Geometrical constraints greatly hinder formin mDia1 activity

AbstractFormins are one of the central players in the assembly of most actin networks in cells. The sensitivity of these processive molecular machines to mechanical tension is now well established. However, how the activity of formins is affected by geometrical constraints related to network architecture, such as filament crosslinking and formin spatial confinement, remains largely unknown. Combining microfluidics and micropatterning, we reconstituted in vitro mDia1 formin-elongated filament bundles induced by fascin, with different geometrical constraints on the formins, and measured the impact of these constraints on formin elongation rates and processivity. When filaments are not bundled, formins can be anchored to static or fluid surfaces, by either end of the proteins, without affecting their activity. We show that filament bundling by fascin reduces both unanchored formin elongation rate and processivity. Strikingly, when filaments elongated by surface-anchored formins are cross-linked together, formin elongation rate immediately decreases and processivity is reduced, up to 24-fold, depending on the cumulative impact of formin rotational and translational freedoms. Our results reveal an unexpected crosstalk between the constraints at the filament and the formin levels. We anticipate that in cells, the molecular details of formin anchoring to the plasma membrane, strongly modulate formin activity at actin filament barbed ends.

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Liquid–Liquid Phase Separation in Crowded Environments

International Journal of Molecular Sciences ◽

10.3390/ijms21165908 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5908 ◽

Cited By ~ 4

Author(s):

Alain A. M. André ◽

Evan Spruijt

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Macromolecular Crowding ◽

Liquid Phase Separation ◽

The Impact ◽

Crowded Environments ◽

Liquid Liquid Phase Separation ◽

In Cells

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

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Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

10.1101/864769 ◽

2019 ◽

Author(s):

Markku Hakala ◽

Hugo Wioland ◽

Mari Tolonen ◽

Antoine Jegou ◽

Guillaume Romet-Lemonne ◽

...

Keyword(s):

Leading Edge ◽

Underlying Mechanism ◽

Actin Dynamics ◽

Dissociation Kinetics ◽

Single Filament ◽

Actin Networks ◽

Capping Protein ◽

Specific Localization ◽

In Cells

AbstractCoordinated polymerization of actin filaments provides force for cell migration, morphogenesis, and endocytosis. Capping Protein (CP) is central regulator of actin dynamics in all eukaryotes. It binds actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. In cells, however, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism has remained enigmatic. We report that a conserved cytoskeletal regulator, twinfilin, is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association of CP with cellular F-actin arrays and its treadmilling throughout leading-edge lamellipodium. These were accompanied by diminished F-actin disassembly rates. In vitro single filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while allowing subsequent filament depolymerization. These results uncover an evolutionary conserved bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

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The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex

Science Advances ◽

10.1126/sciadv.abd5956 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabd5956 ◽

Cited By ~ 1

Author(s):

Artem I. Fokin ◽

Violaine David ◽

Ksenia Oguievetskaia ◽

Emmanuel Derivery ◽

Caroline E. Stone ◽

...

Keyword(s):

Actin Filament ◽

Actin Networks ◽

Capping Protein ◽

Related Proteins ◽

In Cells

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

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Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

10.1101/2020.10.03.322354 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yashar Bashirzadeh ◽

Steven A. Redford ◽

Chatipat Lorpaiboon ◽

Alessandro Groaz ◽

Thomas Litschel ◽

...

Keyword(s):

Network Architecture ◽

Coarse Grained ◽

General Mechanism ◽

Actin Networks ◽

Cellular Processes ◽

Size Dependent ◽

Sensory Projections ◽

Coarse Grained Simulations ◽

Cytoskeletal Networks

AbstractRobust spatiotemporal organization of cytoskeletal networks is crucial, enabling cellular processes such as cell migration and division. α-Actinin and fascin are two actin crosslinking proteins localized to distinct regions of eukaryotes to form actin bundles with optimized spacing for cell contractile machinery and sensory projections, respectively. In vitro reconstitution assays and coarse-grained simulations have shown that these actin bundling proteins segregate into distinct domains with a bundler size-dependent competition-based mechanism, driven by the minimization of F-actin bending energy. However, it is not known how physical confinement imposed by the cell membrane contributes to sorting of actin bundling proteins and the concomitant reorganization of actin networks in intracellular environment. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that the size of such a spherical boundary determines equilibrated structure of actin networks among three typical structures: single rings, astral structures, and star-like structures. We show that α-actinin bundling activity and its tendency for clustering actin is central to the formation of these structures. By analyzing physical features of crosslinked actin networks, we show that spontaneous sorting and domain formation of α-actinin and fascin are intimately linked to the resulting structures. We propose that the observed boundary-imposed effect on sorting and structure formation is a general mechanism by which cells can select between different structural dynamical steady states.

