Name: Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells (Video) | JoVE Journal
Uploaded: 2017-05-16T15:00:00.000+00:00
Duration: 8 min 53 s
Description: Learn a sensitive real-time assay to quantify nucleocytoplasmic transport rates in motor neuron-like NSC-34 cells. This method detects subtle import defects relevant to ALS and other neurodegenerative diseases.

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5 minutes

We thank all the members of the A.I. laboratory for their helpful comments and suggestions. We would like to thank Prof. Mark Hipp (Max Planck Institute for Biochemistry) for providing us with the shuttle-GFP vector. This work was supported by grants from the Israeli Science Foundation (ISF #124/14), the Binational Science Foundation (BSF #2013325), FP7 Marie Curie Career Integration Grant (CIG # 333794), and The National Institute for Psychobiology in Israel (NIPI #b133-14/15).

1. Cell Line Preparation
<ol>
	<li>Thaw approximately 1,000,000 mouse neuroblastoma spinal cord cells (NSC-34 cells) that were stored in a liquid nitrogen container. Use a 37 &#176;C water incubator until the cells are fully thawed.</li>
	<li>Add fresh, warm medium (DMEM medium containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% pen-strep antibiotics).</li>
	<li>Centrifuge thawed cells (SL16R) at 500 x g for 5 min, forming a cell pellet. Discard the medium and add 1 mL of new medium for the cell resuspension.</li>
	<li>Place the resuspended cells in a 15 mL flask and add an extra 5 mL of medium. Incubate the cells overnight in a sterile incubator with 5% CO2 at 37 &#176;C.</li>
	<li>In parallel, place 8 uncoated coverslips (cover glass: 12 mm, 0.13-0.17 mm thick) in a 60-mm culture dish and sterilize using 70% ethanol and exposure to 30 min of UV light until it is dry. Add medium to each dish and dispose to wash off the residual ethanol.</li>
	<li>Add trypsin buffer (2.5 g of trypsin and 0.2 g of EDTA in 1 L of Hanks&#39; Balanced Salt Solution) to the cells after they reach approximately 90% confluence (roughly 6 million cells per flask) to allow adherent cells to dissociate. 
	NOTE: NSC-34 cells are sensitive and only require 15 s of trypsin buffer incubation and 1 additional min of incubation free of trypsin before dissociation using the medium.</li>
	<li>Plate the cells onto the 60 mm culture dishes already containing the coverslips with 3 mL of medium, approximately 350,000 per dish. Leave them in the incubator for 24 h to allow cell attachment onto the coverslips.</li>
</ol>
2. Cell Transfection
NOTE: After approximately 24 h, the NSC-34 cells will reach 40% confluence (approximately 800,000 cells) and will be ready for transfection.
<ol>
	<li>Prepare pure DMEM medium (300 &#181;L of DMEM medium for each 60 mm culture dish) in sterile microcentrifuge tubes.</li>
	<li>Add 2 &#181;g of each plasmid required for transfection to each DMEM-containing microcentrifuge tube. 
	NOTE: The shuttle-GFP plasmid is mandatory in all cell transfections.</li>
	<li>Add transfection reagent to all the microcentrifuge tubes that contain their required plasmids. Here, add 4 &#181;L of the commercial transfection reagent for each 2 &#181;g of DNA.</li>
	<li>Vortex the microcentrifuge tubes containing DMEM, plasmids, and the transfection reagent and incubate them at room temperature for 25 min.</li>
	<li>Add each microcentrifuge tube to a culture dish and stir the dish gently to form a homogenous solution. Place the dish back in the incubator for 48 h. 
