Automated segmentation and quantification of actin stress fibres undergoing experimentally induced changes

Rogge, Henrik; Artelt, Nadine; Endlich, Nicole; Endlich, Karlhans

doi:10.1111/jmi.12593

Cited by 35 publications

(37 citation statements)

References 31 publications

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“…To quantify these differences in F-actin, we used a published MATLAB script for segmentation and analysis of actin fibers (Rogge et al, 2017). This confirmed the visual impression of a reduction in apical actin fibers, and an increase in basal actin fibers ( Fig 5A-E).…”

Section: Trip6 Influences the Organization Of Actin And Myosinsupporting

confidence: 68%

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TRIP6 is required for tension at adherens junctions

Venkatramanan

Valenzuela

Irvine

2020

Preprint

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Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for recruitment of Vinculin and VASP to adherens junctions. Knockdown of TRIP6 or Vinculin, but not of LIMD1, also influences the localization of phosphorylated myosin light chain and F-actin. In TRIP6 knockdown cells actin stress fibers are lost apically but increased basally, and there is a corresponding increase in recruitment of Vinculin and VASP to basal focal adhesions. These observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

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Section: Trip6 Influences the Organization Of Actin And Myosinsupporting

confidence: 68%

“…Actin stress fibers were quantified using the FSegment MATLAB script developed by Rogge et al (2017)…”

Section: Actin Segmentation and Quantitationmentioning

confidence: 99%

TRIP6 is required for tension at adherens junctions

Venkatramanan

Valenzuela

Irvine

2020

Preprint

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“…Cells were stimulated with glucan at 8 µg/ml and imaged in Olympus FV1000 confocal system (optical description similar to contractility assay except for using 60x super-corrected, 1.40 NA, Plan-Apochromat oil immersion objective) with controlled temperature, 37°, 5% CO 2 for 10 minutes. Actin stress fibers were quantified using FSegment matlab script developed by Rogge et al 49 Fraction of actin in linear form was quantified before stimulation and 5 minutes after stimulation.…”

Section: M1 Macrophagesmentioning

confidence: 99%

RHOA mediated mechanical force generation through Dectin-1

Choraghe

Kołodziej

Buser

et al. 2019

Preprint

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Dectin-1 is an innate immune pattern recognition receptor which recognizes β-glucan on the Candida albicans (C. albicans) cell wall. Recognition of β-glucan by immune cells leads to phagocytosis, oxidative burst, cytokine and chemokine production. We looked for specific mechanisms that coordinate phagocytosis downstream of Dectin-1 leading to actin reorganization and internalization of fungus. We found that stimulation of Dectin-1 by soluble b-glucan leads to mechanical force generation and areal contraction in Dectin-1 transfected HEK-293 cells and M1 macrophages. With inhibitor studies, we found this force generation is a SYK-independent, SFK (SRC Family Kinase)-dependent process mediated through the RHOA-ROCK-MLC pathway. We confirmed activation of RHOA downstream of Dectin-1 using G-LISA and stress fiber formation. Through phagocytosis assays, we found direct evidence for importance of RHOA-ROCK-MLC pathway in process of phagocytosis of C. albicans. In conclusion, we found evidence for RHOA-ROCK-MLC mediated mechanical force generation downstream of Dectin-1 for C. albicans phagocytosis.

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“…To investigate the actin cytoskeleton, .150 podocytes were analyzed with the software F_Seg. 19 For quantification of focal adhesion area, cultured podocytes were stained for vinculin and at least 60 cells per group…”

Section: Actin Dynamics and Focal Adhesion Analysismentioning

confidence: 99%

The Role of Palladin in Podocytes

Artelt

Ludwig

Rogge

et al. 2018

JASN

Self Cite

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Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and -actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Palladin has an important role in podocyte function and.

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Automated segmentation and quantification of actin stress fibres undergoing experimentally induced changes

Cited by 35 publications

References 31 publications

TRIP6 is required for tension at adherens junctions

TRIP6 is required for tension at adherens junctions

RHOA mediated mechanical force generation through Dectin-1

The Role of Palladin in Podocytes

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