Name: functional imaging of viral transcription factories using 3d fluorescence microscopy
Uploaded: 2018-01-18T14:00:00
Description: Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

This research was supported by National Institutes of Health grant (R01-DE025985) and by an American Cancer Society Research Scholar Award (RSG-13-383-MPC). This work was also supported by grants from the U.S. Department of Agriculture (2015-67015-23268 and 2014-67015-21787) and New Research Initiative Grant from University of California, Davis.

CAUTION: Cell lines used in this procedure contain infectious viruses, be careful and only proceed in level 2 BSL facility or higher.
1. Cell Preparation and Maintenance
<ol>
	<li>Culture TREx-K-RTA BCBL-1 cells (or another KSHV infected PEL cell lines) in RPMI1640 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin streptomycin glutamine solution.</li>
	<li>Grow cells at 37 &#176;C with 5% carbon dioxide (CO2), making sure to continuously grow and to split cel...

<ol>
	<li>de Laat, W., Grosveld, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grosveld+Spatial+organization+of+gene+expression:+the+active+chromatin+hub.">Grosveld Spatial organization of gene expression: the active chromatin hub.</a> Chromosome Res. 11 (5), 447-459 (2003).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+dynamics:+implications+for+nuclear+architecture+and+gene+expression.">Protein dynamics: implications for nuclear architecture and gene expression.</a> Science. 291 (5505), 843-847 (2001).</li><li>Bernardi, R., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+dynamics+and+functions+of+promyelocytic+leukaemia+nuclear+bodies.">Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.</a> Nat Rev Mol Cell Biol. 8 (12), 1006-1016 (2007).</li><li>Cioce, M., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies:+a+long+history+of+discovery.">Cajal bodies: a long history of discovery.</a> Annu Rev Cell Dev Biol. 21, 105-131 (2005).</li><li>Boisvert, F. M., van Koningsbruggen, S., Navascues, J., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+nucleolus.">The multifunctional nucleolus.</a> Nat Rev Mol Cell Biol. 8 (7), 574-585 (2007).</li><li>Sutherland, H., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factories:+gene+expression+in+unions.">Transcription factories: gene expression in unions.</a> Nat Rev Genet. 10 (7), 457-466 (2009).</li><li>Schmid, M., Speiseder, T., Dobner, T., Gonzalez, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+virus+replication+compartments.">DNA virus replication compartments.</a> J Virol. 88 (3), 1404-1420 (2014).</li><li>Chen, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+Sarcoma-Associated+Herpesvirus+Hijacks+RNA+Polymerase+II+To+Create+a+Viral+Transcriptional+Factory.">Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory.</a> J Virol. 91 (11), (2017).</li><li>Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+and+the+pathogenesis+of+Kaposi+sarcoma:+listening+to+human+biology+and+medicine.">KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine.</a> J Clin Invest. 120 (4), 939-949 (2010).</li><li>Mesri, E. A., Cesarman, E., Boshoff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma+and+its+associated+herpesvirus.">Kaposi's sarcoma and its associated herpesvirus.</a> Nat Rev Cancer. 10 (10), 707-719 (2010).</li><li>Campbell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methyltransferase+1-directed+methylation+of+Kaposi+sarcoma-associated+herpesvirus+latency-associated+nuclear+antigen.">Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen.</a> J Biol Chem. 287 (8), 5806-5818 (2012).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li><li>Darst, R. P., Haecker, I., Pardo, C. E., Renne, R., Kladde, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+diversity+of+Kaposi's+sarcoma-associated+herpesvirus.">Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.</a> Nucleic Acids Res. 41 (5), 2993-3009 (2013).</li><li>Osborne, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myc+dynamically+and+preferentially+relocates+to+a+transcription+factory+occupied+by+Igh.">Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.</a> PLoS Biol. 5 (8), 192 (2007).</li><li>Fanucchi, S., Shibayama, Y., Burd, S., Weinberg, M. S., Mhlanga, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+contact+permits+transcription+between+coregulated+genes.">Chromosomal contact permits transcription between coregulated genes.</a> Cell. 155 (3), 606-620 (2013).</li><li>Borah, S., Darricarrere, N., Darnell, A., Myoung, J., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+nuclear+noncoding+RNA+binds+re-localized+poly(A)+binding+protein+and+is+required+for+late+KSHV+gene+expression.">A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.</a> PLoS Pathog. 7 (10), 1002300 (2011).</li><li>Chang, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylase+JMJD2A+regulates+Kaposi's+sarcoma-associated+herpesvirus+replication+and+is+targeted+by+a+viral+transcriptional+factor.">Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.</a> J Virol. 85 (7), 3283-3293 (2011).</li><li>McAllister, S. C., Hansen, S. G., Messaoudi, I., Nikolich-Zugich, J., Moses, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+efficiency+of+phorbol+ester-induced+lytic+reactivation+of+Kaposi's+sarcoma-associated+herpesvirus+during+S+phase.">Increased efficiency of phorbol ester-induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus during S phase.</a> J Virol. 79 (4), 2626-2630 (2005).</li><li>Miller, G., El-Guindy, A., Countryman, J., Ye, J., Gradoville, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lytic+cycle+switches+of+oncogenic+human+gammaherpesviruses.">Lytic cycle switches of oncogenic human gammaherpesviruses.</a> Adv Cancer Res. 97, 81-109 (2007).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li></ol>

