia3, Alexander Pfeifer4, Attila Aszódi1, Mercedes Costell2 and Reinhard Fässler 1 1Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany 2Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain 3Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel 4Institute for Pharmacology and Toxicology, University of Bonn, Bonn, Germany *Corresponding author. Department of Molecular Medicine, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried 82152, Germany. Tel.: +49 89 8578 2424; Fax: +49 89 8578 2422; E-mail: [email protected] The EMBO Journal (2009)28:1157-1169https://doi.org/10.1038/emboj.2009.58 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis. Introduction Profilins, the most versatile actin monomer-binding proteins, are abundantly expressed in all eukaryotic cells from plants and fungi to mammals. Mammals have four profilin family members; profilin 1, which is ubiquitously expressed, and profilins 2–4, which have a restricted expression pattern (Witke et al, 1998; Di Nardo et al, 2000; Obermann et al, 2005). Genetic and cell biological studies have implicated profilins in many cellular processes such as cell migration, cytokinesis, endocytosis and transcription regulation (reviewed in Witke, 2004). Among these different processes, the role of profilin in actin-driven cellular motility has been most thoroughly studied, and has been very conclusively linked to the biochemistry of profilins with their main ligand, actin (reviewed in Pollard and Borisy, 2003; Witke, 2004). Profilin enhances the rate of actin filament turnover through two molecular mechanisms: profilin-bound ADP–G-actin is rapidly recharged with ATP (Mockrin and Korn, 1980; Goldschmidt-Clermont et al, 1992), and profilin–ATP–actin complexes can be formed from the non-polymerizable thymosin–actin pool to associate productively with free barbed ends (Pollard and Cooper, 1984; Pantaloni and Carlier, 1993). The function of profilins in cytokinesis, though extensively studied, is less clear. Profilin 1 depletion in flies, worms and mice is embryonic lethal. Consequently, studies on cell division in the absence of profilin in higher eukaryotes have mainly been performed at very early stages of embryogenesis, where null alleles cause cell division defects of varying severity (Verheyen and Cooley, 1994; Witke et al, 2001; Severson et al, 2002). In many cases, these defects occur during early cytokinesis and are associated with malformed or non-constricting actomyosin rings. A similar phenotype was reported for profilin deletions in Schizosaccharomyces pombe (Balasubramanian et al, 1994). Profilins are therefore generally considered as a part of the core cytokinesis machinery that is required for actomyosin ring formation (Glotzer, 2005). However, some protists can bypass profilin deficiency by alternative cleavage mechanisms (Dictyostelium discoideum; Haugwitz et al, 1994) or show no requirement of profilin for actomyosin ring assembly (Tetrahymena thermophila; Wilkes and Otto, 2003). In animal cells, contractile ring assembly is mediated by the GTPase RhoA and its effector proteins, formins and Rho-associated kinase (ROCK), which regulate actin nucleation and myosin activation, respectively (Glotzer, 2005). Actin and myosin are two major components of the contractile ring that generate the force for cleavage furrow ingression. ROCK activates myosin II by phosphorylation of the regulatory myosin light chain (MLC) and by inhibition of myosin phosphatase activity (Glotzer, 2005). Profilin function during cytokinesis is connected with the activity of formins, a family of actin regulatory proteins, which have emerged as key ligands for profilins (reviewed in Goode and Eck, 2007). Formins nucleate actin filaments and associate with barbed ends, where various formins slow elongation from as little as 10% to more than 99%. Profilins bind to proline-rich motifs within the formin homology 1 (FH1) domain (Chang et al, 1997) and thereby increase the actin filament elongation rates of formins (Romero et al, 2004; Kovar et al, 2006). The genetic interaction between formins and profilins is well documented for a wide range of organisms (Chang et al, 1997; Watanabe et al, 1997; Severson et al, 2002). However, it is still unclear whether formins require profilins for actin polymerization in mammalian cells. To examine the role of profilin in cytokinesis and formin function in somatic mammalian cells