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. 2013 Dec 9;8(12):e77956.
doi: 10.1371/journal.pone.0077956. eCollection 2013.

Essential function of dynamin in the invasive properties and actin architecture of v-Src induced podosomes/invadosomes

Olivier Destaing  1 , Shawn M Ferguson  2 , Alexei Grichine  3 , Christiane Oddou  3 , Pietro De Camilli  4 , Corinne Albiges-Rizo  5 , Roland Baron  6
Affiliations

Essential function of dynamin in the invasive properties and actin architecture of v-Src induced podosomes/invadosomes

Olivier Destaing et al. PLoS One. .

Abstract

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Dynamin is dynamically associated with actin reorganization and extracellular matrix degradation activity of invadosome rosettes.
A) Extracted images from time serie (min) from representative observations of SKO-v-Src-MEFs expressing Dyn2-pTRFP and spread on gelatin-OregonGreen degradable surface. As shown in the zoom corresponding to the red square, dyn2-pTRFP is localized in rosette (red dash circle), and is present all along the degradative activity of the structure. B–C) Extracted images from time serie (min∶s) from representative observations of SKO-v-Src-MEFs expressing Dyn2-pTRFP in association with GFP-actin and GFP-cortactin. The dynamic of invadosome ring is based on a treadmilling movememnt based on the polymerization of new actin structures at the outer rim and depolymerization of older actin structures at the inner rim of the ring. Dyn2-pTRFP is perfectly colocalized with GFP-actin and GFP-cortactin during expansion of the invadosome ring. D) Zoom on invadosome ring expansion. Quantification of fluorescence intensity (8bits color image coded from 0 to 255 levels) of Dyn2-pTRFP and GFP-actin or GFP-paxillin allowed generating the intensity profile intensity along the yellow line (24 px) in the merged image. Dyn2-pTRFP colocalized either with GFP-actin either with GFP-paxillin present also at the region of actin depolymerization (inner rim). Scale bar = 5 µm (A), 3 µm (B, C) and 0,5 µm (D).
Figure 2
Figure 2. Dynamin is essential for invadosome formation.
A) DKO-v-Src-MEFs cells were obtained 7 days after CRE recombinase expression. Dynamin depletion induced the desorganization of invadosomes (isolated or organized into rings) visualized by phalloidin staining. Endogenous dynamin 2 is concentrated in invadosome and this staining is specific while not present in DKO-v-Src-MEFs cells. Moreover, higher magnification of the areas in the white squares is showing the replacement of invadopodia in DKO-v-Src-MEFs by small cytosolic actin spots. B) Endogenous dynamin 2 is almost not detectable in cells 4 days after expression of the CRE recombinase. C) Quantification of the percentage of cells forming invadosome revealed that almost 95% of SKO-v-Src-MEFs treated with the CRE recombinase are not forming these structures 7 days post infection (n = 650 counted cells per conditions). Scale bar = 10 µm (A).
Figure 3
Figure 3. Desorganization of invadosome induced by dynamin depletion inhibits degradative and invasive properties of DKO-v-Src-MEFs.
A) Extracted images from time serie from representative observations of SKO-v-Src-MEFs and DKO-v-Src-MEFs expressing VASP-RFP and spread on Gelatin-OregonGreen degradable surface. VASP-RFP is localized in adhesion structures as invadosome rings and abolished degradative properties of DKO-v-Src-MEFs (B). C) Invasion is monitored by quantification of the average number of cells per field of observation (32×, 20 fields measured per conditions) of SKO-v-Src-MEFs and DKO-v-Src-MEFs migrating throug a thick layer of matrigel recovering a Boyden chamber in response to a serum chemiotactic gradient. Dynamin depletion inhibits invasive properties of DKO-v-Src-MEFs. Scale bar = 10 µm (A), 50 µm (B).
Figure 4
Figure 4. Dynamin function in invadosome is dependent of a coordinated activity of its GTPase, PH and GED domains.
A) Re-expression of multiple dynamin mutants (450≤n<650 invadosomes) revealed that GTPase, PH domain and PRD domains have cooperative functions to rescue invadosome formation in in DKO-v-Src-MEFs. B) The PRD is only essential domain for dynamin localization in F-actin structures (small actin punctates or invadosomes). C) Amino-acyl sequence of the PRD domain of mouse dynamin 1 where the sites of the multiple stop in the PRD sequence are indicated by an asterisk (*). Increasing PRD length is essential to rescue invadosome formation in DKO-v-Src-MEFs (450≤n<650 invadosomes). D) The ability to rescue invadosome formation is linked with the ability to relocalize to F-actin structures. Scale bar = 5 µm (A, B).
Figure 5
Figure 5. Specific dynamin photoinactivation led to the rapid invadosome disorganization.
A) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-KR and GFP-paxillin. Dyn2-KR is functionnal while localized properly and rescued invadosome formation. KillerRed 45 s light irradiation is followed by the rapid dissociation of invadosome ring (dashed line) stained by GFP-paxillin also localized in focal adhesions (red arrows). B) Extracted images from time serie (min∶s) from representative observations of SKO-v-Src-MEFs expressing KillerRed (KR) alone and GFP-paxillin. Non-localized ROS production after light irradiation of KillerRed alone does not destabilize adhesions structures. C) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-pTRFP (same excitation/emission spectrum but much more photostable than KillerRed) and GFP-paxillin. Light irradiation without ROS production is not sufficient to dissociate invadosome structures. D) Extracted images from time serie (min∶s) from representative observations of SKO-v-Src-MEFs expressing GFP-paxillin and dyn2-KR. In presence of endogenous dynamin, photoinactivation of dyn2-KR has no effect on invadosome organization showing the localized and specificity of ROS produced by the photosensitizer on the proteins fused to it. E) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-GFP and dyn2-KR. ROS production at the level of the GTPase has no effect on dyn2-GFP stability visualizing and confirming the previous experiment. F) Distribution of the percentage of cells where invadosome structures is disorganized×min after light irradiation. The >17,5 min category corresponds to experiments where invadosome presented a life-span superior to 17,5 min (distributed between 20 and 40 min, depending of the duration of each movies) and pulled altogether. 16 to 56 cells per conditions were monitored. Scale bar = 2 µm (A, E), 4 µm (B, C, D).
Figure 6
Figure 6. Photoinactivation revealed a direct and specific function of dynamin in actin organization of invadosome.
A–B) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-KR and GFP-actin. Dynamin photoinactivation leads to a rapid desorganization of actin in invadosome ring and the slow formation of lamellipodia (red arrows) and accumulation of cytosolic actin spots. B) Filopodia (green arrow) and lamellipodia (red arrow) are formed independently of dynamin. Scale bar = 2 µm (A), 4 µm (B, C).
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