ABSTRACT
[This corrects the article DOI: 10.3389/fcell.2022.946335.].
ABSTRACT
Neospora caninum represents a major cause of abortive disease in bovines and small ruminants worldwide. As a typical obligate intracellular apicomplexan parasite, N. caninum needs to modulate its host cell for successful replication. In the current study, we focused on parasite-driven interference with host cell cycle progression. By performing DNA content-based cell cycle phase analyses in N. caninum-infected primary bovine umbilical vein endothelial cells (BUVEC), a parasite-driven S-phase arrest was detected at both 24 and 32 h p. i., being paralleled by fewer host cells experiencing the G0/G1 cell cycle phase. When analyzing S-subphases, proliferation cell nuclear antigen (per PCNA)-based experiments showed a reduced population of BUVEC in the late S-phase. Analyses on key molecules of cell cycle regulation documented a significant alteration of cyclin A2 and cyclin B1 abundance in N. caninum-infected host endothelial cells, thereby confirming irregularities in the S-phase and S-to-G2/M-phase transition. In line with cell cycle alterations, general nuclear parameters revealed smaller nuclear sizes and morphological abnormalities of BUVEC nuclei within the N. caninum-infected host cell layer. The latter observations were also confirmed by transmission electron microscopy (TEM) and by analyses of lamin B1 as a marker of nuclear lamina, which illustrated an inhomogeneous nuclear lamin B1 distribution, nuclear foldings, and invaginations, thereby reflecting nuclear misshaping. Interestingly, the latter finding applied to both non-infected and infected host cells within parasitized BUVEC layer. Additionally, actin detection indicated alterations in the perinuclear actin cap formation since typical nucleo-transversal filaments were consistently lacking in N. caninum-infected BUVEC, as also documented by significantly decreased actin-related intensities in the perinuclear region. These data indicate that N. caninum indeed alters host cell cycle progression and severely affects the host cell nuclear phenotype in primary bovine endothelial host cells. In summary, these findings add novel data on the complex N. caninum-specific modulation of host cell and nucleus, thereby demonstrating clear differences in cell cycle progression modulation driven by other closely related apicomplexans like Toxoplasma gondii and Besnotia besnoiti.
ABSTRACT
Most adult neurons and glia originate from radial glial progenitors (RGs), a type of stem cell typically extending from the apical to the basal side of the developing cortex. Precise regulation of the choice between RG self-renewal and differentiation is critical for normal development, but the mechanisms underlying this transition remain elusive. We show that the non-canonical tubulin Tuba8, transiently expressed in cortical progenitors, drives differentiation of RGs into apical intermediate progenitors, a more restricted progenitor type lacking attachment to the basal lamina. This effect depends on the unique C-terminal sequence of Tuba8 that antagonizes tubulin tyrosination and Δ2 cleavage, two post-translational modifications (PTMs) essential for RG fiber maintenance and the switch between direct and indirect neurogenesis and ultimately distinct neuronal lineage outcomes. Our work uncovers an instructive role of a developmentally regulated tubulin isotype in progenitor differentiation and provides new insights into biological functions of the cellular tubulin PTM "code."
Subject(s)
Cell Differentiation , Cerebral Cortex/cytology , Fibroblast Growth Factor 10/physiology , Neural Stem Cells/cytology , Neuroglia/cytology , Neurons/cytology , Tubulin/physiology , Animals , Cell Lineage , Cells, Cultured , Cerebral Cortex/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Stem Cells/metabolism , Neurogenesis , Neuroglia/metabolism , Neurons/metabolism , Tyrosine/metabolismABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) were identified as hazardous contaminants that are ubiquitous and persistent in aquatic environments, where bryophytes sensu lato (mosses, liverworts and hornworts) are frequently present. Marchantia polymorpha (Class Hepaticae; thalloid liverwort) is known to respond fast to changes in the environment; it accumulates toxic substances in its tissues due to the lack of vascular and radicular systems and a reduced or absent cuticle. The objective of the present study was to quantify the effects of increasing concentrations of anthracene (0, 50â¯100, 280⯵M) on the germination of propagules, plant morphology and chlorophyll content index (CCI) in M. polymorpha under in vitro cultures. The results show that anthracene had no statistical effect on germination or propagula formation. However, plants exposed to anthracene for 30 days showed significantly lowered the content of chlorophyll (measured as CCI), irregular growth patterns and the induction of thalli asexual reproduction as evidenced by the production of multicellular viable propagules in gemmae cups. Results of epifluorescence microscopy also showed concomitant accumulation of anthracene in the cell walls. All of these distinctive morphological and physiological adaptive responses indicators, clearly suggest that M. polymorpha are capable of resisting high (coal tar) anthracene concentrations.