in vivo, we have generated a mouse strain with a profilin-deficient cartilage (Col2pfn1 mice). Cartilage is an attractive model tissue for studying profilin function because (i) cartilage is composed of only one cell type, which can be readily isolated, cultured and studied in vitro (Aszodi et al, 2003); (ii) dynamic F-actin reorganization has an essential function in arranging chondrocytes into characteristic stacks or columns, which are required for the longitudinal growth of bones and (iii) chondrocytes express a single profilin, which is profilin 1. We report that Col2pfn1 mice are grossly normal at birth but later on develop a progressive chondrodysplasia caused by defects in chondrocyte proliferation, actin cytoskeleton organization and the formation of growth plate columns. Furthermore, we found that chondrocytes do not employ profilin for mitosis or actomyosin ring formation and contraction in early cytokinesis, but they require profilin to generate sufficient forces for abscission during late cytokinesis. Results Cartilage-specific deletion of the profilin 1 gene For tissue-specific deletion of profilin 1, we generated a mouse strain in which the promoter and exon 1 of the profilin 1 (pfn1) gene were flanked by loxP sites (pfn1fl/fl). The validity of the targeting strategy was tested by mating pfn1fl/fl mice with Cre deleter mice (Betz et al, 1996). pfn1−/− mice died during the pre-implantation stage, as shown previously (data not shown; Witke et al, 2001). To obtain mice lacking profilin 1 in chondrocytes, pfn1fl/fl mice were crossed with transgenic mice carrying a collagen II promoter-driven Cre recombinase transgene, which deletes floxed genes at the time of chondrogenic differentiation (E11.5–E13) (Sakai et al, 2001) (Col2a1-cre+/pfn1fl/fl, also called Col2pfn1). Western blot analysis of primary rib chondrocytes isolated from Col2pfn1 E15 embryos and newborn mice confirmed the loss of profilin 1 (data not shown and Figure 1A). No other known profilin isoform was detectable in either control or Col2pfn1 cartilage by western blotting for profilin 2 (Figure 1A) or northern blotting for testis-specific profilin 3 and 4 (Figure 1B). These results demonstrate that Col2pfn1 cartilage does not express detectable levels of known profilin isoforms. Figure 1.Morphology of Col2pfn1 mice. (A) Western blot analysis of profilin 1 and 2 expressions in total protein lysates from newborn control brain, newborn control and Col2pfn1 cartilage. (B) Northern blot analysis of total RNA from control testes, control cartilage and Col2pfn1 cartilage. (C) Whole mount Alcian blue/Alizarin red staining of control and Col2pfn1 mouse skeletons. Newborn Col2pfn1 mice are indistinguishable from controls but are shorter at 4 weeks of age. (D) Length of long bones of control and Col2pfn1 mice at birth and at 4 weeks of age (mean±s.d., n=8/8 newborn, n=5/5 P28; *P⩽0.01, **P⩽0.05 analysed by Mann–Whitney test). (E) Body length of control and Col2pfn1 mice from birth to 16 days of age (n=6/6). (F) Body weight of control and Col2pfn1 mice from birth to 18 days of age (n=5/5). Download figure Download PowerPoint Col2pfn1 mice develop progressive chondrodysplasia with abnormal F-actin distribution in chondrocytes Col2pfn1 mice were born at the expected Mendelian ratio, had an intact skeleton (Figure 1C) and were viable. Although 75% of the Col2pfn1 mice had a normal lifespan, 25% of Col2pfn1 mice died within the first 10 days. They developed severe kyphoscoliosis and were significantly weaker and smaller as their wild-type and heterozygote littermates (data not shown). Although the external appearance of newborn Col2pfn1 mice was grossly normal, the length of some long bones such as the femur and humerus was moderately reduced in mutants compared with control animals (Figure 1C and D). At later stages, Col2pfn1 mice progressively developed dwarfism as a consequence of reduced growth of the long bones. Col2pfn1 mice (4 weeks old) have 20–30% shorter long bones compared with control littermates (Figure 1C and D), leading to a corresponding reduction in body length and weight (Figure 1E and F). As the base of the skull also develops through cartilaginous intermediates, the skull growth was also affected in the Col2pfn1 mice (data not shown). To characterize the skeletal phenotype of Col2pfn1 mice, we studied the cartilage structure during the formation of long bones of the appendicular