	NOTE: Since shuttle-GFP should be expressed in all cells, the shuttle-GFP transfection efficiency can be partially examined and pre-tested using fluorescent microscopy after overnight transfection. A full examination of non-fluorescent expressed proteins can be conducted using an immunoblot analysis.</li>
</ol>
3. Microscopy
<ol>
	<li>Attach the coverslips containing transfected NSC-34 cells to a coverslip holder and wash gently with phosphate-buffered saline (PBS) to remove the detached dead cells.</li>
	<li>Identify a suitable cell patch using a green fluorescent filter with an inverted microscope. 
	NOTE: A suitable cell patch is defined as a microscope field of view showing a large number of cells (20-30 cells in a field) and in which the nuclei are clearly defined in the cells. Cell nuclei within the cell patch are first identified by eye and are manually marked by the imaging software.</li>
	<li>Add 200 &#181;L of an exportin-1 inhibitor Leptomycin B (LMB) buffer containing 10 ng/mL LMB in PBS to the coverslips by dripping the buffer on the coverslips under the microscope. 
	NOTE: This point in time is set as interval zero.</li>
	<li>Quantify the fluorescent intensity of the marked nuclei in 3 s intervals for around 400-500 s.</li>
	<li>Test between 3 and 5 cover slips for each transfection group in each individual experiment.</li>
</ol>
4. Analysis
NOTE: Since different cells express different levels of florescent shuttle-GFP, it is important to normalize the nuclear fluorescent intensity of each cell to its own initial fluorescent intensity.
<ol>
	<li>Normalize the fluorescent intensity of the marked nuclei by dividing 3-s fluorescent interval of each nucleus with the average of the initial six intervals of that nucleus.</li>
	<li>After the normalization of each nucleus, produce a fluorescent intensity curve representing the change in nuclei fluorescence as a function of time for each cell patch. To do this, calculate the average of all the cells in each cell-patch (consisting of approximately 20-30 cells in each patch). 
	NOTE: The nuclei fluorescent intensity curve resembles a Michaelis-Menten saturation curve, which is better observed within increased time periods. This is due to a decrease in the cytosolic amounts of shuttle-GFP with time due to nuclear transport. This decrease statistically lowers the probability of a shuttle-GFP to transport into the nucleus, thus reaching saturation in nuclear fluorescence. The slope derived from the linear initial rate of the curve yields the Vmax entry rate into the nucleus.</li>
	<li>Analyze the Vmax slopes of each cell patch, derived from three to five independent experiments, using the graphing software to produce one slope representing each experimental group. Perform quantification using one-way ANOVA statistical analysis.</li>
</ol>
5. Protein Expression Validation
<ol>
	<li>Place 60 mm culture dishes containing the residual cells (not used in the above experiment) from each experimental group on ice.</li>
	<li>Dispose the medium and wash the cells twice with PBS to dilute and wash the remaining medium.</li>
	<li>Treat the cells with 200-400 &#181;L of lysis buffer containing PBS, 0.5% Triton, and protease inhibitor cocktail tablet (PI). Scrape using a cell scraper.</li>
	<li>Collect the cells in lysis buffer in a microcentrifuge tube and homogenize with a homogenizer at 4,000 rpm for 1 min under ice.</li>
	<li>Centrifuge the homogenized cells at 2,500 x g for 10 min. Collect the supernatant, quantify the protein concentration using a Bradford assay, and store until use at -20 &#176;C.</li>
	<li>Collect between 30 and 70 &#181;g of total protein (depending on the protein) and load it into a SDS-PAGE gel to test for protein expression using an immunoblot assay.</li>
</ol>

