Podosome‐like structures of non‐invasive carcinoma cells are replaced in epithelial‐mesenchymal transition by actin comet‐embedded invadopodia

Abstract

Podosomes and invadopodia are actin-based structures at the ventral cell membrane, which have a role in cell adhesion, migration and invasion. Little is known about the differences and dynamics underlying these structures. We studied podosome-like structures of oral squamous carcinoma cells and invadopodia of their invasive variant that has undergone a spontaneous epithelial-mesenchymal transition (EMT). In 3D imaging, podosomes were relatively large structures that enlarged in time, whereas invadopodia of invasive cells remained small, but were more numerous, degraded more extracellular matrix (ECM) and were morphologically strikingly different from podosomes. In live-cell imaging, highly dynamic, invadopodia-embedded actin tails were frequently released and rocketed through the cytoplasm. Resembling invadopodia, we found new club-ended cell extensions in EMT-experienced cells, which contained actin, cortactin, vinculin and MT1-matrix metalloproteinase. These dynamic cell extensions degraded ECM and, in field emission scanning electron microscopy, protruded from the dorsal cell membrane. Plectin, αII-spectrin, talin and focal adhesion kinase immunoreactivities were detected in podosome rings, whereas they were absent from invadopodia. Tensin potentially replaced talin in invadopodia. Integrin α3β1 surrounded both podosomes and invadopodia, whereas integrin αvβ5 localized only to invadopodia heads. Pacsin 2, in conjunction with filamin A, was detected early in podosomes, whereas pacsin 2 was not found in invadopodia and filamin A showed delayed accumulation. Fluorescence recovery after photobleaching indicated faster reorganization of actin, cortactin and filamin A in podosomes compared to invadopodia. In conclusion, EMT affects the invasion machinery of oral squamous carcinoma cells. Non-invasive squamous carcinoma cells constitutively organize podosomes, whereas invasive cells form invadopodia. The club-ended cell extensions, or externalized invadopodia, are involved in ECM degradation and maintenance of contact to adhesion substrate and surrounding cells during invasion.

Tensin 3 is a new partner of Dock5 that controls osteoclast podosome organization and activity

“…Tensin 1 (Tns1), which was initially identified in chicken, has been shown to colocalize with vinculin and to surround cortactin at the podosome belt of chicken osteoclasts (Hiura et al, 1995). Since then, a report mentions the absence of tensin in the podosomes of non-invasive squamous cell carcinoma cell line UT-SCC-43A (Takkunen et al, 2010). Here, we identified tensin 3 as a partner of Dock5 in osteoclasts.…”

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

“…FlnA is composed of an N-terminal tandem calponin-homology actin-binding domain (ABD), followed by 24 immunoglobulin-like repeats, which are structurally separated into a linear rod 1 (repeats 1-15), a compact rod 2 (repeats [16][17][18][19][20][21][22][23], and a C-terminal self-association domain (repeat 24). 36 GST-PACSIN2 F-BAR domain was incubated with His-tagged constructs containing FlnA ABD, repeats 1 to 8, 8 to 15, 16 to 23, or 24, because FlnA interacts with the PACSIN2 F-BAR domain.…”

“…[5][6][7][8][9][10][11] The adaptor protein PACSIN2 (also called syndapin 2, FAP52) is one of the most abundant BAR/F-BAR proteins in human and mouse platelets. 12,13 PACSIN2 has been implicated in a wide range of cellular functions including cell adhesion, spreading, and migration [14][15][16][17] ; receptor internalization 18,19 ; and caveolae biogenesis. [20][21][22] PACSIN2 is composed of an N-terminal F-BAR domain, 3 central Asn-Pro-Phe motifs, which are docking sites for endocytic Eps15 homology domain proteins, 23 and a C-terminal Src homology 3 domain that interacts with actin regulatory and endocytic proteins, such as N-WASP and dynamin 2, 24,25 expression of which is required for normal MK development.…”

“…[20][21][22] PACSIN2 is composed of an N-terminal F-BAR domain, 3 central Asn-Pro-Phe motifs, which are docking sites for endocytic Eps15 homology domain proteins, 23 and a C-terminal Src homology 3 domain that interacts with actin regulatory and endocytic proteins, such as N-WASP and dynamin 2, 24,25 expression of which is required for normal MK development. 26 PACSIN2 is the only BAR/F-BAR protein reported to associate and/or colocalize with the cytoskeletal and scaffold protein filamin A (FlnA), [14][15][16] an essential regulator of platelet formation and function. 27 Heterozygous mutations in the X-linked FLNA gene in humans have been associated with altered DMS formation, giant platelets, and bleeding tendency, 28,29 and a mutation in the PRKACG gene encoding the g regulatory subunit of protein kinase A has been associated with decreased platelet FlnA expression and consequent macrothrombocytopenia in humans.…”

“…[14][15][16] Their interaction was therefore investigated in human platelets, both resting and activated with 10 mg/mL of collagen-related peptide (CRP) for 2 minutes. Platelets were lysed, and PACSIN2 and FlnA were immunoprecipitated, subjected to SDS-PAGE, and probed for FlnA ( Figure 1C).…”