Subject(s)
Anthracenes/pharmacology , Chlorophyll/metabolism , Marchantia/drug effects , Cell Wall/drug effects , Marchantia/anatomy & histology , Marchantia/growth & development , Marchantia/metabolism , Microscopy, FluorescenceABSTRACT
The ability to guide the growth of neurites is relevant for reconstructing neural networks and for nerve tissue regeneration. Here, a biofunctional hydrogel that allows light-based directional control of axon growth in situ is presented. The gel is covalently modified with a photoactivatable derivative of the short laminin peptidomimetic IKVAV. This adhesive peptide contains the photoremovable group 2-(4'-amino-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (HANBP) on the Lys rest that inhibits its activity. The modified peptide is highly soluble in water and can be simply conjugated to -COOH containing hydrogels via its terminal -NH2 group. Light exposure allows presentation of the IKVAV adhesive motif on a soft hydrogel at desired concentration and at defined position and time point. The photoactivated gel supports neurite outgrowth in embryonic neural progenitor cells culture and allows site-selective guidance of neurites extension. In situ exposure of cell cultures using a scanning laser allows outgrowth of neurites in desired pathways.
Subject(s)
Coated Materials, Biocompatible/chemistry , Laminin/chemistry , Neural Stem Cells/metabolism , Neurites/metabolism , Neuronal Outgrowth , Peptide Fragments/chemistry , Peptidomimetics/chemistry , Animals , Hydrogels/chemistry , Mice , Neural Stem Cells/cytologyABSTRACT
Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.
Subject(s)
Biocompatible Materials/pharmacology , Neurogenesis/drug effects , Neurogenesis/radiation effects , Neurons/drug effects , Neurons/radiation effects , Peptides/pharmacology , Amino Acid Sequence , Animals , Biocompatible Materials/chemistry , Cell Adhesion/drug effects , Cell Adhesion/radiation effects , Cells, Cultured , Laminin/chemistry , Laminin/pharmacology , Ligands , Mice, Inbred C57BL , Neurons/cytology , Peptides/chemistry , PhotolysisABSTRACT
Migrating post-mitotic neurons of the developing cerebral cortex undergo terminal somal translocation (ST) when they reach their final destination in the cortical plate. This process is crucial for proper cortical layering and its perturbation can lead to brain dysfunction. Here we present a reductionist biomaterials platform that faithfully supports and controls the distinct phases of terminal ST in vitro. We developed microenvironments with different adhesive molecules to support neuronal attachment, neurite extension, and migration in distinct manners. Efficient ST occurred when the leading process of migratory neurons crossed from low-to high-adhesive areas on a substrate, promoting spreading of the leading growth cone. Our results indicate that elementary adhesive cell-substrate interactions strongly influence migratory behavior and the final positioning of neurons during their developmental journey. This in vitro model allows advanced experimentation to reveal the microenvironmental requirements underlying cortical layer development and disorders.
Subject(s)
Cell Movement , Cellular Microenvironment , Cerebral Cortex/cytology , Neurons/cytology , Actin Cytoskeleton/metabolism , Animals , Cell Adhesion , Growth Cones/metabolism , Mice, Inbred C57BL , Microtubules/metabolismABSTRACT
Engineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively. While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing ß1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties.
Subject(s)
Hydrogels/pharmacology , Laminin/chemistry , Neural Stem Cells/cytology , Neurogenesis , Peptide Fragments/chemistry , Animals , Cells, Cultured , Elasticity , Focal Adhesions/metabolism , Hydrogels/chemistry , Laminin/pharmacology , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/drug effects , Neural Stem Cells/drug effects , Peptide Fragments/pharmacology , Polylysine/chemistry , Polylysine/pharmacology , Tissue Engineering/methodsABSTRACT
Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.
Subject(s)
Acrylic Resins/chemistry , Biocompatible Materials/chemistry , Hydrogels/chemistry , Animals , Cells, Cultured , Laminin/chemistry , Mice , Mice, Inbred C57BL , Polylysine/chemistry , Polymers/chemistryABSTRACT
Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.