<ol><li>Alberts, B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAlberts%2C+B+%22Molecular+biology+of+the+cell%22">Molecular biology of the cell</a>. , 4th edn, Garland Science. (2002).</li>
<li>Freitas, N., Cunha, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+and+signals+for+the+nuclear+import+of+proteins%5BTitle%5D)+AND+%22Curr+Genomics%22%5BJournal%5D)">Mechanisms and signals for the nuclear import of proteins.</a> Curr Genomics. 10, 550-557 (2009).</li>
<li>Kalderon, D., Roberts, B. L., Richardson, W. D., Smith, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+short+amino+acid+sequence+able+to+specify+nuclear+location%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A short amino acid sequence able to specify nuclear location.</a> Cell. 39, 499-509 (1984).</li>
<li>la Cour, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+and+prediction+of+leucine-rich+nuclear+export+signals%5BTitle%5D)+AND+%22Protein+Eng+Des+Sel%22%5BJournal%5D)">Analysis and prediction of leucine-rich nuclear export signals.</a> Protein Eng Des Sel. 17, 527-536 (2004).</li>
<li>Boeynaems, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+screen+connects+nuclear+transport+genes+to+DPR+pathology+in+c9ALS%2FFTD%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD.</a> Sci Rep. 6, 20877(2016).</li>
<li>Freibaum, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GGGGCC+repeat+expansion+in+C9orf72+compromises+nucleocytoplasmic+transport%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.</a> Nature. 525, 129-133 (2015).</li>
<li>Jovicic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modifiers+of+C9orf72+dipeptide+repeat+toxicity+connect+nucleocytoplasmic+transport+defects+to+FTD%2FALS%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.</a> Nat Neurosci. 18, 1226-1229 (2015).</li>
<li>Kwon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poly-dipeptides+encoded+by+the+C9orf72+repeats+bind+nucleoli%2C+impede+RNA+biogenesis%2C+and+kill+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.</a> Science. 345, 1139-1145 (2014).</li>
<li>Rossi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+accumulation+of+mRNAs+underlies+G4C2-repeat-induced+translational+repression+in+a+cellular+model+of+C9orf72+ALS%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS.</a> J Cell Sci. 128, 1787-1799 (2015).</li>
<li>Shi, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxic+PRn+poly-dipeptides+encoded+by+the+C9orf72+repeat+expansion+block+nuclear+import+and+export%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.</a> Proc Natl Acad Sci U S A. , (2017).</li>
<li>Zhang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+C9orf72+repeat+expansion+disrupts+nucleocytoplasmic+transport%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.</a> Nature. 525, 56-61 (2015).</li>
<li>Zhang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C9ORF72+poly%28GA%29+aggregates+sequester+and+impair+HR23+and+nucleocytoplasmic+transport+proteins%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins.</a> Nat Neurosci. 19, 668-677 (2016).</li>
<li>Cleveland, D. W., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+Charcot+to+Lou+Gehrig%3A+deciphering+selective+motor+neuron+death+in+ALS%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS.</a> Nat Rev Neurosci. 2, 806-819 (2001).</li>
<li>DeJesus-Hernandez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanded+GGGGCC+hexanucleotide+repeat+in+noncoding+region+of+C9ORF72+causes+chromosome+9p-linked+FTD+and+ALS%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.</a> Neuron. 72, 245-256 (2011).</li>
<li>Renton, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hexanucleotide+repeat+expansion+in+C9ORF72+is+the+cause+of+chromosome+9p21-linked+ALS-FTD%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.</a> Neuron. 72, 257-268 (2011).</li>
<li>Boeve, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+frontotemporal+dementia+and%2For+amyotrophic+lateral+sclerosis+associated+with+the+GGGGCC+repeat+expansion+in+C9ORF72%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72.</a> Brain. 135, 765-783 (2012).</li>
<li>Haeusler, A. R., Donnelly, C. J., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+expanding+biology+of+the+C9orf72+nucleotide+repeat+expansion+in+neurodegenerative+disease%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">The expanding biology of the C9orf72 nucleotide repeat expansion in neurodegenerative disease.</a> Nat Rev Neurosci. 17, 383-395 (2016).</li>
<li>Woerner, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoplasmic+protein+aggregates+interfere+with+nucleocytoplasmic+transport+of+protein+and+RNA%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.</a> Science. 351, 173-176 (2016).</li>
<li>Tao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleolar+stress+and+impaired+stress+granule+formation+contribute+to+C9orf72+RAN+translation-induced+cytotoxicity%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity.</a> Hum Mol Genet. 24, 2426-2441 (2015).</li>
<li>Wen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antisense+proline-arginine+RAN+dipeptides+linked+to+C9ORF72-ALS%2FFTD+form+toxic+nuclear+aggregates+that+initiate+in+vitro+and+in+vivo+neuronal+death%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.</a> Neuron. 84, 1213-1225 (2014).</li>
</ol>

Authors have nothing to disclose.