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Influence of Microgravity on Apoptosis in Cells, Tissues, and Other Systems In Vivo and In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21249373 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9373

Author(s):

Binod Prasad ◽

Daniela Grimm ◽

Sebastian M. Strauch ◽

Gilmar Sidnei Erzinger ◽

Thomas J. Corydon ◽

...

Keyword(s):

Cosmic Radiation ◽

Current Knowledge ◽

Life Forms ◽

Model Systems ◽

Space Environment ◽

Cellular Damage ◽

The Impact ◽

In Cells

All life forms have evolved under the constant force of gravity on Earth and developed ways to counterbalance acceleration load. In space, shear forces, buoyance-driven convection, and hydrostatic pressure are nullified or strongly reduced. When subjected to microgravity in space, the equilibrium between cell architecture and the external force is disturbed, resulting in changes at the cellular and sub-cellular levels (e.g., cytoskeleton, signal transduction, membrane permeability, etc.). Cosmic radiation also poses great health risks to astronauts because it has high linear energy transfer values that evoke complex DNA and other cellular damage. Space environmental conditions have been shown to influence apoptosis in various cell types. Apoptosis has important functions in morphogenesis, organ development, and wound healing. This review provides an overview of microgravity research platforms and apoptosis. The sections summarize the current knowledge of the impact of microgravity and cosmic radiation on cells with respect to apoptosis. Apoptosis-related microgravity experiments conducted with different mammalian model systems are presented. Recent findings in cells of the immune system, cardiovascular system, brain, eyes, cartilage, bone, gastrointestinal tract, liver, and pancreas, as well as cancer cells investigated under real and simulated microgravity conditions, are discussed. This comprehensive review indicates the potential of the space environment in biomedical research.

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In vitro study of the impact of mechanical tension on the dermal fibroblast phenotype in the context of skin wound healing

Journal of Biomechanics ◽

10.1016/j.jbiomech.2014.07.015 ◽

2014 ◽

Vol 47 (14) ◽

pp. 3555-3561 ◽

Cited By ~ 21

Author(s):

Gwenae¨l Rolin ◽

Delphine Binda ◽

Marion Tissot ◽

Céline Viennet ◽

Philippe Saas ◽

...

Keyword(s):

Wound Healing ◽

Dermal Fibroblast ◽

In Vitro Study ◽

Skin Wound ◽

Vitro Study ◽

Mechanical Tension ◽

Skin Wound Healing ◽

The Impact

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ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells

10.1101/2020.10.17.335380 ◽

2020 ◽

Author(s):

Julia F. Riley ◽

Heidi Hehnly ◽

Carlos A. Castañeda

Keyword(s):

Protein Quality ◽

Wild Type ◽

Disease Mutations ◽

Ubiquitin Binding ◽

Condensate Formation ◽

The Impact ◽

Lateral Sclerosis ◽

Liquid Liquid Phase Separation ◽

In Cells

AbstractMutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging with photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, ectopically expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

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ETS Proto-Oncogene 1 Suppresses MicroRNA-128 Transcription to Promote Osteogenic Differentiation Through the HOXA13/β-Catenin Axis

Frontiers in Physiology ◽

10.3389/fphys.2021.626248 ◽

2021 ◽

Vol 12 ◽

Author(s):

Renyao Li ◽

Ying Dong ◽

Feipeng Li

Keyword(s):

