Work in our lab is supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Israeli Association for Fighting the A-T Disease.

See Table of Materials for details of materials, equipment, and antibodies. Animal procedures were employed under the ethical guidelines of Tel Aviv University&#39;s Ethics Committee after approval. The procedure is carried out on 10-day old mouse pups, regardless of their sex. If needed, genotyping is performed on the previous day using a tail biopsy and standard methods. Solutions are sterile and stored at 4 &#176;C unless indicated otherwise; see Table 1.
1. Preparation of cultures
NOTE: Conduct the procedure within a sterile environment and disinfect the work surface in advance using 70% ethanol.
<ol>
	<li>Add 1 mL of BME to each well of 6-well plates. Using sterile forceps, place a single cell culture insert into each well and incubate the plates in a 37 &#176;C/5% CO2 incubator for a minimum of 2 h prior to the dissection procedure.</li>
	<li>Set up a dissection area within a biological hood, containing the tissue chopper, 30 mm culture plates, a binocular microscope, and a light source.</li>
	<li>Immerse the surgical tools in 70% ethanol throughout the process. Use a urinary cup filled with 70% ethanol for immersion, and a separate urinary cup with 1x PBS to wash off the ethanol before use.</li>
	<li>For each dissection (i.e., for each cerebellum), prepare two 30 mm culture plates filled with cold dissection medium and leave them on ice.</li>
	<li>Spray the animal with 70% ethanol.</li>
	<li>Using sharp scissors, swiftly decapitate the animal. To expose the brain, make a small incision with scissors away from the cerebellum. Using tweezers with rounded ends, gently peel off the skull. Remove the skull in small pieces to avoid damaging the cerebellar tissue.</li>
	<li>Immediately transfer the brain into cold dissection medium or directly remove the cerebellum from within the skull. To do this, expose the cerebellum and separate the cerebellar cortex from the brainstem by disconnecting the three pairs of peduncles, using a fine curved iris spatula. To identify the peduncles, view from rostral to caudal: the paired superior cerebellar peduncles connect the midbrain to each side of the cerebellum; the paired middle cerebellar peduncles connect the pons to each side of the cerebellum; and the paired inferior cerebellar peduncles connect to the medulla to each side of to the cerebellum. Once the cerebellum is visible, separate it from the cerebrum using the same spatula: slide the spatula between the cerebellum, the superior colliculus, and the brainstem. 
	NOTE: Do not make a special effort to remove the meninges as they readily detach after the cerebellum is sliced.</li>
	<li>Set the tissue chopper to a slice thickness of 350 &#181;m.</li>
	<li>Prior to positioning the cerebellum on the chopper&#39;s base, remove excess dissecting buffer from the tissue. This will improve the tissue&#39;s adhesion to the chopper&#39;s base.</li>
	<li>Position the cerebellum on the cutting platform in a vertical orientation and carry out parasagittal slicing at a slice thickness of 350 &#181;m. Gently separate the slices using a fine iris spatula and put them into cold dissection medium.</li>
	<li>Transfer the sliced tissue back to a fresh cold dissection buffer. Use a 1,000 &#181;L tip to delicately guide the tissue slices into the dish containing cold dissection buffer.</li>
	<li>Under the binocular use two curved iris spatulas to carefully separate the tissue slices from each other. For cultures, use slices derived from the cerebellar vermis situated in the medial cortico-nuclear zone of the organ.</li>
	<li>Transfer each tissue slice onto a culture insert using a wide spatula and move each insert carrying a tissue slice into a well in the 6-well plates containing the pre-warmed medium. Leave the plates in the incubator.</li>