This protocol demonstrates a highly sensitive and quantitative new assay to evaluate minute changes in nucleocytoplasmic transport. Using this system, it is possible to observe and measure nucleocytoplasmic transport and its dysfunction in real time, not only large defects that result in dramatic changes in protein distribution. This assay not only has a sensitivity advantage, but it also requires little preparation, is inexpensive, and is a very easy and low-engagement assay.
To test the effi...

The nuclear pore complex (NPC) controls the import and export of large molecules into and out of the cell nucleus. In contrast to small molecules, which can enter the nucleus without regulation1, the transport of larger molecules is strongly controlled by the NPC. These large molecules, such as proteins and RNA, associate with transport factors, including importins and exportins, to be imported and exported from the nucleus2. In order to be imported, proteins must contain a small peptide motif, generally called a nuclear localization signal (NLS), which is bound by importins2. These sequences of amino acids act as a tag and are diverse in composition3,4. Proteins can be exported from the nucleus to the cytoplasm due to their association with exportins, which bind a signal sequence, generally called a nuclear export signal (NES)2. Both importins and exportins are able to transport their cargo due to the regulation of the small Ras related nuclear protein GTPase (Ran)2. Ran has been shown to exist in different conformational states depending on whether it is bound to GTP or GDP. When bound to GTP, Ran can bind to importins or exportins. Upon binding to RanGTP, importins release their cargo, while exportins must bind RanGTP to form a complex with their export cargo. The dominant nucleotide binding state of Ran depends on its location in the nucleus (RanGTP) or the cytoplasm (RanGDP).
Recently, a number of studies have shown a connection between impairments in the nucleocytoplasmic pathway and both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)5,6,7,8,9,10,11,12. ALS is a progressive and fatal neurodegenerative disease affecting both upper and lower motor neurons (MNs)13 and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are classified as sporadic (SALS), lacking any apparent genetic linkage, but 10% are inherited in a dominant manner (familial ALS; FALS). Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified14,15 as a genetic cause of ALS and FTD. These mutants account for 30-40% of FALS cases16, and different studies have contended that they cause toxicity by affecting nucleocytoplasmic transport5,6,7,8,9,10,11,12.
These studies have mostly shown the final outcomes and manifestations of HREs on nucleocytoplasmic transport, but they do not demonstrate nucleocytoplasmic disruption in real time. As a result, only severe nucleocytoplasmic transport deficiency has been evaluated11,17,18.
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 done using an exportin inhibitor, as described previously18, thus allowing the shuttle-GFP only to enter the nucleus. Using florescent microscopy and a software capable of quantifying fluorescent changes in real time, it is possible to quantify gradual changes in the fluorescence intensity of the shuttle-GFP, located in the nucleus. As a result, a Michaelis-Menten-like saturation curve of the fluorescence-to-time axis, representing the amount of nuclear shuttle-GFP at any given time, can be made. By using the initial linear rate of the curves, it is possible to generate a slope, which represents the rate of entry into the nucleus before fluorescence saturation.
To validate the assay, the translated C9orf72 dipeptide repeats, poly(GR) and poly(PR), which have been previously reported to disrupt nucleocytoplasmic transport, were used5,7,8,10,12. Using this 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. Moreover, the ability of different factors to rescue a nucleocytoplasmic transport defect can be determined.

