This work was supported by grants from the Wellcome Trust (091549 to S.G and 109093/Z/15/A to S.M.), the Wellcome Trust Centre for Cell Biology core grant (092076) and Medical Research Council Non-Clinical Senior Research Fellowship (MR/R008205/1 to S.G.), the European Molecular Biology Organization under a long-term postdoctoral fellowship (ALTF 1070-2017 to R.A.C), and the Independent Research Fund Denmark (T.H.J).

A. Langford and W. Worboys are affiliated with UVO3, a commercial company. They had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

The &#967;CRAC method, combined with the new cross-linking and cell harvesting devices, has great potential because it is applicable to a wide range of model organisms and therefore should be of general interest to the RNA field. There are many areas in which &#967;CRAC can be utilized. For example, the method could be used to measure the hierarchical assembly of proteins into large macromolecular complexes, such as the spliceosome and the ribosome, which often involves dynamic interactions between proteins and RNA molecules. We also now routinely use it to monitor interactions between RNA decay factors and their substrates when cells are subjected to diverse kind of stresses. This enables us to determine at what stage of the adaptive response these factors are most active, what substrates they bind to, and how dynamic these interactions are. Such data should enable researchers to determine the relative contribution of each factor in adaptation to environmental changes.
&#967;CRAC uses dual affinity purification tags (HTF or HTP) to purify the protein under highly stringent and denaturing conditions. This ensures that the copurified RNA is highly enriched for RNAs that were covalently cross-linked to the protein of interest. However, relying on affinity tags has disadvantages. For example, the tag could interfere with protein function, which could give a distorted readout of its RNA-binding interactome. Additionally, for some model organisms it may not always be possible to utilize tags because the genetic tools to integrate DNA fragments into the genome or to transform expression plasmids are not yet available. However, it is straightforward to alter some parts of the &#967;CRAC protocol to make it compatible with CLIP-based protocols that rely on antibodies for purification of the RBP. Indeed, this study showed that it is possible to combine iCLIP-based purifications with our crosslinker. We are now in the process of developing CLIP protocols to study the temporal association of human RNA-binding proteins with nascent RNA transcripts.
When performing &#967;CRAC on a new protein, the UV exposure must be optimized in order to induce maximal cross-linking. This is important&#160;because high UV exposures can reduce the recovery of RNA during the purification step. Cells expressing the recombinant RBP were exposed to various UV doses, 100 mJ/cm2, 250 mJ/cm2, 500 mJ/cm2, and 1 J/cm2. The RNPs were then captured and the RNAs were fragmented and radiolabeled. Afterwards, the RNPs were resolved by SDS-PAGE and an autoradiogram was taken in order to deduce which exposure gave the most intense signal (i.e. the maximal cross-linking).
Once the experimental conditions are optimized, several control experiments are recommended when performing &#967;CRAC. First, a UV irradiated, untagged sample can be used to monitor background binding to the purification beads. Second, when applying &#967;CRAC during a shift experiment, a second time series where the cells are shifted back into the original medium enables investigation into whether the filtration of the cells itself induces changes in RNA levels or protein-RNA interactions.
As mentioned in the Introduction, numerous recently published papers suggest a number of optimizations to the CLIP protocol. This includes the use of fluorescently labeled adapters for detecting the protein-RNA complex through infrared scanning10 as well as optimizations to various nucleic acid purification and size selection steps shown to increase the complexity of the resulting libraries12,45. We are currently implementing some of these improvements to further refine the &#967;CRAC protocol. The protocol presented here already contains a number of improvements to the original CRAC and &#967;CRAC protocols that increase the complexity of the data. For example, previously, after resolving the cross-linked, radioactive protein-RNA complexes on SDS-PAGE gels, they were transferred to a nitrocellulose membrane and the cross-linked RNA was isolated from the blot. However, the transfer of the RNP and subsequent RNA extraction can be very inefficient, particularly when dealing with large RBPs such as RNA polymerase subunits. This can result in a significant reduction in the recovery of the cross-linked RNA. In the current protocol, the cross-linked RNA is extracted directly from SDS-PAGE gel slices as illustrated in Figure 1. This increased the recovery of cross-linked RNAs. Additionally, after PCR amplification of the cDNAs the product was originally resolved on 3%, low melting temperature agarose gels, and then 175&#8211;300 bp PCR products were extracted from the gel. However, these gels can be easily overloaded, resulting in very poor separation of the DNA. Replacing agarose gels with precast TBE gels resulted in more consistent size separation and better recovery of PCR products.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61027">Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC</a>. The Authors section was updated from:
Stuart W. McKellar1 
Ivayla Ivanova1 
Robert W. van Nues2 
Ross A. Cordiner3 
Will Worboys4 
Andrew Langford4 
Torben Heick Jensen3 
Sander Granneman1 
1Centre for Synthetic and Systems Biology, University of Edinburgh 
2Institute of Cell Biology, University of Edinburgh 
3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd.