There are some aspects of the protocol that can be altered to accommodate unusual circumstances. The choice of fixation and permeabilization buffers can also be altered. For fixation buffers, paraformaldehyde is also effective and ethanol can be used for permeabilization. 
Quenching the formaldehyde with glycine PBS is recommended as it prevents formaldehyde from disabling the antibodies in absence of bovine serum albumin (BSA), but if the coverslips are washed thoroughly enough, the glycine P...

It has become increasingly clear that the spatiotemporal organization of the nucleus plays an important role in modulating finely-tuned gene expression in eukaryotes. Most tissue specific genes are distributed over many chromosomes and need to be regulated synchronously in order to respond to specific stimuli in a coordinated manner1. Cells construct active chromatin hubs (ACH) as a way of bringing together genes and their cis-regulatory components to a specific nuclear space1.
It has also been demonstrated by various studies that although the nucleus seems very dense and viscous, biologically active molecules can traverse the nucleus rather quickly via diffusion2. As a consequence of this ephemeral property, most DNA binding proteins &#39;jump&#39; from binding site to binding site, feeling their way around the nuclear space, which allows for a highly adaptive and versatile nucleus2.
Despite this dynamic behavior of biomolecules within the nucleus, nuclear bodies without membranes such as (but not limited to) the nucleolus, Cajal bodies, and promyelocytic leukemia nuclear bodies (PML-NB) still exist. It is through a variety of mechanisms such as tandem DNA repeats (nucleolus), rRNA (nucleolus) and structural proteins such as coilin (Cajal bodies) or PML proteins (PML-NB) that hold these constructs together3,4,5. These structures and other congregations such as transcription factories serve as scaffolds that not only increase the local concentration of required components, but also regulate the composition of proteins and nucleic acids within them to ultimately create a central site for efficient cellular functions6.
Visualizing when and where nuclear structures form provides a wealth of information to researchers studying epigenetics. From a virology perspective, the reactivation of latently infected viruses, such as KSHV, significantly alters the landscape of the nucleus and distribution of nuclear enzymes to shift transcription primarily from cellular genes to viral genes, ultimately to produce fully functional viral progeny7,8. How does KSHV manipulate the cellular gene expression machinery to facilitate viral gene expression? Such information could also shed light on temporal cellular gene regulatory mechanisms.
Like all other herpesviruses, KSHV has two life cycles called lytic replication and latency. KSHV primarily resides in the latency stage, in which most of its viral gene expression is silenced, except latency associated genes9,10. During latency KSHV produces latency associated nuclear antigen (LANA), which constitutively binds the viral genomes and tethers viral chromatin to the human chromosome11. Because of LANA&#39;s intimate relationship with the viral genome, it is possible to use IFA and DAPI to stain and locate where the viral episomes were in relation to the host chromatin.
