The analysis of F-actin structure of mesenchymal stem cells by quantification of fractal dimension

The actin cytoskeleton is indispensable for the motility and migration of all types of cells; therefore, it plays a crucial role in the ability of the tissues to repair. Mesenchymal stem cells are intensively used in regenerative medicine, but usually relatively low percent of transplanted cells reaches the injury. To overcome this evident limitation, researchers try to enhance the motility and migration rate of the cells. As one of the approaches, co-cultivation and preconditioning of stem cells with biologically active compounds, which can cause actin cytoskeleton rearrangements followed by an increase of migratory properties of the cells, could be applied. The observed changes in F-actin structure induced by the compounds require quantitative estimation, and measurement of fluorescence intensity of the F-actin image captured by various microscopic techniques is commonly used nowadays. However, this approach could not always accurately detect the observed changes in the shape and structure of actin cytoskeleton. At this time, the image of F-actin has an irregular geometric pattern, and thus could be considered and characterized as a fractal object. To quantify the re-organization of cellular F-actin in terms of fractal geometry Minkovsky’s box-counting method is suitable, but it is not widely used nowadays. We modified and improved the previously described method for fractal dimension measurement, and successfully applied it for the quantification of the F-actin structures of human mesenchymal stem cells.

Mechanically induced nuclear shuttling of β-catenin requires co-transfer of actin

Mesenchymal stem cells (MSC) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin and Yes-Associated Protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles x 2%) and static (1 x 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3h after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid (LPA) did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms utilized for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin and YAP1.

Changes in Cell Morphology and Actin Organization in Embryonic Stem Cells Cultured under Different Conditions

The cellular cytoskeleton provides the cell with a mechanical rigidity that allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events such as motility or the establishment of cell polarity. From the earliest stages of development, as represented by the ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology, and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super-resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal, and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting, respectively, a homogeneous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two culturing conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.

Decoding natural astrocyte rhythms: dynamic actin waves result from environmental sensing by primary rodent astrocytes

Astrocytes are key regulators of brain homeostasis, which is essential for proper cognitive function. The role of cytoskeletal dynamics in this critical regulatory process is unknown. Here we find that actin is dynamic in certain subcellular regions, especially near the cell boundary. Our results further indicate that actin dynamics concentrates into “hotspot” regions that selectively respond to certain chemophysical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes actin dynamics more frequent yet weaker, and it also alters actin network structure. Superresolution images analyzed with a filament extraction algorithm demonstrate that surface topography is associated with a predominant perpendicular alignment of actin filaments near the cell boundary whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Thus, actin structure and dynamics together integrate information from different aspects of the environment that might steer the operation of neural cell networks.TeaserAstrocytes display dynamic actin that is modulated by combinations of chemophysical stimuli and environmental topographies.

Changes in Cell Morphology and Actin Organization in Embryonic Stem Cells Cultured Under Different Conditions

The cellular cytoskeleton provides the cell with a mechanical rigidity which allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events like motility, or establishment of cell polarity. From the earliest stages of development, as represented by ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting a homogenous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two media conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.

Bioinformatics Analysis of Actin Molecules: Why Quantity Does Not Translate Into Quality?

It is time to review all the available data and find the distinctive characteristics of actin that make it such an important cell molecule. The presented double-stranded organization of filamentous actin cannot explain the strong polymorphism of actin fibrils. In this work, we performed bioinformatics analysis of a set of 296 amino acid actin sequences from representatives of different classes of the Chordate type. Based on the results of the analysis, the degree of conservatism of the primary structure of this protein in representatives of the Chordate type was determined. In addition, 155 structures of rabbit actin obtained using X-ray diffraction analysis and electron microscopy have been analyzed over the past 30 years. From pairwise alignments and the calculation of root-mean-square deviations (RMSDs) for these structures, it follows that they are very similar to each other without correlation with the structure resolution and the reconstruction method: the RMSDs for 11,781 pairs did not exceed 3 Å. It turned out that in rabbit actin most of the charged amino acid residues are located inside the protein, which is not typical for the protein structure. We found that two of six exon regions correspond to structural subdomains. To test the double-stranded organization of the actin structure, it is necessary to use new approaches and new techniques, taking into account our new data obtained from the structural analysis of actin.

Syk facilitates phagosome-lysosome fusion by regulating actin-remodeling in complement-mediated phagocytosis

Effective phagocytosis is crucial for host defense against pathogens. Macrophages entrap pathogens into a phagosome and subsequently acidic lysosomes fuse to the phagosome. Previous studies showed the pivotal role of actin-remodeling mediated by phosphoinositide-related signaling in phagosome formation, but the mechanisms of phagosome-lysosome fusion remain unexplored. Here we show that in complement-mediated phagocytosis, phagosome-lysosome fusion requires the disappearance of F-actin structure surrounding the phagosome and a tyrosine kinase Syk plays a key role in this process. Using macrophage-like differentiated HL60 and Syk-knockout (Syk-KO) HL60 cells, we found that Syk-KO cells showed insufficient phagosome acidification caused by impaired fusion with lysosomes and permitted the survival of Candida albicans in complement-mediated phagocytosis. Phagosome tracking analysis showed that during phagosome internalization process, F-actin surrounding phagosomes disappeared in both parental and Syk-KO cells but this structure was reconstructed immediately only in Syk-KO cells. In addition, F-actin-stabilizing agent induced a similar impairment of phagosome-lysosome fusion. Collectively, Syk-derived signaling facilitates phagosome-lysosome fusion by regulating actin-remodeling.

E-cadherin: Unexpected actor of invadopodia formation in pancreatic cancer

Invasion/metastasis axis is the major driver of cancer mortality. By maintaining tissue cohesion, E-cadherin is considered as a protective marker for tumoral progression. However, recent studies suggested that the appearance of hybrid epithelial-mesenchymal (E/M) cells still expressing E-cadherin is favourable for the establishment of metastasis. This hypothesis is consistent with the observation that most pancreatic ductal adenocarcinomas (PDAC) are invasive and express E-cadherin in primary tumour and metastases. By using a data constituted by a series of patient-derived xenografts (PDX) from the PaCaOmics multi-centric clinical trial, we show that E-cadherin expression is not associated with stages of the pathology, prognosis, and overall survival in PDAC. The role of E-cadherin in PDAC aggressiveness was tested both in vitro and in cancer cell implantation models. We show that E-cadherin is a key component of membrane protrusions implicated in the extracellular matrix remodelling and degradation, called invadopodia. E-cadherin downregulation in a pancreatic model of E/M hybrid cells reveals that E-cadherin downregulates Arp2/3 complex expression. As this complex is essential for branched actin structure, E-cadherin depletion strongly impaired invadopodia formation. On the other hand, we demonstrate that E-cadherin interacts with the membrane protease MT1-MMP at the in-vadopodial membrane. E-cadherin could be recycled back to the invadopodial membrane simultaneously with MT1-MMP. Indeed, both Rab7 vesicle-dependant and/or a Rab11 vesicle-dependant pathway are required for both E-cadherin and MT1-MMP trafficking. This new localization of E-cadherin and its implication in cell invasion shines a new light on hybrid EMT features in tumoral invasion.