to:
Stuart W. McKellar1 
Ivayla Ivanova1 
Robert W. van Nues2 
Ross A. Cordiner3 
Mehak Chauhan1 
Niki&#160;Christopoulou1 
Will Worboys4 
Andrew Langford4 
Torben Heick Jensen3 
Sander Granneman1 
1Centre for Engineering Biology, University of Edinburgh 
2Institute of Cell Biology, University of Edinburgh 
3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61027">Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC</a>. The Authors section was updated.

Erratum: Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC

RNAs often rely on RBPs to exert their function, which has led to great interest in understanding the dynamics between these molecules. Many RBPs have been identified in a wide variety of organisms. However, it has always been notoriously difficult to study RBP-RNA interactions in vivo. A major breakthrough in studying such interactions came with the emergence of CLIP1. This method utilizes ultraviolet (UV, 254 nm) irradiation to induce covalent bonds between RBPs and their directly bound RNAs (i.e., zero-distance cross-linking). Subsequently, the RBP of interest is immunopurified under stringent conditions to ensure that only RNAs covalently cross-linked to the proteins are identified. Bound RNAs are then partially digested with RNases and subsequently converted into cDNA libraries for sequencing. The high purification stringency is important as it greatly increases the specificity of protein and RNA recovery, which is also further enhanced through SDS-PAGE purification of the cross-linked ribonucleoprotein (RNP) complex. CLIP and related methods also provide nucleotide resolution insight into the protein binding site, because during the preparation of the sequencing library, amino acids that cross-linked to the RNA frequently terminate the reverse transcriptase or cause the enzyme to introduce mutations at this site1,2,3.
Since its introduction, the original CLIP protocol has produced a remarkable variety of derivative methodologies. A particularly important breakthrough came with the development of HITS-CLIP (or CLIP-seq), which merges high-throughput sequencing with the CLIP approach3. This has since been adopted by all CLIP-based methodologies. iCLIP introduced improvements in the RNase-mediated trimming and adaptor ligation techniques that facilitate more accurate mapping of the RBP binding sites4. PAR-CLIP combined 4thio-uridine/uracil labeling with cross-linking at 365 nm, making it possible to map cross-linking sites by analyzing T-C substitutions5. CRAC, urea-iCLIP, dCLIP, and uvCLAP introduced denaturing conditions and dual affinity purification steps that further reduce background binding to the affinity resin and further increase the specificity of protein capture2,6,7,8,9. Additionally, CRAC, uvCLAP, and dCLIP introduced tagging the RBP of interest with an affinity tag, thus overcoming the need to generate specific antibodies.
Several optimizations have also been made to expedite the CLIP methodology. The original CLIP protocol utilized radiolabeling of the captured RNAs in order to visualize the RBP-RNA complexes after SDS-PAGE. However, the use of radioactivity can be problematic for laboratories not set up for such work. irCLIP incorporates a fluorophore-coupled adaptor that facilitates visualization through infrared imaging10 and sCLIP utilizes biotinylation of captured RNAs in order to visualize them through streptavidin-conjugated HRP11. Furthermore, eCLIP completely forgoes RNA labeling; instead the protein is excised based solely on its known size12. Streptavidin-based purification has also been used to speed up the process of library preparation in FAST-iCLIP, where a biotinylated 3&#8217; adaptor is ligated onto the RNAs and used to enable purification after reverse transcription and circularization13. Additional enhancements to the iCLIP protocol also greatly increased the complexity of the libraries4.
Finally, CLIP has been modified to enable capture of RBPs from different cellular subcompartments14,15, to visualize newly transcribed RNAs using pulsed induction of photoactivable ribonucleosides5,16,17, to capture methylated RNAs18,19,20, to examine RNA-RNA interactions21,22, and to map 3&#8217; ends23,24.