Subject(s)
Cell Communication/genetics , Gene Regulatory Networks , Heme Oxygenase-1/metabolism , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Pseudopodia/metabolism , Animals , Cell Communication/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Coculture Techniques , Crystallography, X-Ray , Culture Media, Conditioned/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks/drug effects , Humans , Male , Mice , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms/genetics , Protein Binding/drug effects , Proteomics , Pseudopodia/drug effects , Sequence Analysis, RNA , Tandem Mass Spectrometry , Transcriptome/drug effects , Transcriptome/geneticsABSTRACT
UNLABELLED: By placing stiff structures under soft materials, prior studies have demonstrated that cells sense and prefer to position themselves over the stiff structures. However, an understanding of how cells migrate on such surfaces has not been established. Many studies have also shown that cells readily align to surface topography. Here we investigate the influence of these two aspects in directing cell migration on surfaces with 5 and 10µm line stiffness patterns (a cellular to subcellular length scale). A simple approach to create flat, stiffness-patterned surfaces by suspending a thin, low modulus polydimethylsiloxane (PDMS) film over a high modulus PDMS structure is presented, as well as a route to add undulations. We confirm that cells are able to sense through the thin film by observation of focal adhesions being positioned on stiff regions. We examine migration by introducing migration efficiency, a quantitative parameter to determine how strongly cells migrate in a certain direction. We found that cells have a preference to align and migrate along stiffness patterns while the addition of undulations boosts this effect, significantly increasing migration efficiency in either case. Interestingly, we found speed to play little role in the migration efficiency and to be mainly influenced by the top layer modulus. Our results demonstrate that both stiffness patterns and surface undulations are important considerations when investigating the interactions of cells with biomaterial surfaces. STATEMENT OF SIGNIFICANCE: Two common physical considerations for cell-surface interactions include patterned stiffness and patterned topography. However, their relative influences on cell migration behavior have not been established, particularly on cellular to subcellular scale patterns. For stiffness patterning, it has been recently shown that cells tend to position themselves over a stiff structure that is placed under a thin soft layer. By quantifying the directional migration efficiency on such surfaces with and without undulations, we show that migration can be manipulated by flat stiffness patterns, although surface undulations also play a strong role. Our results offer insight on the effect of cellular scale stiffness and topographical patterns on cell migration, which is critical for the development of fundamental cell studies and engineered implants.
Subject(s)
Cell Movement , Dimethylpolysiloxanes/chemistry , Membranes, Artificial , Cell Line , Humans , Surface PropertiesABSTRACT
Poly(acrylamide) P(AAm) gels have become relevant model substrates to study cell response to the mechanical and biochemical properties of the cellular microenvironment. However, current bioconjugation strategies to functionalize P(AAm) gels, mainly using photoinduced arylazide coupling, show poor specificity and hinder conclusive studies of material properties and cellular responses. We describe methylsulfonyl-containing P(AAm) hydrogels for cell culture. These gels allow easy, specific and functional covalent coupling of thiol containing bioligands in tunable concentrations under physiological conditions, while retaining the same swelling, porosity, cytocompatibility, and low protein adsorption of P(AAm) gels. These materials allow quantitative and standardized studies of cell-materials interactions with P(AAm) gels.
Subject(s)
Acrylic Resins/chemistry , Cell Culture Techniques , Hydrogels/chemistry , Hydrogels/chemical synthesis , Sulfhydryl Compounds/chemistry , HeLa Cells , Humans , Molecular Structure , Tumor Cells, CulturedABSTRACT
Cell detachment and migration from the endothelium occurs during vasculogenesis and also in pathological states. Here, we use a novel approach to trigger single cell release from an endothelial monolayer by in-situ opening of adhesive, fibril-like environment using light-responsive ligands and scanning lasers. Cell escapes from the monolayer were observed on the fibril-like adhesive tracks with 3-15 µm width. The frequency of endothelial cell escapes increased monotonically with the fibril width and with the density of the light-activated adhesive ligand. Interestingly, treatment with VEGF induced cohesiveness within the cell layer, preventing cell leaks. When migrating through the tracks, cells presented body lateral reduction and nuclear deformation imposed by the line width and dependent on myosin contractility. Cell migration mode changed from mesenchymal to amoeboid-like when the adhesive tracks narrowed (≤5 µm). Moreover, cell nucleus was shrunk showing packed DNA on lines narrower than the nuclear dimensions in a mechanisms intimately associated with the stress fibers. This platform allows the detailed study of escapes and migratory transitions of cohesive cells, which are relevant processes in development and during diseases such as organ fibrosis and carcinomas.