	<li>Replace the medium every other day: aspirate the used medium using a sterile glass pipette, and with&#160;a 5 mL serological pipette, add 1 mL of fresh MBE with the pipette&#39;s tip leaning against the well&#39;s wall. Make sure not to direct the medium flow onto the tissue. 
	NOTE: The tissue slice will exhibit an inflammation-like appearance for the first 5-7 days in culture and will be ready for experiments as soon as this phenomenon disappears and up to 2 weeks after culture establishment.</li>
</ol>
2. Treatment with DNA damaging agents, fixation, staining, and microscopic imaging
<ol>
	<li>Add DNA-damaging chemicals directly to the culture medium at appropriate final concentrations, for predetermined durations. After the exposure period, remove the chemicals by replacing the medium with fresh culture medium.</li>
	<li>If the DDR readout involves PAR staining, add an inhibitor of poly(ADP-ribose) glycohydrolase (PARG) to the culture medium at a final concentration of 10 &#181;M, 30 min prior to fixation. 
	NOTE: This treatment is critical as the PAR modification is short-lived and continuously degraded by PARG.</li>
	<li>Immediately prior to fixation, aspirate the medium and replace it with 1 mL of 0.1 M phosphate buffer at room temperature. 
	NOTE: Cold buffer might cause detachment of the tissue slices from the insert.</li>
	<li>Immediately fix the slices by replacing the buffer with precooled (-20 &#176;C) acetone:methanol mix (1:1) such that the tissue slice is submerged in this mix&#160;and leave for 20 min at -20 &#176;C. 
	NOTE: It is possible to stop at this stage and proceed with immunostaining later. To do so, wash the tissues 3x with 0.1 M phosphate buffer precooled to 4 &#176;C, making sure the tissue is submerged in the buffer. Tissues can be stored now in 0.1M phosphate buffer for up to 2 weeks at 4 &#176;C.</li>
	<li>Transfer the tissue slice into a well in a 48-well plate.</li>
	<li>Use a scalpel and a cutting board to remove the margins of the insert, cutting around the tissue slice. Place each slice in a well of a 48-well plate. Add 100 &#181;L of 0.1 M phosphate buffer.</li>
	<li>For permeabilization, aspirate the buffer, add to the well 100 &#181;L of 0.1 M phosphate buffer containing 1% Triton X-100, and leave for 5 min at room temperature.</li>
	<li>Aspirate the solution and add 100 &#181;L of blocking solution (see Table 1) per well. Shake gently at room temperature for 2 h.</li>
	<li>During the blocking step, dilute the primary antibody or anti-PAR reagent (1:800) in the blocking solution.</li>
	<li>Remove the blocking solution (no need to wash) and add 100 &#181;L of primary antibody mix to each well. Apply gentle shaking for 2 h at room temperature.</li>
	<li>Dilute the secondary antibodies in 0.1 M phosphate buffer containing 0.2% Triton X-100 (1:500) while minimizing their exposure to light.</li>
	<li>Aspirate the primary antibody solution and wash the slices 3 x 5 min with 0.1 M phosphate buffer containing 0.2% Triton X-100, at room temperature, with gentle shaking.</li>
	<li>Replace the wash solution with 100 &#181;L of secondary antibody solution (1:500 dilution). Continue gentle shaking for 2 h at room temperature in the dark (cover the plates with aluminum foil).</li>
	<li>To prepare a DAPI solution, dilute the stock solution (2 mg/mL) to 1 &#181;g/mL in 1x PBS.</li>
	<li>Twenty minutes before the end of the 2 h incubation with the secondary antibody, add 20 &#181;L of the final DAPI solution to each well and continue shaking for 20 min. Aspirate the liquid and wash with the wash solution for 3 x 5 min with gentle shaking.</li>
	<li>For mounting, place the tissue on a microscope glass slide with the tissue facing up, add mounting medium, and cover with a cover glass. Place the slides on a flat surface and let them dry overnight in the dark, at room temperature (avoid using mounting medium containing DAPI to prevent blurriness).</li>
	<li>Store the slides at 4 &#176;C in a lightproof box.</li>
	<li>Capture microscopic images using a confocal microscope and appropriate filters.</li>
</ol>