Despite the great contributions of CLIP-based techniques in aiding our understanding of the interactions between RBPs and RNAs, it has been limited by the inefficiency of UV cross-linking. Although culture cells grown in a monolayer are generally relatively easy to cross-link, this is significantly more challenging in tissues or cells in solution. Tissues can require multiple rounds of UV exposure in order to penetrate to the required cell layers, while microbial cells are often grown in rich mediums that contain aromatic, UV-absorbing compounds25. Indeed, UV irradiation times of up to 30 min have been used to generate sufficient cross-linking between RBPs and their bound RNAs for such samples26,27,28. This extended UV exposure induces stress responses within the cell, such UV-induced DNA damage, which can contaminate the final data in some applications.
The majority of CLIP studies have focused on generating single &#34;snapshots&#34; of specific protein-RNA interactions in a cell. However, protein-RNA interactions are inherently dynamic, particularly when cells are subject to changes in their environment. This can include a sudden reduction in the availability of essential nutrients or rapid changes in temperature. As such, to truly understand the role of an RBP during stress, it is best to perform time-resolved analyses because they can capture the full spectrum of RBP targets during stress and determine at what stage of the stress response the chosen RBP is active. In particular, studies in yeast showed that the first few minutes of adaptation are absolutely crucial for survival and RNA half-lives in bacteria can vary from minutes to seconds29,30,31,32,33. Therefore, such time-resolved analyses should ideally be performed at high temporal resolution. However, the long cross-linking times make the study of early stage adaptive responses particularly challenging.
In order to overcome these issues, we recently developed an improved method that is capable of cross-linking and harvesting cells on minute-long timescales. Our &#967;CRAC method allows quantitative measurement of dynamic changes in RBP-RNA interactions at previously unwitnessed resolution. Crucial to this method was the development of a novel UV irradiation device32 that reduces the required cross-linking time in yeast and bacteria in solution around 10-fold, effectively freezing RBP-RNA interactions instantaneously. In addition, in order to rapidly harvest the cells after UV irradiation, we developed a vacuum filtration device that can harvest exponentially growing yeast in a 0.5 L culture in around 30 s32. These technological innovations allow the study of RBP-RNA dynamics at minute-scale resolution. Additionally, we also introduced several optimizations to the original CRAC protocol2 in order to increase its practicality.
Using &#967;CRAC, we recently studied the targetome of a yeast nuclear RBP, Nab3, in response to glucose deprivation. In Saccharomyces cerevisiae, Nab3 can form a complex with Nrd1, an RBP, and the RNA helicase Sen1 to form the NNS complex. NNS binding to the RNA polymerase and the nascent transcript can trigger transcriptional termination34. This complex is mostly involved in removing cryptic noncoding RNA transcripts but has also been shown to regulate expression of protein-coding genes. The study showed differential targeting of Nab3 to noncoding and coding transcripts after only a minute of stress32. We demonstrated that co-transcriptional termination by Nab3 results in a very transient, pulse-like expression of retrotransposon genes, which would have been difficult to detect using traditional CLIP-based approaches. Additionally, the short UV irradiation times in our UV cross-linker also significantly increased the recovery of short-lived noncoding RNAs32. &#967;CRAC will likely be a crucial tool in elucidating not only how RBPs shape the response to stress on immediate timescales but also their changing roles during the whole lifecycle of a response. This manuscript provides a detailed overview of all the steps in the &#967;CRAC protocol. For illustrative purposes, the method was used to study the yeast Nrd1 protein, which is involved in transcriptional termination and RNA decay35,36, and its RNA targetome in response to glucose deprivation across a multitude of timepoints. Finally, we also demonstrate that our UV irradiation unit can rapidly cross-link RBPs to RNA in HeLa cells, making it possible to also perform high-resolution time-resolved analyses in adherent cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:876px;" width="876">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1,4-dithioreitol</td>
			<td style="width:85px;">Merck</td>
			<td style="width:101px;">10708984001</td>
			<td style="width:495px;">Buffer component in mammalian cell lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1.5 mL tubes</td>
			<td style="width:85px;">Eppendorf</td>
			<td style="width:101px;">0030 120.086</td>
			<td>General reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">2 mL tubes</td>
			<td style="width:85px;">Eppendorf</td>
			<td style="width:101px;">0030 123.344</td>
			<td>For holding columns and collection of waste</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">32P-yATP</td>
			<td style="width:85px;">Perkin Elmer</td>
			<td style="width:101px;">NEG502Z-250</td>