skeleton. At E15.5, mutant bones showed no gross histological abnormalities (data not shown). At the newborn stage, some Col2pfn1 growth plates exhibited an increased height of the hypertrophic zone accompanied by a disorganization of the columnar structure of the proliferating zone and a more rounded morphology of the normally flat proliferating chondrocytes (Figure 2A and B). The columnar arrangement of these cells was disturbed, indicating a defect in chondrocyte polarity and/or movement (Aszodi et al, 2003) (Figure 2B). In 4-week-old Col2pfn1 mice, this phenotype became more pronounced (Figure 2C). The height of the proliferative zone was reduced to a few cells, whereas many chondrocytes in the hypertrophic zone were unusually enlarged and appeared binucleated (Figure 2D). Interestingly, already at newborn stage 1.7% of the cells in profilin-deficient growth plates were binucleated compared with 0.45% of control chondrocytes (Figure 2E). Although the number of binucleated cells in control growth plates remained at a low level, the percentage of multinucleated profilin-deficient chondrocytes increased to 15% at 4 weeks of age. Figure 2.Growth plate cartilage defects in Col2pfn1 mice. (A) Haematoxylin/eosin (H/E)-stained sections of the knee joint region of newborn control and Col2pfn1 mice. (B) H/E-stained sections of the proliferative zone of the tibial epiphyseal growth plate of newborn control and Col2pfn1 mice. (C) H/E-stained sections of the knee joint region of 4-week-old Col2pfn1 and control mice. (D) H/E-stained sections of the tibial epiphyseal growth plate of 4-week-old control and Col2pfn1 mice. Arrows point towards binucleated cells. (E) Quantification of binucleated cells in newborn and 4-week-old growth plates of control and Col2pfn1 mice (mean±s.d., n=3/3, *P<0.05, **P<0.001 analysed by Student's t-test). (F) Quantification of BrdU incorporation (mean±s.d., n=2/3 newborn, n=3/4 P28, *P⩽0.01 analysed by Mann–Whitney test). (G) Sections from the growth plate of 10-day-old control and Col2pfn1 mice stained with fluorescently labelled phalloidin to visualize F-actin. Confocal sections and projections are shown. Abbreviations: r, resting zone; p, proliferative zone; h, hypertrophic zone. Download figure Download PowerPoint The decreased size of the proliferative zone in the postnatal growth plate as well as the presence of binucleated cells pointed to a proliferation defect in Col2pfn1 cartilage. To analyse chondrocyte proliferation in Col2pfn1 cartilage, we performed a bromodeoxyuridine (BrdU) incorporation assay to label cells in the S phase of the cell cycle. At the newborn stage, no differences were detectable in the proportion of proliferating chondrocytes in the proliferative zone between control and Col2pfn1 cartilage. However, in 4-week-old Col2pfn1 growth plates, significantly less chondrocytes proliferated compared with control growth plates (Figure 2F). Importantly, we found no indication for an increase in chondrocyte apoptosis in growth plates of newborn or 2-week-old Col2pfn1 mice or for altered expression of chondrocyte differentiation markers in the different growth plate zones at E16.5 (Supplementary Figure 1A and B). Finally, we analysed F-actin distribution in chondrocytes of the different growth plate zones in tissue sections. Overall, Col2pfn1 chondrocytes had a less intense and more punctated F-actin staining and the membrane protrusions of Col2pfn1 chondrocytes appeared shorter compared with control cells (Figure 2G). This defect was most pronounced in the prehypertrophic and hypertrophic zones, suggesting a deficiency of these cells in efficient F-actin assembly. In line with the in vivo results, rib chondrocytes isolated from newborn Col2pfn1 mice expressed around 75% of total cellular actin compared with chondrocytes of control littermates (data not shown) and the F/G actin ratio was reduced to 60% in profilin-deficient cells compared with control cells (data not shown). In summary, analysis of Col2pfn1 cartilage indicates a role for profilin 1 in proliferation, arranging chondrocytes into columns and actin cytoskeleton organization, whereas chondrocyte differentiation and survival do not require profilin. Col2pfn1 chondrocytes assemble actomyosin rings but fail to complete abscission To determine whether the proliferation defects in Col2pfn1 chondrocytes is caused by a failure in mitosis and/or cytokinesis, we