Osteogenic Differentiation ◽

Molecular Mechanisms ◽

Phosphate Concentration ◽

Osteoblast Cell ◽

Osteoblast Cell Line ◽

Specific Antagonist ◽

Alkaline Phosphate ◽

The Impact ◽

In Cells

ETS proto-oncogene 1 (ETS1) has been implicated in osteoporosis (OP), but the exact molecular mechanisms are complex. This work focuses on the impact of ETS1 on the osteogenic differentiation and the molecules involved. A mouse pre-osteoblast cell line MC3T3-E1 was used for in vitro experiments. ETS1 was upregulated during the process of osteogenic differentiation of MC3T3-E1 cells. Overexpression of ETS1 promoted expression of osteogenic markers, alkaline phosphate concentration, and calcareous accumulation in cells. ETS1 was found to specifically bind to miR-128 promoter to suppress its transcription, while miR-128 could target homeobox A13 (HOXA13). Therefore, ETS1 suppressed miR-128 transcription to upregulate HOXA13 expression. Overexpression of HOXA13 promoted the osteogenic differentiation ability of cells and increased the protein level of β-catenin. Either overexpression of miR-128 or downregulation of β-catenin by CWP232228, a β-catenin-specific antagonist, blocked the promoting roles of ETS1 in cells. To conclude, this study provided evidence that ETS1 suppresses miR-128 transcription to activate the following HOXA13/β-catenin axis, therefore promoting osteogenic differentiation ability of MC3T3-E1 cells. This finding may offer novel ideas for OP treatment.

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The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex

10.1101/2020.05.29.123372 ◽

2020 ◽

Author(s):

Artem I. Fokin ◽

Violaine David ◽

Ksenia Oguievetskaia ◽

Emmanuel Derivery ◽

Caroline E. Stone ◽

...

Keyword(s):

Actin Filament ◽

Actin Networks ◽

Capping Protein ◽

Related Proteins ◽

In Cells

AbstractDendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the Capping Protein for unclear reasons. Here we show that that the WASH complex interacts with Dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In Dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap Dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.One Sentence SummaryDendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of Dynactin.

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364 QUANTIFICATION OF DEVELOPMENTALLY IMPORTANT GENE TRANSCRIPTS EXPRESSED IN EMBRYOS PRODUCED IN VIVO AND IN VITRO USING SEX-SORTED AND NON-SORTED RAM SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab364 ◽

2010 ◽

Vol 22 (1) ◽

pp. 338

Author(s):

K. H. Beilby ◽

S. Wilkening ◽

S. P. de Graaf ◽

C. Wrenzycki ◽

C. G. Grupen

Keyword(s):

Gene Expression ◽

Epigenetic Modification ◽

Reproductive Technologies ◽

Embryo Research ◽

Cumulative Impact ◽

Altered Expression ◽

Stage Of Development ◽

The Impact

The AI of sex-sorted spermatozoa results in decreased levels of fertility in most species. This is not the case in sheep, where low-doseAI of sex-sorted ram spermatozoa produces similar, if not superior levels of fertility to non-sorted controls (Beilby et al. 2009 Theriogenology, 71, 829-835). In an effort to provide insight into the molecular basis for this difference in fertility between species, the aim of the present study was to examine the impact of sex-sorting technology on ovine embryo gene expression. After semen collection, ejaculates (n = 8) were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen (non-sorted control). Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched spermatozoa, or non-sorted control spermatozoa, and collected by flushing uterine horns on Day 6 after AI. Embryos were produced in vitro by using established oocyte in vitro maturation and in vitro fertilization procedures (using X, Y or non-sorted spermatozoa), and cultured in vitro for 6 days. The relative abundance of Glut-3, G6PD, SUV39H1, DnMT3a, and HSP70 was measured in high grade blastocysts (in vivo Day 6: n = 23; in vitro Day 6: n = 21) using quantitative, real-time PCR (iCycler5TM, BioRad, Hercules, CA, USA). Blastocyst cell numbers were quantified to ensure embryos were at a similar stage of development. The sex of all embryos was identified by PCR to allow comparison between treatments. Fold differences in gene expression, acquired through the A ACt method, were calculated and compared by an ANOVA. The expression of HSP70 was up-regulated in in vitro embryos derived from sex-sorted spermatozoa compared to those produced in vivo (P < 0.05). For all other genes examined, there was no effect of sex or sperm treatment on gene expression. Glut-3, G6PD, SUV39H1, andDnMT3a were all up-regulated in in vitro embryos compared with in vivo embryos (P < 0.05). These results suggest that fertilization with sex-sorted ram spermatozoa does not result in aberrant patterns of gene expression in embryos produced in vivo. The data from the present study provide further evidence that in vitro culture induces epigenetic modification within the embryonic genome, when compared to the in vivo physiological standard. It would be of interest to conduct a similar in vivo study in cattle, where sex-sorting technology has not been as biologically successful. The altered expression of HSP70, which is associated with cellular stress, may demonstrate a cumulative impact of in vitro reproductive technologies on the preimplantation embryo. Research supported by XY, Inc.

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