The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics

Berginski, Matthew E.; Gomez, Shawn M.

doi:10.3410/f1000research.2-68.v1

Cited by 29 publications

(36 citation statements)

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“…In particular, contrast was adjusted, and images smoothened and aligned using the rigid body algorithm. Focal adhesion (FA) dynamics were then analysed using the Focal Adhesion Analysis Server (Berginski & Gomez, 2013). Only FA with lifetime minimum of 10 frames was analysed, and FA that was assembled and disassembled during the course of imaging was used for measurement of FA kinetics.…”

Section: Immunofluorescencementioning

confidence: 99%

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

et al. 2016

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SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

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Section: Immunofluorescencementioning

confidence: 99%

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

et al. 2016

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“…Three different channels were acquired: blue for DAPI, green for phalloidin (actin cytoskeleton) and red for Cy3 (vinculin). Vinculin images were uploaded to the Focal Adhesion Analysis Server [39] using the actin cytoskeleton as cell mask. The images of binarized focal adhesions were measured using the Analyze particles tool in ImageJ (ImageJ 1.5b, National Institutes of Health (NIH)).…”

Section: Image Analysismentioning

confidence: 99%

PLLA/ZnO nanocomposites: Dynamic surfaces to harness cell differentiation

Trujillo

Lizundia

Vilas

et al. 2016

Colloids and Surfaces B: Biointerfaces

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Graphical abstract2 Highlights  Biodegradable poly(L-lactide) matrix loaded with ZnO nanorods. Dynamic presentation of nanorods triggered by PLLA degradation. Changes in surface properties as a function of time control cell behaviour. Nanorods exposure promotes cell differentiation.3 Keywords: PLLA, ZnO nanoparticle, C2C12 myoblast, nanocomposite, cell differentiation AbstractThis work investigates the effect of the sequential availability of ZnO nanoparticles, (nanorods of ~ 20 nm) loaded within a degradable poly(lactic acid) (PLLA) matrix, in cell differentiation. The system constitutes a dynamic surface, in which nanoparticles are exposed as the polymer matrix degrades. ZnO nanoparticles were loaded into PLLA and the system was measured at different time points to characterise the time evolution of the physicochemical properties, including wettability and thermal properties. The micro and nanostructure were also investigated using AFM, SEM and TEM images. Cellular experiments with C2C12 myoblasts show that cell differentiation was significantly enhanced on ZnO nanoparticles -loaded PLLA, as the polymer degrades and the availability of nanoparticles become more apparent, whereas the release of zinc within the culture medium was negligible. Our results suggest PLLA/ZnO nanocomposites can be used as a dynamic system where nanoparticles are exposed during degradation, activating the material surface and driving cell differentiation.4

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“…Cell areas were estimated by the cut-and-weigh method using images of cells stained by Alexa-Fluor 488 phalloidin. Focal adhesions were analyzed using the Focal Adhesion Analysis Server to quantify images of cells stained by immunofluorescence for vinculin (56,57).…”

Section: Methodsmentioning

confidence: 99%

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

Liu

Chang

Grinnell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomeraseimmortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.thoracic aortic aneurysms | ACTA2 mutation | vascular disease | cell migration | human fibroblasts

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The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics

Cited by 29 publications

References 0 publications

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

PLLA/ZnO nanocomposites: Dynamic surfaces to harness cell differentiation

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

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