scholarly journals Site-Search Process for Synaptic Protein-DNA Complexes

2021 ◽  
Vol 23 (1) ◽  
pp. 212
Author(s):  
Sridhar Vemulapalli ◽  
Mohtadin Hashemi ◽  
Yuri L. Lyubchenko
Keyword(s):  
Dna Cleavage ◽  
High Speed ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Time Lapse ◽  
Single Site ◽  
Search Process ◽  
Synaptic Protein ◽  
Dna Complexes ◽  
Site Search

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM (HS-AFM) to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualised sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.

scholarly journals Insight into dynamics of APOBEC3G protein in complexes with DNA assessed by high speed AFM

2019 ◽  
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
High Speed ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Structural Information ◽  
Dynamic Structure ◽  
Time Lapse ◽  
Ssdna Binding ◽  
Binding Domains ◽  
Ssdna Binding Protein ◽  
Dumbbell Structure

AbstractAPOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zing-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has lacked, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built computational model for the full-length A3G monomer and validated its structure by data obtained from time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM was applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we developed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

scholarly journals Module to Support Real-Time Microscopic Imaging of Living Organisms on Ground-Based Microgravity Analogs

2021 ◽  
Vol 11 (7) ◽  
pp. 3122
Author(s):  
Srujana Neelam ◽  
Audrey Lee ◽  
Michael A. Lane ◽  
Ceasar Udave ◽  
Howard G. Levine ◽  
...  
Keyword(s):  
Real Time ◽  
High Speed ◽  
Simulated Microgravity ◽  
Image Acquisition ◽  
Time Lapse ◽  
Moving Frames ◽  
Cell Functions ◽  
Microgravity Simulation ◽  
Division Cell ◽  
Over Time

Since opportunities for spaceflight experiments are scarce, ground-based microgravity simulation devices (MSDs) offer accessible and economical alternatives for gravitational biology studies. Among the MSDs, the random positioning machine (RPM) provides simulated microgravity conditions on the ground by randomizing rotating biological samples in two axes to distribute the Earth’s gravity vector in all directions over time. Real-time microscopy and image acquisition during microgravity simulation are of particular interest to enable the study of how basic cell functions, such as division, migration, and proliferation, progress under altered gravity conditions. However, these capabilities have been difficult to implement due to the constantly moving frames of the RPM as well as mechanical noise. Therefore, we developed an image acquisition module that can be mounted on an RPM to capture live images over time while the specimen is in the simulated microgravity (SMG) environment. This module integrates a digital microscope with a magnification range of 20× to 700×, a high-speed data transmission adaptor for the wireless streaming of time-lapse images, and a backlight illuminator to view the sample under brightfield and darkfield modes. With this module, we successfully demonstrated the real-time imaging of human cells cultured on an RPM in brightfield, lasting up to 80 h, and also visualized them in green fluorescent channel. This module was successful in monitoring cell morphology and in quantifying the rate of cell division, cell migration, and wound healing in SMG. It can be easily modified to study the response of other biological specimens to SMG.

scholarly journals BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin–β-catenin interactions

2006 ◽  
Vol 174 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Shernaz X. Bamji ◽  
Beatriz Rico ◽  
Nikole Kimes ◽  
Louis F. Reichardt
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Time Lapse ◽  
Synapse Density ◽  
Vesicle Mobility ◽  
Small Clusters ◽  
Vertebrate Central Nervous System

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin–β-catenin adhesion complexes that occurs after tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin–β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin–β-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

scholarly journals Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

2004 ◽  
Vol 11 (1-2) ◽  
pp. 45-57 ◽  
Author(s):  
Floriana Volpicelli ◽  
Claudia Consales ◽  
Massimiliano Caiazzo ◽  
Luca Colucci-D'Amato ◽  
Carla Perrone-Capano ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Sonic Hedgehog ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ventral Mesencephalon ◽  
Mes Cells ◽  
Dopaminergic Differentiation

We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, allTH+neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction.

scholarly journals Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jenny I Aguilar ◽  
Mary Hongying Cheng ◽  
Josep Font ◽  
Alexandra C Schwartz ◽  
Kaitlyn Ledwitch ◽  
...  
Keyword(s):  
High Speed ◽  
Molecular Mechanisms ◽  
Motor Coordination ◽  
Neurodegenerative Disorder ◽  
Expression System ◽  
Distinct Type ◽  
Underlying Disease ◽  
Deficiency Syndrome ◽  
Motor Deficits ◽  
Therapeutic Implications

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

A FE Modelling Method for the Thermal Characteristics of High-Speed Motorized Spindle

2016 ◽  
Vol 693 ◽  
pp. 3-10
Author(s):  
Jia Rui Wang ◽  
Ping Fa Feng ◽  
Zhi Jun Wu ◽  
Ding Wen Yu ◽  
Jian Fu Zhang
Keyword(s):  
High Speed ◽  
Thermal Contact ◽  
Convective Heat Transfer Coefficient ◽  
Thermal Characteristics ◽  
Experimental System ◽  
Research Field ◽  
Motorized Spindle ◽  
Fe Modelling ◽  
Simulation Accuracy ◽  
Modelling Method

Finite element simulation is an effective method to study the thermal characteristics of high-speed motorized spindle, how to improve the simulation accuracy has become the key point of this research field. This paper presents a FEA method using ANSYS to precisely predict the thermal characteristics of high-speed spindle. Firstly, the heating and cooling characteristics of high-speed spindle are analyzed, main heating source, convective heat transfer coefficient, and thermal contact resistance are calculated. Secondly, FEA model of the machine center is built, the temperature field and thermal deformation of the spindle system are simulated. Thirdly, an experimental system to test thermal characteristics is designed, simulation results are compared with the experimental results. The result shows that the simulation errors are controlled in a relative low range, the FE modelling method can precisely predict the thermal characteristics of the motorized spindle.

scholarly journals Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 431
Author(s):  
Antonella D’Agostino ◽  
Anna Virginia Adriana Pirozzi ◽  
Rosario Finamore ◽  
Fabrizia Grieco ◽  
Massimiliano Minale ◽  
...  
Keyword(s):  
Tissue Repair ◽  
Molecular Mechanisms ◽  
Pharmaceutical Preparation ◽  
Time Lapse ◽  
Biological Properties ◽  
Protein Quantification ◽  
Matrix Remodeling ◽  
Tissue Elasticity ◽  
Triticum Vulgare

Background: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. Methods: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose–response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. Results: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. Conclusions: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment.

scholarly journals Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

2016 ◽  
Vol 113 (11) ◽  
pp. E1545-E1554 ◽  
Author(s):  
Samiran Mondal ◽  
Nasim A. Begum ◽  
Wenjun Hu ◽  
Tasuku Honjo
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Bimolecular Fluorescence Complementation ◽  
Functional Requirements ◽  
C Terminus ◽  
Gradient Fractionation

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

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