وقد أيد هذا المشروع من المنحة لتعزيز القدرات البحثية (جامعة بولينغ غرين ستيت).

<ol>
	<li>Ma, J., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Snapshots+of+Three-Dimensional+Distribution+of+Transient+Interactions+in+Nuclear+Pore+Complex.">Single Molecule Snapshots of Three-Dimensional Distribution of Transient Interactions in Nuclear Pore Complex.</a> Proc. Natl. Acad. Sci. USA. , (2010).</li><li>Sarma, A., Goryaynov, A., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Imaging+of+the+Calcium-Regulated+Nuclear+Pore+Permeability.">Single Molecule Imaging of the Calcium-Regulated Nuclear Pore Permeability.</a> , .</li><li>Yang, W., Musser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single+molecules+transiting+through+nuclear+pore+complexes+using+narrow-field+epifluorescence+microscopy.">Visualizing single molecules transiting through nuclear pore complexes using narrow-field epifluorescence microscopy.</a> Methods. 39, 316-328 (2006).</li><li>Yang, W., Gelles, J., Musser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+single-molecule+translocation+through+nuclear+pore+complexes.">Imaging of single-molecule translocation through nuclear pore complexes.</a> Proc. Natl. Acad. Sci. USA. 101, 12887-12892 (2004).</li></ol>

الإعلان عن أي تضارب في المصالح.

يجب توخي الحذر لضمان اقامة colocalization السليم للجزيئات واحد NE أثناء التجربة. من المهم أيضا أن تقدم إشارة ارتفاع نسبة الضوضاء ، وخصوصا عندما إشارة منخفضة نظرا لوضع العلامات أو photobleaching الفقراء. يقتصر دقة الترجمة عن الضوضاء الخلفية (التي تنشأ من خارج التركيز مضان ، CCD الضوض?...

single-molecule imaging of nuclear transport

وقد ثبت جدوى النهج جزيء المجهري مضان واحد لتكون ذات جدوى كبيرة في فهم التفاعلات البيولوجية على مدى العقد الماضي. وظل التحقيق في التفاعلات الجزيئية مع ارتفاع قرارات الزمانية والمكانية عميقة داخل الخلايا صعبة بسبب إشارات ضعيفة بطبيعتها الناشئة من الجزيئات الفردية. يعمل يانغ مؤخرا وآخرون. أثبتت أن ضيق المجال المجهري epifluorescence التصور يسمح للنقل النواة للهيولى على مستوى الجزيء واحد. من النهج جزيء واحد ، حركية مهمة ، مثل النقل النووي الوقت والكفاءة ، لجزيئات البضائع التي تعتمد على إشارة ومستقلة قد تم الحصول عليها. وصف نحن هنا بروتوكولا للنهج المنهجي مع القرار ، أي spatiotemporal تحسين نانومتر و 12 مللي 0.4. مكن قرار تحسين لنا لالتقاط النقل النشط والأحداث العابرة نشر السلبي من خلال مسام المجمعات النووية (مجلس الشعب) في شبه سليمة الخلايا. نتوقع أن يستخدم هذا الأسلوب في توضيح الأحداث الأخرى ملزمة والاتجار داخل الخلايا.

Watch this Scientific Journal Video about Single-Molecule Imaging of Nuclear Transport at JoVE.com

Single-Molecule Imaging of Nuclear Transport

approch قدم واحدة جزيء المجهري رؤى الرواية إلى النقل النووي.

المفرد جزيء التصوير النقل النووية

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

single-molecule-imaging-of-nuclear-transport

Research

JoVE Journal

Biology

13.3K Views.  Bowling Green State University. approch قدم واحدة جزيء المجهري رؤى الرواية إلى النقل النووي.

Video: Single-Molecule Imaging of Nuclear Transport

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.

This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.

سباتيوتيمبورالي الخاضعة لسيطرة النووية إزفاء من الضيوف في الخلايا الحية باستخدام الغراء الجزيئية محبوس كالعلامات فوتواكتيفاتابل

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is ...

Chemistry

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

اختبار تصوير أحادي الجزيء في المختبر لتحليل حركية الترجمة المعتمدة على الغطاء

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Biochemistry

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

تصور داخل الخلايا المطوقة الاتجار بواسطة عمر Fluorescence التصوير المجهري

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Immunology and Infection

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

فحص لقياس نوكلوسيتوبلاسميك النقل في الوقت الحقيقي داخل موتور الخلايا العصبية مثل نسك-34 الخلايا

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Neuroscience

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

في فيفو تتبع في محطة Presynaptic الأعصاب الحركية المورفولوجية جزيء واحد

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

التصوير والتحليل لنقل الليفية العصبية في العصب الذبي الماوس المخزّن

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

الجمع بين التلاعب جزيء واحدة والتصوير لدراسة التفاعلات-DNA البروتين

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

التصوير تحت الخلوي للتعامل مع الكالسيوم العصبي في الجسم الحي

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Directionality of Nuclear Transport

Ras-related nuclear protein or Ran is a small G protein that cycles between its GTP and GDP bound states. Ran specific regulators, a Ran GTPase Activating Protein or RanGAP present in the cytosol and a Ran guanine nucleotide exchange factor or RanGEF present inside the nucleus regulate GTP/GDP exchange. A high concentration of GTP inside the cells, in addition to this asymmetric distribution of&#160; Ran-specific regulators, leads to a higher RanGTP concentration inside the nucleus. This concentration gradient of RanGTP formed across the nucleus helps in the unidirectional trafficking of nuclear proteins.
During nuclear export, RanGTP binds exportin and increases its affinity to bind the cargo.&#160; The Ran GTP-cargo-exportin complex transits out of the nucleus through the nuclear pore complex or NPCs.&#160; RanBP1 and RanBP2 present near the cytosolic side of the NPCs stimulate RanGAP activity, helping Ran undergo GTP hydrolysis to RanGDP. This weakens the cargo-exportin affinity, eventually leading to cargo release in the cytosol. Unlike RanGTP, RanGDP cannot bind receptors alone and releases the exportins to be recycled back into the nucleus for a second round of cargo export.
The continuous efflux and hydrolysis of RanGTP in the cytosol lowers the RanGTP levels in the nucleus. Nuclear transport factor 2 (NTF2) binds hydrolyzed RanGDP in the cytosol transporting them back to the nucleus to compensate for the loss. Inside the nucleus, RanGDP binds to the Regulators of Chromatin Condensation 1 (RCC1), a RanGEF, and undergoes a GDP/GTP exchange to restore the nuclear RanGTP concentrations.

اتجاه النقل النووي

Ras-related nuclear protein or Ran is a small G protein that cycles between its GTP and GDP bound states. Ran specific regulators, a Ran GTPase Activating ...