observed the division of isolated primary cells by video microscopy. The sum of mitosis and cytokinesis duration measured as the time between nuclear envelope breakdown and abscission was increased in Col2pfn1 chondrocytes (Supplementary Figure 2A). Therefore, we analysed the succession of mitotic stages to identify possible mitotic defects in Col2pfn1 chondrocytes. The duration of prophase and metaphase was unchanged. The combined interval of anaphase and telophase measured as the time between the onset of sister chromatid separation and nuclear envelope reformation was not significantly longer in Col2pfn1 chondrocytes compared with control cells (Supplementary Figure 2B), indicating a defect during cytokinesis. We also analysed mitotic spindle formation by immunofluorescence but found no obvious defects in Col2pfn1 chondrocytes (Supplementary Figure 2C). Thus, profilin appears to be dispensable for mitosis in chondrocytes but critically involved in cytokinesis. Time-lapse video microscopy showed that primary Col2pfn1 chondrocytes were less spread but displayed equatorial furrowing and midbody formation on the same timescale as control chondrocytes (Figure 3A–C; Supplementary Videos S1, S2, S3). However, most Col2pfn1 chondrocytes remained rounded up, failed to form and extend lamellipodia, and showed a marked delay in abscission (Figure 3B; Supplementary Video S2), whereas some fused following constriction (Figure 3C; Supplementary Video S3). In line with this finding, 20.1% of Col2pfn1 chondrocytes isolated from newborn growth plates and kept for 4 days in culture were binucleated compared with 4.9% of primary control chondrocytes. Similarly, 2.8% of immortalized control chondrocytes were binucleated, whereas around 12% of immortalized profilin-deficient cells were binucleated (data not shown). To verify that the observed cell division in profilin-deficient cells was actomyosin based, we expressed Lifeact, a 17-amino-acid peptide fused to GFP, in immortalized control and Col2pfn1 chondrocytes, which stains F-actin structures in eukaryotic cells without interfering with actin dynamics (Riedl et al, 2008). In both cell types, Lifeact is equally distributed around the cell cortex at late metaphase and becomes enriched at the cell equator after anaphase onset. As cytokinesis proceeds, Lifeact localizes strongly to the constricting furrow (Figure 3D; Supplementary Videos S4 and S5). Despite an increase in blebbing during cytokinesis in Col2pfn1 chondrocytes, the furrow diameter of the cortical actin ring and the timing of its constriction were similar to control cells (Figure 3D and E; Supplementary Videos S4 and S5). The unchanged localization of F-actin and contractile ring markers RhoA and anillin in control and profilin-deficient chondrocytes were confirmed in fixed cells (Supplementary Figure 3). In line with these observations, cytokinesis in Col2pfn1 chondrocytes was still sensitive to myosin II inhibition by blebbistatin and to Latrunculin B treatment, an actin-sequestering agent (data not shown). Taken together, these results suggest that equatorial furrowing in Col2pfn1 chondrocytes is caused by an actomyosin ring and is not obviously altered by the lack of profilin. Figure 3.Profilin-deficient cells assemble contractile rings. (A–C) Montage of phase-contrast video of mitotic primary control and Col2pfn1 chondrocytes; (A) control chondrocyte, (B) dividing Col2pfn1 chondrocyte and (C) fusing Col2pfn1 chondrocyte. Bar, 10 μm. (D) Montage showing Lifeact localization to the cleavage furrow region of dividing immortalized control and profilin-deficient cells. Selected time points were taken from time-lapse recordings of live cells. Upper panel, confocal section; lower panel, pseudocoloured intensity profile. Time (in minutes and seconds) starts at the onset of anaphase. Bar, 10 μm. (E) Plots of furrow diameter over time after anaphase onset. The furrow diameter is shown for three representative examples of control and profilin-deficient cells. Download figure Download PowerPoint Prolonged and failed cytokinesis can be frequently linked to an abnormal midbody structure (Gromley et al, 2005; Zhao et al, 2006). Therefore we analysed the midbody of control and Col2pfn1 chondrocytes by immunofluorescence for known midbody proteins and by electron microscopy. Midbodies of primary Col2pfn1 chondrocytes show normal MKLP-1 and Aurora B localization (Supplementary