<table><tbody><tr><td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td><td>tebu-bio</td><td>CLU140-A</td><td></td></tr><tr><td>DMEM 4.5 g/L L-glucose</td><td>Biological Industries</td><td>01-055-1A</td><td></td></tr><tr><td>FBS European Grade</td><td>Biological Industries</td><td>04-007-1A</td><td></td></tr><tr><td>SL 16R Centrifuge</td><td>Thermo Scientific</td><td>75004030</td><td></td></tr><tr><td>Thermo Forma Series ii Water Jacketed CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>3111</td><td></td></tr><tr><td>Cover glass 12 mm dia., 0.13-0.17 mm thick</td><td>Bar Naor LTD</td><td></td><td></td></tr><tr><td>TurboFect Transfection Reagent</td><td>Thermo Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/R0531&quot;&gt;R0531&lt;/a&gt;</td><td></td></tr><tr><td>Axiovert 100 inverted microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Imaging Workbench 2</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Leptomycin B</td><td>Sigma</td><td>L2913</td><td></td></tr><tr><td>PBS</td><td>Biological Industries</td><td>02-023-1A</td><td></td></tr><tr><td>Homogenizer motor/control/chuck/90 degree clamp</td><td>Glas-Col</td><td>099C K5424CE</td><td></td></tr><tr><td>MicroCL 17R Centrifuge, Refrigerated</td><td>Thermo Scientific</td><td>75002455</td><td></td></tr></tbody></table>

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

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.

Using the procedure presented here, motor neuron-like NSC-34 cells were transfected with a NLS-NES-GFP (shuttle-GFP) protein. This protein, which has NLS and NES signal sequences (Figure 1), can shuttle between the nucleus and the cytoplasm. Generic GFP can enter or exit the nucleus in the absence of any localization signal tag only by diffusion and is therefore distributed evenly between the nucleus and the cytoplasm (Figure 2A). In contrast, the shuttle...

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.