To study KSHV reactivation at the single episome level and the association with other viral episomes in an infected cell, a strategy to locate actively transcribing viral chromatin in situ has been established. Accordingly, LANA and RNAPII IFA with intron RNA-FISH were combined, by generating RNA-FISH probes that bind to the intron region (exact probe sequences can be found in the Izumiya lab&#39;s most recent publication8) of KSHV K-Rta-the key viral protein that is essential and sufficient for KSHV reactivation-it was possible to identify where transcription was actually taking place8,11,12,13,14,15. This intron RNA-FISH technique enables researchers to visualize where mRNA is being transcribed immediately before it is spliced and exported to cytoplasm16,17.
KSHV can be reactivated by various chemical stimuli including phorbol esters such as 12-O-Tetradeconoyl-phorbol-13acetate (TPA) and histone deacetylase inhibitors such as sodium butyrate, additionally KSHV can be induced to reactivate by overexpression of the viral transcription factor, K-Rta19. Researchers have successfully increased the efficiency of KSHV reactivation by synchronizing the cell&#39;s cycles prior to inducing reactivation18. Thus, for these particular studies, cells were synchronized using a double thymidine block (protocol described below) and incubated with TPA and doxycycline (Dox) for a short time. Doxycyline was used because the cell line utilized in these experiments has a doxycycline-inducible K-Rta cassette, which was cloned from cDNA and does not include the intron region of K-Rta. Although it is possible to reactivate KSHV using only induced K-Rta expression, it has been proven by other researchers that due to a variety of different biochemical factors K-Rta expression alone proves to be a feeble reactivation stimuli20. By combining all of these, and by limiting the drug incubations to a short period of time, a robust but not overly artificial KSHV reactivation was achieved for imaging.
After labeling LANA, RNAPII, K-Rta introns, and DNA as described in this paper, 3D fluorescence imaging was performed using a widefield deconvolution microscope. After processing with imaging software, the spatial distribution of active viral episomes can be properly evaluated. Using this technique, the central questions regarding the fundamental nature of the formation of active chromatin hubs and other nuclear structures can be studied. Having identical viral episomes in a single cell that processes the same regulatory elements may represent a unique research tool to deepen the understanding of spatiotemporal gene regulatory mechanisms.
A limitation of imaging multiple cell specimens fixed at different time points to characterize an inherently dynamic molecular process is that subtle or small-scale changes in fluorescence distribution are undetected or deemed insignificant. This is true unless in the rare instance, every cell observed displays the same subtle change. Thus, the full spatiotemporal relationship of active viral transcription and other nuclear structures cannot be critically evaluated using fixed imaging. To address these technical challenges, the best approach is to image live cells that have marked viral episomes and to follow the location of key cellular enzymes over time.

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			<td height="85" style="height:85px;width:216px;">RPMI medium 1650 GlutaMAX</td>
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functional imaging of viral transcription factories using 3d fluorescence microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

A slightly abridged protocol was performed where BCBL-1 cells were used and only LANA and K-Rta RNA were stained (Figure 1). This experiment allows us to examine where actively transcribing viral episomes are and the heterogeneity of response to KSHV reactivation stimuli in a population of cells. In the cell indicated by the arrow in Figure 1, the distinct K-Rta fluorescence in regions that closely match the distribution of viral...