			<td>For radiolabelling the 5&#39; end of the RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">4-12% Bis-Tris gel</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:101px;">NP0321BOX</td>
			<td>SDS-PAGE gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">4X loading buffer</td>
			<td style="width:85px;">Novex</td>
			<td style="width:101px;">NP0008</td>
			<td>Protein loading dye concentrate</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">50 bp ladder</td>
			<td style="width:85px;">New England Biolabs</td>
			<td style="width:101px;">N3236</td>
			<td>Reference ladder for excising region of interest from the amplified cDNA library</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">50% PEG</td>
			<td style="width:85px;">NEB</td>
			<td style="width:101px;">B100045</td>
			<td>For the L5 linker ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">6% TBE gel</td>
			<td style="width:85px;">Invitrogen</td>
			<td>EC6265BOX</td>
			<td>For separation and purification of the cDNA library</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Acetone</td>
			<td style="width:85px;">ACROS Organics</td>
			<td style="width:101px;">423245000</td>
			<td>Washing of TCA-precipitated proteins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">anti-FLAG beads</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">M8823-1ML</td>
			<td>For purifcation of FLAG-tagged RBPs</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">ATP (100 mM)</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">R0441</td>
			<td>For ligation of the L5 linker onto the 5&#39; end of captured RNAs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Beta-mercaptoethanol</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">M3148-100ML</td>
			<td>Buffer component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Biomax MS intensifying screen</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">Z363162-1EA</td>
			<td>For intensifying the autoradiogram signal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Chloroform</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">1010219</td>
			<td>For phenol-chloroform extraction following RNA purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">cOmplete EDTA-free protease inhibitor cocktail</td>
			<td style="width:85px;">Roche</td>
			<td style="width:101px;">11873580001</td>
			<td>For inhibition of cellular proteases after lysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Complete supplement mixture -TRP</td>
			<td style="width:85px;">Formedium</td>
			<td style="width:101px;">DCS0149</td>
			<td>For preparation of synthetic defined medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Costar Spin-X 0.22 &#181;m filters</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">CLS8160</td>
			<td>For isolating the excised cDNAs following gel extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">DNase RQ1</td>
			<td style="width:85px;">Promega</td>
			<td style="width:101px;">M6101</td>
			<td>For DNA digest following cell lysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">dNTPs (10 mM)</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">4638956001</td>
			<td>For reverse transcription and PCR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Ethanol</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">10041814</td>
			<td>For phenol-chloroform extraction following RNA purification and DNA precipitation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:101px;">AM9261</td>
			<td>For protease K buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Exonuclease I</td>
			<td style="width:85px;">New England Biolabs</td>
			<td style="width:101px;">M0293</td>
			<td>For degradation of primers following PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass microfiber filters</td>
			<td style="width:85px;">Whatman</td>
			<td style="width:101px;">1823-010</td>
			<td>For isolating the excised cDNAs following gel extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glucose</td>
			<td style="width:85px;">Formedium</td>
			<td style="width:101px;">GLU03</td>
			<td>For preparation of glucose-containing, synthetic defined medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glycogen (20 mg/mL)</td>
			<td style="width:85px;">Roche</td>
			<td style="width:101px;">10901393001</td>
			<td>Precipitation of proteins, RNA and DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">GST-TEV</td>
			<td style="width:85px;">Homemade</td>
			<td style="width:101px;"></td>
			<td>Construct and purification protocol is available upon request</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Guanidium hydrochloroide</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">10071503</td>
			<td>Required for pulldown denaturing conditions and washing buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">IgG beads</td>
			<td style="width:85px;">GE Healthcare</td>
			<td style="width:101px;">17-0969-01</td>
			<td>For purification of protein A-tagged RBPs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Imidazole</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">I2399-100G</td>
			<td>For elution of captured proteins from Nickel beads</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Isoamyl alcohol</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">A393-500</td>
