Dynamin function in invadosome is dependent of a coordinated activity of its GTPase, PH and GED domains.

<p>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).</p

Desorganization of invadosome induced by dynamin depletion inhibits degradative and invasive properties of DKO-v-Src-MEFs.

<p>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).</p

Electron microscopy of CHO-K1 cells (a–d) incubated with HAdV5F35 at 10,000 vp/cell in the presence of FX (8 µg/ml), and harvested after 2 h at 37°C.

<p>(<b>a</b>), Representative CHO-K1 cell section showing a cytoplasmic vesicle containing abundant electron dense material. (<b>b–d</b>), Cell surface-bound HAdV5F35 particles. (<b>e</b>), CHO-CD46 cells incubated with HAdV5F35 in the absence of FX (w/o FX). Note the difference in size and sharpness of the viral contour between HAdV5F35 particles seen in (e) and in (b–d).</p

Dynamin is dynamically associated with actin reorganization and extracellular matrix degradation activity of invadosome rosettes.

<p>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).</p

Cellular uptake and extracellular release of HAdV5wt and HAdV5F35 vectors by CHO-K1 cells.

<p>(<b>A</b>), Cell attachment of vector (MOI 5,000) was performed at 4°C for 1 h, and cellular internalization at 37°C for 1 h, respectively, with or without FX (8 µg/ml), as indicated on the <i>x</i>-axis. The number of viral genome copies was determined by qPCR of the fiber gene, normalized to the ß-actin gene. (<b>B</b>), Extracellular vectors associated with microvesicles (MVs) or exosomes (EXOs) recovered from the extracellular medium at 72 h post transduction, were determined as above.</p

Specific dynamin photoinactivation led to the rapid invadosome disorganization.

<p>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).</p

Dynamin is essential for invadosome formation.

<p>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).</p

SPR analysis of the <i>in vitro</i> binding of chimeric HAdV5F35 vector to (A) surface-immobilized FX, or (B, C) immobilized HS with or without FX or FXGL.

<p>(<b>A</b>), Representative sensorgrams for HAdV5wt vector (discontinuous lines) injected at 2×10⁹ and 4×10⁹ vp/ml, or for HAdV5F35 injected at the same doses (solid lines). (<b>B</b>), Comparison of binding to HS of HAdV5wt and HAdV5F35 vector particles (2×10⁹ vp/ml) in the presence of FX or FXGL at 720 copies per vector particle. Controls shown are FX and FXGL alone. For better clarity, the sensorgrams for virions alone, which superimposed those of FX and FXGL, are not shown (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018205#pone-0018205-g001" target="_blank">Fig. 1C</a>). (<b>C</b>), Dose-response effect of FX on HAdV5F35 binding to immobilized HS. Note that a detectable signal was observed for 120 copies of FX per virion, and reached the maximal value for 480 copies/vp.</p

Photoinactivation revealed a direct and specific function of dynamin in actin organization of invadosome.

<p>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).</p

Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000 vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml) for 2 h at 37°C.

<p>(<b>A</b>), (i) and (ii): General views of cell sections showing (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of the space between the vector particle and the inner leaflet of the vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a tangential section. (<b>B</b>), (i): vector particle within an endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core seen in the process of traverse of the nuclear pore.</p