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

The overall goal of combining nascent RNA-FISH with amino staining of viral latent proteins is to visualize active transcription coupled to viral transcription factory formation. This method can be used to help answer key questions in the fields of genetics, cell biology, virology, and cell biology virology. The main advantage of this technique is it utilizes an inducible viral mini-chromosome model to study the molecular details behind genomic architectural alterations.Collect cells in a 1.5 milliliter micro-centrifuge tube. Centrifuge the cells for two minutes at 200 Volt gravity. Wash the cells three times with one milliliter of sterile DEPC PBS.Next, re-suspend the cells in 1.2 milliliters of DEPC PBS. Place the cover slips into the bottoms of a six-well plate. Pipette 200 microliters of the cell DEPC PBS mixture onto each cover slip.After the cells have settled for two minutes, aspirate excess DEPC PBS, leaving only a thin layer of cells. Carefully add one milliliter of fixing solution to each cover slip. Allow the cells to fix for 10 minutes.Wash the cover slips three times with DEPC PBS. After washing, carefully add one milliliter of glycine DEPC PBS to a final concentration of glycine of 100 millimolar to each cover slip. Allow the cells to quench for five minutes.Add 1.5 milliliters of DEPC PBS and place on a shaker for five minutes, and shake firmly but gently in hand for one minute. Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Carefully add one milliliter of permeabilizing solution to each cover slip.Allow the cells to permeabilize for 15 minutes. Add 1.5 milliliters of DEPC PBS. Place the cells on a shaker for five minutes, and shake firmly but gently in hand for one minute.Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Label the microscope slides, and moisten a paper towel then line the bottom of a sealable plastic container with the towel. Next, prepare the primary antibody solution and multiply the recipe for each cover slip present.Place 30 microliters of primary antibody solution onto each labeled microscope slide. Ensure the primary antibody concentration is correct by holding the corner of the cover slip and gently flicking it. Additionally, the corner of a tissue can be used to wick excess moisture.Then place the cover slip onto the labeled microscope slides, being careful not to introduce bubbles. Cover the exterior of the container with plastic wrap. Move the container into an incubator set to 37 degrees Celsius for one hour.During incubation, prepare the necessary reagents. After incubation, wash the cells three times with DEPC PBS in a six-well plate for five minutes each. Then, wash the cells with FISH wash buffer three times for five minutes each.And keep the cover slip in FISH wash buffer until the secondary antibody, an intron probe mixture has been prepared. Next, clean the glass slides for primary antibody incubation for hybridization with soapy water. After they're dry, clean them with ethanol and a lab tissue.Place 40 microliters of the secondary antibody and FISH intron probe with solution onto each glass slide. Place the cover slips with the cells onto the slides with the cells side facing down on top of the buffer. And be careful not to introduce bubbles.Place the slides into a plastic container with a moist paper towel on the bottom. Wrap the plastic container in first plastic wrap, and then aluminum foil. And incubate the container with the slides.After incubation, carefully slide the cover slip from the edge of the glass slide and place it back into six-well plates, facing up. Wash the cells with FISH wash buffer three times in a six-well plate for five minutes each. Next, wash the cells two times with two-fold SSC.Add DAPI at one to 1000 concentration into the two-fold SSC and allow the solution to sit for five minutes at room temperature. Wash the cells twice with two-fold SSC. Clean the glass slides used for hybridization and add 10 microliters of mounting solution onto the glass slides.Place the cover slips face down onto the mounting solution. With a laboratory tissue, gently remove the excess mounting solution, being careful not to squash the cells. Using nail polish, apply an ample layer around the edge of the cover slips and allow the polish to dry.The sample is now ready for fluorescence microscopy. BCBL-1 cells were stained for LANA and K-Rta RNA to examine where actively transcribing viral episomes are and the heterogeneity of response to Kaposi's Sarcoma-associated Herpesvirus, or KSHV reactivation stimuli. The distinct K-Rta fluorescence in regions that closely match the distribution of viral episomes is evidence for active transcription taking place close to KSHV genomes.In the same sample, cells can be observed which exhibit much weaker and diffuse K-Rta fluorescence that does not significantly overlap with LANA, illustrating the considerable variation and the degree of response to reactivation stimuli within a population of cells. Next, the effectiveness of thymidine synchronization was examined by visualizing the expression of viral protein which clearly demonstrated that synchronized cells responded more to the reactivation stimuli than the unsynchronized population. DAPI staining can serve several functions, this example clearly shows that there are three separate cells instead of one.3D microscopy alongside this technique revealed ring-like RNA PII structures seen with KSHV genomes dotted on the periphery. With its development, the mini viral chromosome would provide unique tools for researchers to explore the special temporal organization of gene-expression and the regression of the cell nucleus.

functional-imaging-viral-transcription-factories-using-3d

Research

JoVE Journal

Biology

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.