In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis

Pijuan, Jordi; Barceló, Carla; Moreno, David F.; Maiques, Óscar; Sisó, Pol; Martí, Rosa M.; Macià, Anna; Panosa, Anaïs

doi:10.3389/fcell.2019.00107

Cited by 380 publications

(297 citation statements)

References 24 publications

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“…To minimize the risk of phototoxicity, we restricted image acquisition of the fluorescent mTurquoise2 channel for our transfected cells to the last frame alone. We utilized a similar individual cell tracking data analysis approach as described in (Pijuan et al ., 2019). Cell motility was tracked using the manual tracking function in ImageJ.…”

Section: Methodsmentioning

confidence: 99%

The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

Pedrosa

Nogueira

Thwaites

et al. 2018

Preprint

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29The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. 30We observed that infection of various cell types, including fibroblasts and epithelial cells resulted 31 in the formation of unusually stable focal adhesions that resisted disassembly induced by the 32 myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the 33 vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while 34 vinculin remained in its normal position within the force transduction layer. The candidate type III 35 effector TarP which localized to focal adhesions during infection and when expressed 36 ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant 37 phenotypes. These effects of TarP, including its localization to focal adhesions required 38 interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. 39The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and 40 enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia 41 inserts TarP within focal adhesions to alter their organization and dynamics. 48attachment to the extracellular matrix, and their assembly and turnover are exquisitely regulated 49 at multiple levels, by kinases, phosphatases, protein-protein interactions, internalization of 50 components, and degradation (Borradori and Sonnenberg, 1999; Geiger et al., 2001; Rosenblatt 51 et al., 2001; Zaidel-Bar et al., 2007). Disruption of one or more of these regulatory processes 52 alters the adhesion dynamics and properties of the cells. 53One strategy employed by bacteria to neutralize exfoliation relies on the precise 54 targeting of one or more components of the focal adhesion proteome. The best-characterized 55 example is that of Shigella, which neutralizes epithelial extrusion to colonize the epithelium 56 efficiently (Kim et al., 2009). It does so by delivering the OspE effector by the type III secretion 57 system (T3SS). This protein reinforces host cell adherence to the basement membrane by 58 interacting with integrin-linked kinase (ILK), a serine/threonine kinase that is part of the focal 59 adhesome (Kim et al., 2009; Zaidel-Bar et al., 2007). A consequence of the OspE-ILK 60 interaction is an increased surface expression of β1-integrin, which in turn promotes focal 61 adhesion (FA) assembly. In addition, the OspE-ILK complex stabilizes the focal adhesions (FAs) 62 by reducing phosphorylation of focal adhesion kinase (FAK) at a functionally important Tyr397 63 residue and of paxillin. Inhibition of both phosphorylation events has been shown to induce FA 64 disassembly (Kim et al., 2009). Interestingly, some EPEC and EHEC strains, as well as 65 Citrobacter rodentium possess the effector EspO, which shares strong homology with OspE 66 (reviewed in Vossenkämper, Macdonald and Marchès, 2011; Morita-Ishihara et al., 2013). As 67 such, it is conceivable that these pathogens also reinforce ...

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Section: Methodsmentioning

confidence: 99%

The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

Pedrosa

Nogueira

Thwaites

et al. 2018

Preprint

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“…Maiques and co-workers assessed the effect of TTCC blockers on the migration and invasion of melanoma cells using wound-healing, single-cell, and transwell assays [66]. We showed that TTCC blockers reduce the migration rates and invasive potential of BRAF V600E -mutant melanoma cells due to autophagy inhibition, with no significant effect in NRAS-mutant melanoma cells ( Table 1; Figure 2) [20,66]. Melanoma cell invasion and subsequent metastasis are hallmarks of melanoma dissemination [67].…”

Section: Migration and Invasionmentioning

confidence: 97%

T-Type Calcium Channels: A Potential Novel Target in Melanoma

Barceló

Sisó

Maiques

et al. 2020

Cancers

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T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAF V600E -mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.

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“…The lower chamber was lled with 600 μl of 1640 medium supplemented with 20% FBS. After 24 hours of incubation at 37°C with 5%CO 2 , cells remaining in the upper membrane surface were removed with a cotton swab, whereas invaded cells were xed and stained with 0.5% crystal violet [8].…”

Section: Transwell Assaymentioning

confidence: 99%

The role of long noncoding RNA AL161431.1 in the development and progression of pancreatic cancer

Fan

et al. 2020

Preprint

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Background: Pancreatic cancer is known for its notorious fast progression and poor prognosis. Various long noncoding RNAs (lncRNAs) have been shown to be involved in the pathogenesis processes of pancreatic cancer.Methods: We first identified lncRNA AL161431.1 through bioinformatic analysis. Then, we explored the role of lncRNA AL161431.1 in the development and progression of pancreatic cancer by in vitro and in vivo experiments, including qRT-PCR, Western blot, immunofluorescence and immunohistochemistry assays, and flow cytometry, in BxPC-3 and SW1990 cells, as well as clinical samples. Results: We found that lncRNA AL161431.1 was highly expressed in patients with pancreatic cancer. Knock down of lncRNA AL161431.1 led to increased cancer cell death and cell cycle arrest. Xenograft growth of SW1990 cells with stable knockdown of lncRNA AL161431.1 in mice was significantly slower than that of SW1990 cells with scrambled control shRNA. Finally, we showed the involvement of lncRNA AL161431.1 in pancreatic cancer was related to its promotion of the epithelial mesenchymal transition pathway.Conclusions: LncRNA AL161431.1 is involved in the progression of pancreatic cancer through its promotion of the epithelial mesenchymal transition pathway.

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In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis

Cited by 380 publications

References 24 publications

The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

T-Type Calcium Channels: A Potential Novel Target in Melanoma

The role of long noncoding RNA AL161431.1 in the development and progression of pancreatic cancer

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