The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts

Abstract

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src(-/-) OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src(-/-) OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src(-/-) podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src(-/-) OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src(-/-) OCLs by Src inhibitors or by expressing dominant-negative Src(K295M) induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.

Figure 1.

Src controls podosome formation and actin organization in osteoclasts. (A) F-actin staining with phalloidin revealed that Src^−/− OCLs form fewer podosomes. Podosome numbers were determined by counting 20 different cells per condition (cell size normalized to the number of nuclei). Phalloidin staining of Src^+/− OCLs revealed the presence of the actin cloud around podosome cores (indicated by red arrow). (B) Src^−/− OCLs do not form podosome belts but form actin ruffles at the top of the cell (4 μm above the cell substrate interface), which are absent from Src^+/− OCLs (scale bar, 10 μm).

Figure 2.

Src activity regulates the actin cloud. (A) Left panels, Src^+/− osteoclasts, Src^−/− osteoclasts, and Src^−/− osteoclasts treated with the Src family kinase inhibitor PP1 were fixed and stained for vinculin (green) and F-actin (red). The actin channel (center panels, magnification of areas in green square in left panels) clearly shows the extensive presence of the less dense actin cloud between individual podosome cores in the Src^+/− cells. Processing the actin images with the “3D surface plot” plug-in (right panels) revealed many podosomes with an associated cloud (blue arrow) and some without (red arrow). The actin clouds were present but much less extensive in the Src^−/− cells, and treatment with PP1 essentially eliminated the clouds. (B) Time series sequence extracted from observation of Src^−/− OCLs microinjected with GFP-actin and empty vector (top) or GFP-actin and Src^WT (bottom, scale bar, 3 μm).

Figure 3.

Src regulates podosome life span and the actin flux in podosomes. (A) Time series (in min) extracted from representative observations of Src^+/− (top) or Src^−/− (bottom) OCLs expressing GFP-actin. Red arrows indicate individual podosomes. The absence of c-Src induced the presence of long-lived podosomes (scale bar, 4 μm). (B) Distribution of podosome life span for Src^+/− and Src^−/− OCLs. The life spans of a total 500 podosomes were measured in 3–5 independent experiments (*p < 0.001). (C) FRAP analysis was performed on Src^+/− and Src^−/− OCLs expressing GFP-actin. The change in fluorescence intensity of a podosome (red square) within the bleached area (green square) was quantified over 2 min, and the characteristic time of recovery (τ) was determined. GFP-actin fluorescence recovered faster in the core of the Src^+/− podosome (arrow, τ = 13.72 s) than in the core of Src^−/− podosomes (arrow, τ = 28.16 s; scale bar 2 μm). (D) 15–20 τ values for Src^+/− OCLs and Src^−/− OCLs expressing GFP-actin + empty vector or GFP-actin + Src^WT were determined in two independent experiments. Values are presented as a percentage of the average τ of Src^−/− podosomes (*p < 0.05).

Figure 4.

Src kinase activity is essential for normal podosome organization. (A) Microinjection of the cDNA coding for GFP-actin did not affect podosome organization in Src^−/− OCLs. Re-expression of Src^WT in Src^−/− OCLs restored normal podosome organization. In contrast, expression of kinase-dead Src (Src^K295M/Y527F) induced the formation of isolated podosomes with little or no actin cloud (see inset). The Src mutant with both SH2 and SH3 disabled, Src^W118K/R175L, did not restore podosome organization, and was localized in the actin ruffles at the top of the Src^−/− OCLs, not around podosomes (scale bar, 10 μm). (B) To determine whether the Src^W118K/R175L mutant can phosphorylate substrates in vivo, Src^−/− OCLs were comicroinjected with paxillin-GFP (as a reporter of microinjected cell) and Src^WT or Src^W118K/R175L. After 6 h, cells were fixed and stained for phosphotyrosine. Expression of Src^WT induced an increase of phosphotyrosine within the cell, whereas Src^W118KR175L did not (scale bar, 10 μm).

Figure 5.

Re-expression of Src^Y527F, Src^W118K, or Src^R175L in Src^−/− OCLs restored normal podosome organization. In contrast, expression of kinase-dead Src (Src^K295M) induced the formation of isolated podosomes (scale bar, 10 μm).

Figure 6.

Src kinase activity is essential to rescue dynamic podosome properties in Src^−/− OCLs. GFP-actin was comicroinjected into Src^−/− OCLs with the indicated Src mutant. The life spans of an average of 500 podosomes per condition in 6–9 cells from two to four independent experiments were determined. Expression of Src^WT, Src^Y527F, Src^R175L, or Src^W118K restored podosome life span to values comparable to those of podosomes observed in Src^+/− OCLs. Src^W118K/R175L did not affect life span of podosomes in Src^−/− OCLs. In contrast, expression of Src kinase-dead mutants induced formation of long-lived podosomes, with an average life span greater than 25 min (*p < 0.001 relative to Src^+/−; #p < 0.001 relative to Src^−/−).

Figure 7.

Complete inhibition of Src family kinase activities in Src^−/− OCLs induces the collapse of the components of the cloud into the podosome core. (A) Src^−/− OCLs were microinjected with cDNAs encoding Src^WT or Src^K295M/Y527F and stained for F-actin (with phalloidin) or Src (anti-avian Src). In the cells expressing Src^WT (top panel), podosome cores were surrounded by a well-formed cloud, and Src staining was restricted to the cloud. In the cells expressing Src^K295M/Y527F(bottom panel), no clouds were detected and Src was localized in the outer part of the podosome cores (scale bar, 1 μm). Right panels, fluorescence intensities of F-actin (red) and Src (green) measured along length of the white line indicated in the merged figures. (B) Src^−/− OCLs were injected with cDNAs encoding dynamin2-GFP, RFP-actin, and either Src^WT or Src^K295M/Y527F and fixed. In the cells expressing Src^WT (top panel), podosome cores were surrounded by a well-formed cloud, and dynamin2-GFP fluorescence was largely outside the core. In the cells expressing Src^K295M/Y527F(bottom panel), clouds were not detected and dynamin2-GFP was colocalized with the podosome cores, although it was also present in some areas between cores where no actin was detected (scale bar, 5 μm). Right panels, fluorescence intensities of RFP-actin (red) and dynamin2-GFP (green) measured along length of the white line indicated in the merged figures. (C) Fluorescence recovery was normalized and quantified by measuring the characteristic time of recovery (τ). GFP-actin fluorescence recovers extremely rapidly in the core of long-lasting podosomes. τ values were measured in two independent experiments for Src^−/− OCLs expressing GFP-actin + empty vector, or GFP-actin + Src^WT, GFP-actin + Src^K295M/Y527F, or GFP-actin + empty vector treated with PP1 10 μM (p < 0.05).