scholarly journals Analysis of Actin and Focal Adhesion Organisation in U2OS Cells on Polymer Nanostructures

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jakob B. Vinje ◽  
Noemi Antonella Guadagno ◽  
Cinzia Progida ◽  
Pawel Sikorski
Keyword(s):  
Quantitative Analysis ◽  
Actin Cytoskeleton ◽  
Flat Glass ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Flat Surfaces ◽  
Pillar Arrays ◽  
U2os Cells ◽  
Glass Surfaces ◽  
Nanopillar Arrays

Abstract Background In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays. This change is analogous to similar responses for cells seeded on soft substrates. Results In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays. This change is analogous to similar responses for cells seeded on soft substrates. Conclusion Overall, we show that the combination of high throughput nanofabrication, advanced optical microscopy, molecular biology tools to visualise cellular processes and data analysis can be used to investigate how cells interact with nanostructured surfaces and will in the future help to create culture substrates that induce particular cell function. Graphic Abstract

scholarly journals Actin cytoskeletal and focal adhesion organisation in U2OS cells on polymer nanostructures

2020 ◽  
Author(s):  
Jakob Vinje ◽  
Noemi Antonella Guadagno ◽  
Cinzia Progida ◽  
Pawel Sikorski
Keyword(s):  
Flat Glass ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Complex Situation ◽  
Flat Surfaces ◽  
Physiological Relevance ◽  
Pillar Arrays ◽  
U2os Cells

Cells in their natural environment are embedded in a complex surrounding consisting of biochemical and biomechanical cues directing cell properties and cell behaviour. Nonetheless, in vitro cell studies are typically performed on flat surfaces, with clear differences from the more complex situation cells experience in vivo. To increase the physiological relevance of these studies, a number of advanced cellular substrates for in vitro studies have been applied. One of these approaches include flat surfaces decorated with vertically aligned nanostructures. In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays.

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.

scholarly journals Cell response to substrate rigidity is regulated by active and passive cytoskeletal stress

2020 ◽  
Vol 117 (23) ◽  
pp. 12817-12825 ◽  
Author(s):  
Bryant L. Doss ◽  
Meng Pan ◽  
Mukund Gupta ◽  
Gianluca Grenci ◽  
René-Marc Mège ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Tumor Formation ◽  
Cell Stiffness ◽  
Matrix Rigidity ◽  
Cytoskeleton Organization ◽  
Local Rigidity ◽  
Rigidity Sensing ◽  
Pillar Arrays

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.

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 GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli

2021 ◽  
Author(s):  
Nathan Hodson ◽  
Michael Mazzulla ◽  
Maksym N. H. Holowaty ◽  
Dinesh Kumbhare ◽  
Daniel R. Moore
Keyword(s):  
Skeletal Muscle ◽  
Focal Adhesion ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Focal Adhesions ◽  
Immunofluorescent Staining ◽  
Whole Body ◽  
Vastus Lateralis Muscle ◽  
Cell Periphery ◽  
Rps6 Phosphorylation

Following anabolic stimuli (mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or prior to translocation (i.e. in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7;23±5yrs;76.8±3.6kg;13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7;22±2yrs;78.1±3.6kg;12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

scholarly journals Different translation dynamics of β-and γ-actin regulates cell migration

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pavan Vedula ◽  
Satoshi Kurosaka ◽  
Brittany MacTaggart ◽  
Qin Ni ◽  
Garegin Papoian ◽  
...  
Keyword(s):  
Cell Migration ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Level ◽  
Focal Adhesions ◽  
Mesenchymal Cell ◽  
Cell Periphery ◽  
Pronounced Difference ◽  
Coding Sequence

β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequence, however it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using the β-actin 3′UTR, β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at the focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

scholarly journals Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

2021 ◽  
Author(s):  
Erik S Linklater ◽  
Emily Duncan ◽  
Ke Jun Han ◽  
Algirdas Kaupinis ◽  
Mindaugas Valius ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Migration And Invasion ◽  
Matrix Remodeling ◽  
Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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