			<td>For phenol-chloroform extraction following RNA purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Luna Universal One-Step RT-qPCR</td>
			<td style="width:85px;">NEB</td>
			<td style="width:101px;">E3005S</td>
			<td>For qPCR of the cDNA in order to calculate required number of PCR cycles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Magnesium chloride</td>
			<td style="width:85px;">Fluka Analytical</td>
			<td style="width:101px;">63020-1L</td>
			<td>For PNK buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Membrane filters</td>
			<td style="width:85px;">Millipore</td>
			<td style="width:101px;">AAWP09000 for yeast or HAWP09000 for bacteria</td>
			<td>For vacuum filtration of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micro bio-spin columns</td>
			<td style="width:85px;">Biorad</td>
			<td style="width:101px;">732-6204</td>
			<td>For collecting eluate after gel extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Ni-NTA beads</td>
			<td style="width:85px;">Qiagen</td>
			<td style="width:101px;">30210</td>
			<td>For secondary protein capture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">NP-40</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">I8896-100ML</td>
			<td>Buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Pfu polymerase</td>
			<td style="width:85px;">Promega</td>
			<td style="width:101px;">M7741</td>
			<td>For amplification of the cDNA library</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Phenol</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">P4682-400ML</td>
			<td>For phenol-chloroform extraction following RNA purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Pierce spin columns</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">69725</td>
			<td>For on-column enzymatic reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Protease K</td>
			<td style="width:85px;">Roche</td>
			<td style="width:101px;">3115887001</td>
			<td>For degradation of the RBP following gel extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Quartz Petri dish</td>
			<td style="width:85px;">UVO3</td>
			<td style="width:101px;">N/A</td>
			<td>For cross-linking of adherent cells. Available from https://www.vari-x-link.com for 400 GBP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Radiography films</td>
			<td style="width:85px;">Amersham</td>
			<td style="width:101px;">28906843</td>
			<td>For autoradiography visualisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RNAClean XP beads</td>
			<td style="width:85px;">Beckmann</td>
			<td style="width:101px;">A63987</td>
			<td>SPRI beads for clean up of RNAs and cDNAs</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">RNase H</td>
			<td style="width:85px;">New England Biolabs</td>
			<td style="width:101px;">M0297</td>
			<td>For degradation of RNAs following reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RNase-It</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:101px;">400720</td>
			<td>For RNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">rRNasin</td>
			<td style="width:85px;">Promega</td>
			<td style="width:101px;">N2511</td>
			<td>For inhibition of any contaminating RNases during enzymatic reaction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium acetate</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">S2889-1KG</td>
			<td>For phenol-chloroform extraction following RNA purification and DNA precipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Sodium chloride</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">7647-14-5</td>
			<td>Buffer component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium deoxycholate</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">D6750-100G</td>
			<td>Buffer component in mammalian cell lysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium dodecylsulfate</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">L3771-1KG</td>
			<td>For protease K buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">SUPERase-In</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:101px;">AM2694</td>
			<td>For inhibition of cellular RNases after lysis</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">SuperScript IV</td>
			<td style="width:85px;">Thermo Fisher Scientific</td>
			<td style="width:101px;">18090010</td>
			<td>For reverse transcription</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">T4 PNK</td>
			<td style="width:85px;">New England Biolabs</td>
			<td style="width:101px;">M0201</td>
			<td>For radiolabelling the 5&#39; end of the RNA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">T4 RNA ligase 1</td>
			<td style="width:85px;">New England Biolabs</td>
			<td style="width:101px;">M0204</td>
			<td>For ligation of the L5 adaptor onto the RNA 5&#39; end</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">T4 RNase ligase 2, truncated K222Q</td>
			<td style="width:85px;">NEB</td>
			<td style="width:101px;">M0351S</td>
			<td>For ligation of the App_PE linker onto the 3&#39; end of captured RNAs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TBE buffer (10X)</td>
			<td style="width:85px;">Invitrogen</td>
			<td>15581-028</td>
			<td>For running TBE gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TEV protease</td>
			<td style="width:85px;">Homemade</td>
			<td style="width:101px;"></td>