Subject(s)
Cell Adhesion/physiology , Cell Movement/physiology , Cellular Microenvironment/physiology , Endothelial Cells/physiology , Extracellular Matrix/metabolism , Oligopeptides/metabolism , Adhesiveness/radiation effects , Cell Adhesion/radiation effects , Cell Movement/radiation effects , Cells, Cultured , Cellular Microenvironment/radiation effects , Endothelial Cells/cytology , Endothelial Cells/radiation effects , Endothelium/cytology , Endothelium/physiology , Endothelium/radiation effects , Extracellular Matrix/chemistry , Extracellular Matrix/radiation effects , Humans , Light , Oligopeptides/chemistry , Oligopeptides/radiation effectsABSTRACT
The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvß3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.
Subject(s)
Cell Adhesion/physiology , Integrins/metabolism , Quartz Crystal Microbalance Techniques , Cell Adhesion/radiation effects , Cell Line , Cell Membrane/metabolism , Focal Adhesions , Humans , Ligands , Light , Oligopeptides/metabolism , Protein BindingABSTRACT
BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.
Subject(s)
Axons/metabolism , Receptor, EphA3/biosynthesis , Retinal Ganglion Cells/metabolism , Tectum Mesencephali/metabolism , Animals , Axons/physiology , Blotting, Western , Cells, Cultured , Chick Embryo , Chickens , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Microscopy, Confocal , Phosphorylation , Receptor, EphA3/genetics , Receptor, EphA3/metabolism , Retina/embryology , Retina/growth & development , Retina/metabolism , Superior Colliculi/embryology , Superior Colliculi/growth & development , Superior Colliculi/metabolism , Tectum Mesencephali/embryology , Tectum Mesencephali/growth & development , Time Factors , Time-Lapse Imaging , Tissue Culture Techniques , Tyrosine/metabolism , Visual PathwaysABSTRACT
We report the synthesis and properties of a photoactivatable caged RGD peptide and its application for phototriggering integrin- and cell-binding to surfaces. We analysed in detail 1) the differences in the integrin-binding affinity of the caged and uncaged forms by quartz crystal microbalance (QCM) studies, 2) the efficiency and yield of the photolytic uncaging reaction, 3) the biocompatibility of the photolysis by-products and irradiation conditions, 4) the possibility of site, temporal and density control of integrin-binding and therefore human cell attachment, and 5) the possibility of in situ generation of cell patterns and cell gradients by controlling the UV exposure. These studies provide a clear picture of the potential and limitations of caged RGD for integrin-mediated cell adhesion and demonstrate the application of this approach to the control and study of cell interactions and responses.
Subject(s)
Integrins/metabolism , Peptides, Cyclic/metabolism , Cell Adhesion , Cells, Cultured , Humans , Protein Binding , Quartz Crystal Microbalance Techniques , Ultraviolet RaysABSTRACT
The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is â¼ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.
Subject(s)
Mechanical Phenomena , Melanosomes/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Animals , Biomechanical Phenomena , Cell Survival , Dynactin Complex , Elasticity , Microtubule-Associated Proteins/metabolism , Models, Biological , Protein Transport , Viscosity , Xenopus laevisABSTRACT
We report the synthesis, characterization and applications of a ruthenium-bipyridine based caged nicotine. The complex [Ru(bpy)(2)(nic)(2)](2+) (where bpy = 2,2' bipyridine and nic = nicotine (3-[(2S)-1-methylpyrrolidin-2-yl] pyridine)) releases nicotine with a quantum yield Ï = 0.23 upon irradiation with biologically harmless, blue (473 nm) or green (532 nm) light. The photolysis reaction is clean and very fast, with a time constant of 17 ns. The synthesis is simple and the obtained compound is characterized by NMR, UV-Vis spectroscopy and cyclic voltammetry. We find that this compound is active in biological systems, being able to elicit action potentials in leech neurons.
Subject(s)
Light , Nicotine/chemistry , Nicotine/pharmacology , Animals , Electrophysiology , Leeches/cytology , Magnetic Resonance Spectroscopy , Models, Chemical , Neurons/drug effects , Neurons/metabolism , Photochemistry/methods , Photolysis , Ruthenium/chemistryABSTRACT
We introduce a new caged glutamate, based in a ruthenium bipyridyl core, that undergoes heterolytic cleavage after irradiation with visible light with wavelengths up to 532nm, yielding free glutamate in less than 50ns. Glutamate photorelease occurs also efficiently following two-photon (2P) excitation at 800nm, and has a functional cross section of 0.14GM.