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

The overall goal of this assay is to detect minute changes in the nucleocytoplasmic transport rate of cells in real time in the absence or presence of potential transport rate-suppressive molecules. This method can help answer key questions about specific neurogenetic diseases such as ALS, that have been proposed to negatively impact nucleocytoplasmic transport. Some advantages of this technique are that it is highly sensitive and quantitative and that it allows the real-time analysis of minute changes in the nucleocytoplasmic transport rate.Demonstrating the procedure will be Tom Shani, a graduate student from my laboratory. Begin by thawing approximately one times 10 to the sixth mouse neuroblastoma spinal cord cells from liquid nitrogen storage in a 37 degree Celsius water bath. When the cells are fully thawed, collect them by centrifugation, and resuspend the pellet in one milliliter of fresh medium.Then transfer the cells into 15-milliliter flask containing five milliliters of cell culture medium for an overnight incubation at 37 degrees Celsius in 5%carbon dioxide. When the cells reach 90%confluency, place eight uncoated cover slips into individual 60-millimeter culture dishes and sterilize the cover slips with 70%ethanol. Dry the cover slips under a UV light for 30 minutes.When the glass is dry, wash the cover slips with fresh medium to remove the residual ethanol. When the cover slips are ready, dissociate the neuroblastoma cells with trypsin-EDTA for about 15 seconds in the cell culture incubator. Then rest the sells for one minute at room temperature.Collect the cells by centrifugation. After counting, resuspend the pellet at a 3.5 times 10 to the fifth cells per three milliliters of medium concentration and seed three milliliters of cells per dish. Then place the cultures in the incubator for 24 hours to allow the cells to attach to the cover slips.The next day, add two micrograms of each transfection plasmid to one sterile microcentrifuge tube containing 300 microliters of DMEM medium per 60-millimeter culture dish. Add four microliters of commercial transfection reagent per two micrograms of plasmid to each tube. And vortex the DNA transfection solutions.Alternatively in cell populations where the shuttle-GFP nucleus to cytoplasmid ratio is close to a one-to-one ratio, the cells can be pre-labeled with a nuclear marker prior to their transfection to facilitate nuclear distinction. After a 25 minute incubation at room temperature, add the plasmids to each dish. And gently stir to obtain a homogenous solution.Return the cultures to the incubator for another 48 hours. Then attach the coverslips to a coverslip holder for a gentle PBS wash to remove any detached dead cells. Next place the coverslip under an inverted microscope and identify a patch of 20 to 30 GFP-expressing cells in which the nuclei are clearly defined.Use the imaging software to mark the nuclei within the patch. Then add 200 microliters of exportin-1 inhibitor leptomycin B buffer dropwise to the coverslip and quantify the fluorescence intensity of the marked nuclei in three-second intervals for around 400 to 500 seconds. After all the cover slips have been imaged, normalize the fluorescence intensity of each cell nuclei and generate a fluorescence intensity curve representing the changes in nuclear fluorescence as a function of time for each cell patch.Then analyze the maximum reaction velocity of the slope of each cell patch derived from three to five independent experiments to generate one slope per experimental group. For protein expression validation 48 hours after transfection, place the culture dishes on ice and wash the cells two times with PBS. Treat the cells in each dish with 200 to 400 microliters of lysis buffer.And use a cell scraper to remove the cells from the cover slips. Transfer the detached cells from each plate into individual microcentrifuge tubes. And further dissociate the cells with a homogenizer under ice.After one minute, pellet the homogenized cell pieces by centrifugation and measure the protein concentration in each supernatant by Bradford assay. Then pool 30 to 70 micrograms of protein per group into new microcentrifuge tubes. Store the tubes negative 20 degrees Celsius until SDS page protein expression analysis.Generic GFP can enter or exit the cell nucleus in the absence of a localization signal tag by diffusion only, resulting in an even distribution of the protein between the nucleus and the cytoplasm. In contrast, shuttle-GFP protein is mostly observed in the cytoplasm due to the nuclear export and nuclear localization signals directing its dispersion. Treatment with leptomycin B, a GFP protein export inhibitor, induces the accumulation of shuttle-GFP within the nucleus, allowing the shuttle-GFP import rate to be measured.These changes in the nuclear fluorescence intensity in the absence or presence of protein export inhibition can then be quantified in real time to assess the nuclear cytoplasmic transport rate of the shuttle-GFP overtime. To confirm that the activity of nucleolar stress-inducing dipeptide repeat proteins is not affected by protein export inhibition GFP, GFP-tagged dipeptide repeat proteins were visualized by fluorescence microscopy after leptomycin B treatment revealing poly(PR)expression in the nucleus as inclusions only, excluding the possibility of the generation of a false positive nuclear import signal. Poly(GR)expression however was observed homogenously in the cytosol and as inclusions within the nucleus.Indeed, neuroblastoma spinal cord cell transfection with poly(GR)only followed by leptmycin B treatment results in no observed shuttle movement of the expressed poly(GR)Further analysis of the nuclear cytoplasmic shuttle-GFP transport rate reveals the singular expression of either poly(PR)or poly(GR)dipeptide repeats indicating an about 50%decrease in the total nuclear import rate compared to control empty plasmid-transfected cells. We first had the idea for this method when we tried to find the quantitative assay for detecting small changes in nucleocytoplasmic dysfunction in real time. Once mastered, the microscopic analysis of the cells can be performed in approximately 10 minutes per coverslip.The data analysis for each coverslip can be completed in a few minutes. After its development, this technique paves the way for researchers in the field of neurodegenerative diseases to explore defects in nucleocytoplasmic transport using cellular models. After watching this video, you should have a good understanding of how to detect and quantify minute changes in cellular nucleocytoplasmic transport rate in real time.

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Research

JoVE Journal

Neuroscience

Single molecule microscopy approch provided novel insights into nuclear transport.

Single-Molecule Imaging of Nuclear Transport

This protocol outlines the extraction of proteins from neuronal cancer cells. The cells are washed with a buffer and lysed via enzymatic and mechanical disruption. The extracted protein-rich supernatant is collected after centrifugation.

Protein Extraction from Neuronal Cells

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

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.

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

This protocol describes how to efficiently set up and perform automated analyses of live cell imaging data obtained from tracking the nucleocytoplasmic transport via optogenetic tools.

Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Learn a sensitive real-time assay to quantify nucleocytoplasmic transport rates in motor neuron-like NSC-34 cells. This method detects subtle import defects relevant to ALS and other neurodegenerative diseases.

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

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