			<td>For eluting captured proteins following FLAG capture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Thermosensitive alkaline phosphatase</td>
			<td style="width:85px;">Promega</td>
			<td style="width:101px;">M9910</td>
			<td>For 5&#39; and 3&#39; dephosphorylation of RNAs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trichloroacetic acid (100%)</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">T0699-100ML</td>
			<td>For precipitation of RBP-RNA complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tris hydrochloride</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:101px;">15504-020</td>
			<td>Buffer component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Triton X-100</td>
			<td style="width:85px;">Sigma Aldrich</td>
			<td style="width:101px;">T8787-100ML</td>
			<td>Buffer component in mammalian cell lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Vari Filter</td>
			<td style="width:85px;">UVO3</td>
			<td style="width:101px;">N/A</td>
			<td>Device for vacuum harvesting cells. Available from https://www.vari-x-link.com for 100 GBP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Vari-X-Linker</td>
			<td style="width:85px;">UVO3</td>
			<td style="width:101px;">N/A</td>
			<td>Cross-linker for cross-linking cells. Available from https://www.vari-x-link.com for 16,000 GBP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Yeast nitrogen base</td>
			<td style="width:85px;">Formedium</td>
			<td style="width:101px;">CYN0410</td>
			<td>For preparation of synthetic defined medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zirconia beads</td>
			<td style="width:85px;">Thistle</td>
			<td>11079105Z for yeast or 11079101Z for bacteria</td>
			<td>For cell lysis via bead beating</td>
		</tr>
	</tbody>
</table>

monitoring protein-rna interaction dynamics in vivo at high temporal resolution using &#967;crac

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

To demonstrate the efficacy of the &#967;CRAC method, a time-course experiment with yeast strains expressing an HTP-tagged Nrd1 protein was performed. A detailed schematic representation describing how the method works is provided in Figure 1. Like Nab3, Nrd1 is involved in nuclear RNA decay of a variety of RNA transcripts37. Previous work from the Corden lab suggested that Nrd1 binding to its RNA targets changes significantly when cells are subjected to glucose starvation28,38. As such, cells growing exponentially in medium containing glucose (SD-TRP) were shifted to the same medium without glucose (S-TRP) over a time-course to monitor dynamic changes in Nrd1-RNA interactions. Samples were taken and cross-linked in the Vari-X-linker chamber (Figure 3A) before the shift and then after 1, 2, 4, 8, 14, and 20 min. The medium used for cell growth was deliberately deficient in tryptophan to reduce UV absorption by this aromatic amino acid. Note that it is best to use synthetic medium that is filter sterilized as&#160;because autoclaving the medium can lead to caramelization of the sugars. This then&#160;reduces the cross-linking efficiency.
Figure 4A shows a representative autoradiograph from a &#967;CRAC experiment. Note that in this example, the samples were not pooled together. Instead, each was run individually on the gel. This is recommended for initial experimental tests to show that the protein cross-links effectively to RNA at all of the tested timepoints. A particularly intense signal was observed at the expected molecular weight of the RBP, representing the protein bound to very short, radiolabeled RNAs not amenable for sequencing. Therefore, the smeary signal above this band, which is the protein crosslinked to longer RNA fragments, was isolated. The fragment was cut from just above the protein band plus around 30 kDa. Figure 4B shows an autoradiogram following excision, with the protein cross-linked to short RNAs left in the gel and the previously smeary signal now excised.
After reverse transcription, the cDNA library must be amplified using PCR. However, overamplification of the library must be avoided as this can introduce bias towards sequences preferentially amplified by the polymerase and generate PCR artefacts. Overamplified libraries also contain a large number of duplicate sequences that waste reads on the sequencer. In order to calculate the ideal number of PCR cycles for amplification of the final library, an aliquot of the cDNA was amplified through qPCR using the P5 and BC oligonucleotides. The first cycle at which the library reached peak fluorescence was chosen as the PCR cycle count. Figure 4C gives an example of a qPCR from a typical cDNA library, which yielded a peak cycle count of 16. This value was then used for the final &#967;CRAC PCR. In order to process the sequenced data, we used software previously developed in our lab (pyCRAC) and the corresponding pipeline for analysis of the kinetic CRAC data (Nues et al., 2017; https://git.ecdf.ed.ac.uk/sgrannem/pycrac, https://bitbucket.org/sgrann/kinetic_crac_pipeline/src/default/). These open source software tools enable demultiplexing and trimming of the data, removal of PCR duplicates, identification of statistically significant peaks, cluster reads into contiguous sequences, and identify binding motifs39. Further details of how these tools operate are found on their respective webpages.
