scholarly journals The Role of Palladin in Podocytes

2018 ◽  
Vol 29 (6) ◽  
pp. 1662-1678 ◽  
Author(s):  
Nadine Artelt ◽  
Tim A. Ludwig ◽  
Henrik Rogge ◽  
Panagiotis Kavvadas ◽  
Florian Siegerist ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Kidney Biopsy ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Early Developmental Stage ◽  
Zebrafish Embryos ◽  
Filtration Barrier ◽  
Biopsy Specimens

Background 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.Methods 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.Results 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 α-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.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals Identification of Novel Recognition Motifs and Regulatory Targets for the Yeast Actin-regulating Kinase Prk1p

2003 ◽  
Vol 14 (12) ◽  
pp. 4871-4884 ◽  
Author(s):  
Bo Huang ◽  
Guisheng Zeng ◽  
Alvin Y.J. Ng ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Yeast Genome ◽  
Three Dimensional Model ◽  
Recognition Sequence ◽  
Yeast Saccharomyces Cerevisiae

Prk1p is a serine/threonine kinase involved in the regulation of the actin cytoskeleton organization in the yeast Saccharomyces cerevisiae. Previously, we have identified LxxQxTG as the phosphorylation site of Prk1p. In this report, the recognition sequence for Prk1p is investigated more thoroughly. It is found that the presence of a hydrophobic residue at the position of P-5 is necessary for Prk1p phosphorylation and L, I, V, and M are all able to confer the phosphorylation at various efficiencies. The residue flexibility at P-2 has also been identified to include Q, N, T, and S. A homology-based three-dimensional model of the kinase domain of Prk1p provided some structural interpretations for these substrate specificities. The characterization of the [L/I/V/M]xx[Q/N/T/S]xTG motif led to the identification of a spectrum of potential targets for Prk1p from yeast genome. One of them, Scd5p, which contains three LxxTxTG motifs and is previously known to be important for endocytosis and actin organization, has been chosen to demonstrate its relationship with Prk1p. Phosphorylation of Scd5p by Prk1p at the three LxxTxTG motifs could be detected in vitro and in vivo, and deletion of PRK1 suppressed the defects in actin cytoskeleton and endocytosis in one of the scd5 mutants. These results allowed us to conclude that Scd5p is likely another regulatory target of Prk1p.

scholarly journals mDia1 Targets v-Src to the Cell Periphery and Facilitates Cell Transformation, Tumorigenesis, and Invasion

2010 ◽  
Vol 30 (19) ◽  
pp. 4604-4615 ◽  
Author(s):  
Masahiro Tanji ◽  
Toshimasa Ishizaki ◽  
Saman Ebrahimi ◽  
Yuko Tsuboguchi ◽  
Taiko Sukezane ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Focal Adhesions ◽  
Small Gtpase ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Morphological Transformation ◽  
Focus Formation

ABSTRACT The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.

Effects of cadmium on the actin cytoskeleton in renal mesangial cells

2013 ◽  
Vol 91 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Douglas M. Templeton ◽  
Ying Liu
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Mesangial Cells ◽  
Focal Adhesions ◽  
Filamentous Actin ◽  
Human Mesangial Cells ◽  
Dependent Protein

We provide an overview of our studies on cadmium and the actin cytoskeleton in mesangial cells, from earlier work on the effects of Cd2+ on actin polymerization in vivo and in vitro, to a role of disruption or stabilization of the cytoskeleton in apoptosis and apoptosis-like death. More recent studies implicate cadmium-dependent association of gelsolin and the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with actin filaments in cytoskeletal effects. We also present previously unpublished data concerning cadmium and the disruption of focal adhesions. The work encompasses studies on rat, mouse, and human mesangial cells. The major conclusions are that Cd2+ acts independently of direct effects on cellular Ca2+ levels to nevertheless activate Ca2+-dependent proteins that shift the actin polymerization–depolymerization in favour of depolymerization. Cadmium-dependent translocation of CaMK-IIδ, gelsolin, and a 50 kDa gelsolin cleavage fragment to the filamentous (F-)actin cytoskeleton appear to be involved. An intact filamentous actin cytoskeleton is required to initiate apoptotic and apoptotic-like death, but F-actin depolymerization is an eventual result.

scholarly journals Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

1991 ◽  
Vol 114 (3) ◽  
pp. 481-491 ◽  
Author(s):  
F M Pavalko ◽  
K Burridge
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Site ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Stress Fibers ◽  
Actin Binding ◽  
Cell Periphery

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.

scholarly journals MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kung-Chi Chang ◽  
Sarah D. Diermeier ◽  
Allen T. Yu ◽  
Lily D. Brine ◽  
Suzanne Russo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Actin Cytoskeleton ◽  
Cancer Progression ◽  
Mammary Tumor ◽  
Focal Adhesions ◽  
Breast Cancer Progression ◽  
Migration And Invasion ◽  
Mammary Tumor Cell

AbstractMisregulation of long non-coding RNA (lncRNA) genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin1 (Tns1) to regulate its expression in trans. The Tns1 protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. We identify LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and metastasis. Our findings demonstrate that LINC01271 represents a potential therapeutic target to alter breast cancer progression.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals The Revolutionary Roads to Study Cell–Cell Interactions in 3D In Vitro Pancreatic Cancer Models

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 930
Author(s):  
Donatella Delle Cave ◽  
Riccardo Rizzo ◽  
Bruno Sainz ◽  
Giuseppe Gigli ◽  
Loretta L. del Mercato ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Response ◽  
Three Dimensional ◽  
Cellular Models ◽  
Common Cancer ◽  
Cancer Models ◽  
Anti Cancer ◽  
Tumor Environment

Pancreatic cancer, the fourth most common cancer worldwide, shows a highly unsuccessful therapeutic response. In the last 10 years, neither important advancements nor new therapeutic strategies have significantly impacted patient survival, highlighting the need to pursue new avenues for drug development discovery and design. Advanced cellular models, resembling as much as possible the original in vivo tumor environment, may be more successful in predicting the efficacy of future anti-cancer candidates in clinical trials. In this review, we discuss novel bioengineered platforms for anticancer drug discovery in pancreatic cancer, from traditional two-dimensional models to innovative three-dimensional ones.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

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