Figure 4A). Electron microscopy images indicate similar morphology of midbody from control and profilin-deficient chondrocytes (Supplementary Figure 4B–E). Thus, defects in midbody structure do not account for the abscission defects in Col2pfn1 chondrocytes. On the basis of these observations, we hypothesized that Col2pfn1 chondrocytes could be impaired in developing the traction forces required for daughter cell separation. This hypothesis is further supported by the analysis of F-actin distribution in control and Col2pfn1 chondrocytes. Actin filament polymerization and reorganization are important factors for traction force generation (reviewed in Wang and Lin, 2007). Although control cells exhibit a defined actin cytoskeleton with stress fibres during later stages of cytokinesis, stress fibres are largely absent in Col2pfn1 chondrocytes (Figure 4A). To analyse traction forces semiquantitatively, we performed traction force microscopy (TFM) on dividing immortalized control and profilin-deficient cells cultured on elastic polyacrylamide substrates (Dembo and Wang, 1999). Similar to primary Col2pfn1 chondrocytes (Figure 3A–C), immortalized profilin-deficient chondrocytes show also a strongly reduced re-spreading during cell division and failure to form lamellipodia (Figure 4B). Two representative deformation diagrams determined over 40 min following complete cleavage furrow constriction are shown in Figure 4B; raw data to this figure are provided in Supplementary Videos S6 and S7. Active substrate deformation by the control cell occurred predominantly at the leading edges of the lamellipodia of the two daughter cells, whereas relaxation took place in the area comprising the contracting rear ends and the midbody. In contrast, deformation forces were smaller and less directional in profilin-deficient cells. We next measured the marker displacement amplitude in a defined perimeter around the cells as an indicator for the total deformation force over the observation period. We found that the displacement amplitude was shifted to significantly lower values in profilin-deficient cells (Figure 4C). Quantification of the mean displacement amplitude showed that this parameter is three times as large for control cells as for profilin-deficient cells (Figure 4D). Thus, Col2pfn1 chondrocytes can assemble and constrict actomyosin rings, but fail to generate sufficient traction force to achieve a timely daughter cell separation. Figure 4.Col2pfn1 chondrocytes fail to complete abscission during late cytokinesis. (A) Control and Col2pfn1 chondrocytes during late cytokinesis stained with an antibody against tubulin and fluorescently labelled phalloidin to visualize F-actin. Bar, 10 μm. (B) Representative deformation maps of control and profilin-deficient cells on a flexible polyacrylamide substrate during cytokinesis. Arrow direction and colour indicate deformation direction and magnitude (blue<green<yellow 100 cells, ***P⩽0.001 analysed by Mann–Whitney test). (C) Quantification of spreading area of the indicated immortalized cell lines seeded on fibronectin for 24 h (mean±s.d., n>100 cells, NS=not significant, ***P⩽0.001 analysed by Mann–Whitney test). (D) Trajectories of individual primary control and Col2pfn1 cells from the frame-by-frame analysis of time-lapse recordings during a 90-min observation period. The migration velocities of the respective primary cells are indicated (mean±s.d., migration data of over 110 cells from four independent control–Col2pfn1 pairs). (E) Quantification of cell migration velocity of the indicated immortalized cell lines (mean±s.d., migration data of over 140 cells for each cell line from three independent experiments were pooled, NS=not significant, ***P⩽0.001 analysed by Mann–Whitney test). Rescue of profilin-deficient cells with wild-type profilin 1 restores the migratory behaviour. Download figure Download PowerPoint The examination of FA and actin stress fibre formation revealed that both structures critically depend on profilin. Although FAs formed 3 h after seeding on fibronectin in control cells, they could only be observed after 12 h in Col2pfn1 chondrocytes. Stress fibres in Col2pfn1 chondrocytes were reduced in numbers, were short and thick, and oriented in a random manner (Figure 6A). In contrast, wild-type chondrocytes developed a robust stress fibre system that was
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