We also started to develop a &#967;CRAC protocol for mammalian cells. The majority of mammalian cell lines are grown as a monolayer and the tray in our crosslinker with the UV-permeable bag is not suitable for experiments with adherent cells. To overcome this problem, we developed a stage where users can UV irradiate 1&#8211;2 Petri dishes (150 mm diameter and 25 mm in depth) with adherent cells (Figure 3B). As a first test, the efficiency of the cross-linker for mammalian cells was measured through cross-linking and capture of stably tagged GFP-RBM7 using anti-GFP antibodies and a traditional CLIP-based purification. As shown in Figure 5A, the cross-linker was able to recover protein-RNA complexes from mammalian cells grown as a monolayer using 254 nm UV irradiation at efficiencies comparable to a widely used UV irradiation device. However, standard cell culture plasticware normally used for UV cross-linking experiments is impenetrable to 254 nm UV. Therefore, in our cross-linker the cells would only receive irradiation from the upper bank of UV lamps. To overcome this, we developed a UV-permeable quartz Petri dish for cell growth and cross-linking. Use of the quartz cultureware displayed robust recovery of protein-RNA complexes with as few as 2 s of UV irradiation (Figure 5B). When combined with RBP capture methods for mammalian cells such as CLIP technologies, these short cross-linking times are amenable with timecourses to recover spatiotemporal RNA-binding profiles of RBPs in response to genotoxic stresses or rapid depletions of protein factors, or in parallel with transcriptional or cell cycle synchronization.
Figure 6 shows several examples of the Nrd1 data processed by the &#967;CRAC pipeline. This figure was prepared using the bedgraph files generated by the pipeline and the python GenomeBrowser package (https://pypi.org/project/GenomeBrowser/1.6.3/), which we designed to simplify making publication-quality genome browser images of the data. The grey rectangles represent genomic regions that expressed noncoding RNAs, such as the cryptic unstable transcript (CUTs), stable uncharacterized transcripts (SUTs)40, and Xrn1-sensitive unstable transcripts (XUTs)41. The data in Figure 6 show that Nrd1 binds to many of these noncoding RNA transcripts, consistent with the idea that this protein is involved in degradation of this class of transcripts42. Figure 6A shows a ~15 kb region on chromosome IV. Here there was a significant increase in binding of Nrd1 to transcripts encoding the high-affinity glucose transporters HXT6 and HXT7, both of which are upregulated during glucose starvation. It is likely that transcription termination by the NNS complex may influence the induction kinetics of these genes during glucose starvation. Figure 6B shows an example of Nrd1 cross-linking to the Imd3 transcript, which is known to be regulated by Nab343. In this case the data demonstrated a significant reduction in binding upon glucose starvation. Previous work showed&#160;decreased binding of Nab3 to the Tye7 transcript during glucose starvation44. Consistent with this observation,the &#967;CRAC data suggest that binding of Nrd1 decreased during glucose starvation and Nrd1 cross-linking to Tye7 was at its lowest after 8 min of stress (Figure 4C). However, it appears that this effect was only transient, because after 14 min of glucose starvation, Nrd1 binding went back to starting levels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61027/61027fig1v2.jpg" /> 
Figure 1: Schematic representation of the &#967;CRAC protocol. Tagged strains were grown until the desired density. RBP indicates RNA-binding protein. Afterwards, a reference sample was taken and cross-linked with 254 nm UV light. The remaining cells were harvested by filtration and then rapidly shifted to the stress-inducing medium. For the &#967;CRAC experiment described here, samples were taken and cross-linked 1, 2, 4, 8, 14, and 20 min after the shift (1). The RBP of interest was then purified using a highly stringent two-step affinity purification (2). Next, the captured cross-linked RNAs were partially digested with RNases, radiolabeled at the 5&#8217; end and adapters were ligated onto them (3). The 5&#8217; adapters contained unique &#8220;in-read&#8221; barcode sequences so that the individual samples could be separated bioinformatically after sequencing. The RBP-RNA complexes were then eluted, pooled, and precipitated together (4), resolved by SDS-PAGE and visualized through autoradiography (5). Subsequently, a single gel slice containing the radioactive signal just above the main band, illustrated with dashed red box in the autoradiography image, was cut from the gel (5). The gel slices were treated with protease K and the RNA was subsequently extracted (6), converted into cDNAs, and amplified through PCR (7). The PCR step introduced additional barcodes (yellow block introduced by P7 oligo) so that many libraries could be multiplexed into a single lane. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61027/61027fig2v2.jpg" /> 
Figure 2: Cross-linking and vacuum filtration. (A) The cross-linker. The cell suspension is poured into a funnel located in the top right of the machine (also see Figure 3A for a close-up) and held in a UV-transparent bag located in the middle tray. This bag is flanked by two shutters that remain closed until the user instructs the machine to start the irradiation step. The cells are irradiated with UV light from the trays both above and below. The machine comes supplied with 254 and 365 nm UV lamps, with the latter being applicable for PAR-CLIP experiments. The machine is operated through a touchscreen panel located on the top right which allows one to control UV dosage or exposure time. (B) Following cross-linking, the cells are drained from the left-hand side of the machine. Cell suspensions are recovered through vacuum and drained into a glass flask where they can be subsequently poured into a vacuum filtration device for harvesting. (C) Vacuum filtration devices. These are opened and closed via a clip and a filter is inserted between it. Four filtration devices were used in parallel for very short time series to not lose any time as a result of changing filters. (D) Following filtration, the media supernatant was drained into flasks for subsequent disposal. Valves were installed below the vacuum filtration devices to maintain the vacuum in the system when the filter is removed. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61027/61027fig3v2.jpg" /> 
Figure 3: Cross-linking suspended vs. adherent cells. (A) The cross-linker with the Vari-X-linker chamber for suspension cells. The cell culture is poured into the sample inlet (funnel) located in the top right of the tray. (B) Tray that can hold plastic or quartz Petri dishes for cross-linking adherent cells or small volumes of suspension cells. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61027/61027fig4v2.jpg" /> 
Figure 4: Library preparation. (A) Example of an autoradiogram from a Nrd1-HTP &#967;CRAC experiment. The strong, concentrated signal represents the protein crosslinked to very short RNAs, while the smear above represents the protein cross-linked to RNAs of sufficient length for sequencing. (B) The smear was excised as shown in an autoradiogram taken after gel excision. (C) A representative qPCR from a &#967;CRAC cDNA library. In this example, maximum amplification of the cDNA was reached at 16 cycles. Thus, 16 cycles were used for the final amplification. The error bar represents the standard deviation of three technical qPCR replicates. (D) Example of a phosphorimage from a cDNA library on a 6% TBE gel. (E) cDNA length and quality analysis from a chip-based capillary electrophoresis. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61027/61027fig5v2.jpg" /> 
Figure 5: High RNase test iCLIP experiment to test crosslinking in mammalian cells. Shown are autoradiograms from GFP-RBM7 iCLIP experiments which tested the efficiency of RNP recovery across various cross-linking energies. Immunoprecipitations were performed using anti-GFP antibodies coupled to magnetic beads on cross-linked cells which stably expressed GFP-RBM7. Immunoprecipitates were incubated with high concentrations of RNase I in order to trim associated RNAs to short, uniform lengths. RNPs were visualized by 32P labelling and SDS-PAGE and migrate as a defined band, close to the migration of the non-cross-linked protein. Quantification indicates the results of densitometric analyses of radiolabeled RBM7-RNA signal normalized to the anti-GFP western blot signal. (A) Cross-linking time-course of the commonly used UVP cross-linker versus our cross-linker (Vari-X-linker; VxL). (B) Cross-linking time-course of our cross-linker on quartz (left) and plastic (right) cultureware. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61027/61027fig6v2.jpg" /> 
Figure 6: Example genome browser plots showing the power of &#967;CRAC to show differential, temporal binding of Nrd1 to its targets. Each box shows plots for individual genomic regions. The arrows indicate on which strand the genes are encoded (left pointing arrow = minus strand; right pointing arrow = plus strand). The timepoints (min) are indicated by t0, t1, t2, etc on the y-axes of each subplot. Roman numerals indicating the chromosomes and the coordinates are shown. (A) Upon glucose deprivation, Nrd1 binds two high-affinity glucose transporters, HXT6 and HXT7, which are both upregulated in this condition. (B) Nrd1 is observed to bind to Imd3, an already validated target of Nab344, with reducing intensity following glucose starvation. (C) Nrd1 binding of Tye7 exhibits a dynamic and transient nature, decreasing after glucose starvation to a minimum after 8 min of stress. However, binding subsequently returns to basal levels after 14 min. Reads were normalized to &#8220;reads per million&#8221; (RPM; y-axis). Grey boxes indicate regions encoding noncoding RNAs. <a href="https://www.jove.com/files/ftp_upload/61027/61027fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using Ãâ€¡CRAC at JoVE.com

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

monitoring-protein-rna-interaction-dynamics-vivo-at-high-temporal

Research

JoVE Journal

Biochemistry

4.8K Views.  University of Edinburgh. Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

Video: Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.