Cerebellar Purkinje Cells: DNA Damage Response in Organotypic Cultures

The authors declare no conflicts of interest related to this study.

General comments 
The major advantage of the organotypic culture system is that it facilitates studies using the cerebellar cortex tissue, preserving its structural organization for several weeks in the culture dish setup. This system is useful for conducting in-depth morphological analyses of Purkinje cells (PCs), including detailed examinations of dendritic spines and ultrastructural features52,53,54...

The integrity of cellular DNA is constantly under threat from DNA damaging agents, predominantly metabolic by-products like reactive oxygen species, which inflict tens of thousands of DNA lesions per cell daily1. The persistent upkeep of genome stability is essential for cellular homeostasis2,3. The cornerstone of this maintenance is the DNA damage response (DDR) - an intricate, layered signaling network that initiates specific DNA repair pathways while carefully adjusting many other cellular processes4,5. Deficits in the DDR are commonly manifested as &#39;genome instability syndromes,&#39; marked by chromosomal instability, progressive tissue deterioration, impaired growth or development, a predisposition to cancer, and heightened sensitivity to particular DNA-damaging agents6,7,8,9. Notably, neurodegeneration, which often includes cerebellar atrophy, is a distinct feature of many genome instability syndromes7,10,11,12.
The autosomal recessive disorder, ataxia-telangiectasia (A-T), is a well-documented example of a genome instability disorder13,14,15. This condition arises from null mutations in the ATM (A-T, mutated) gene, responsible for coding the pivotal protein kinase, ATM, which is known primarily as a DDR mobilizer in response to DNA double-strand breaks (DSBs)16,17. A-T manifests as a multisystem disorder, predominantly characterized by progressive cerebellar degeneration, leading to acute motor impairments, immunodeficiency, gonadal atrophy, cancer predisposition, and extreme sensitivity to ionizing radiation. Cultured cells from individuals with A-T show chromosomal instability and increased sensitivity to genotoxic agents, especially those causing DSBs15,18,19. Importantly, ATM also plays a role in repairing other DNA lesions, underscoring its broad significance in maintaining genome stability20,21,22.
Despite thorough research into ATM&#39;s numerous roles, the specific mechanism leading to cerebellar degeneration in A-T remains a topic of active debate, with various models proposed to elucidate this process23,24,25,26,27,28,29,30. Our model28 suggests that cerebellar degeneration in A-T patients begins with the dysfunction and eventual loss of Purkinje cells (PCs). Considering ATM&#39;s critical role in preserving genome stability in the face of ongoing DNA damage, PCs are particularly vulnerable to the absence of ATM. We attribute this vulnerability to the combination of their high metabolic activity, distinctive chromatin structure, and extensive transcriptional activity. Ultimately, it is suggested that the loss of PC function, and hence, their degeneration, is due to the stochastic, functional inactivation of genes, a consequence of producing defective transcripts28.
The study of PC biology in the laboratory is impeded by challenges in cultivating isolated PCs, as these cells rely heavily on their natural milieu and neighboring cells for survival and function, rendering them incompatible with dissociated culture growth. Nonetheless, PCs can remain viable for extended periods in tissue slice cultures. Cerebellar organotypic cultures, which are tissue slices typically derived from rodent cerebella, maintain the tissue&#39;s structural organization and support various experimental manipulations analogous to those possible with cultured cells. Therefore, these cultures allow for cerebellar studies within a controlled setting31,32,33,34,35,36,37,38,39,40,41,42,43. Specifically, in the context of A-T, murine cerebellar organotypic cultures have proven to be instrumental in exploring the DDR in Atm-deficient PCs40,41,42,43. While Atm-deficient mice display only a subtle cerebellar phenotype44,45,46,47, presumably due to differences between human and mouse cerebellar physiology, the assumption is that ATM&#39;s roles are largely conserved across these species. This notion is supported by our observations that the deficient response to DNA DSBs in Atm-/- murine PCs aligns with that observed in other murine and human ATM/Atm-deficient cell types40,41,42,43.
A limitation in analyzing PC responses to various stimuli or stresses within organotypic cultures is the necessity to rely on microscopic imaging for readouts. The DDR is usually studied using bulk biochemical readouts, although common immunofluorescent markers are utilized as well, such as following the dynamics of formation and resolution of nuclear foci of phosphorylated histone H2AX (&#947;H2AX) and the 53BP1 protein, which are considered indicators of DSBs48,49. A broader measure is the fluorescent imaging of poly(ADP-ribose) (PAR) chain formation on proteins, a rapid and robust early DNA damage response, particularly to strand breaks7,50. We modified the protocol by Komulainen et al.51 for PAR staining in cerebellar organotypic cultures. We observed a pronounced PAR response in the sizeable nuclei of PCs. Presented here is our refined protocol for establishing murine cerebellar organotypic cultures and for visualizing the PAR response under genotoxic stress.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>150 x 30 cm Petri dish</td>
			<td>Corning Inc.</td>
			<td>3261</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm Petri dish</td>
			<td>Corning Inc.</td>
			<td>3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>270725-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Marienfeld</td>
			<td>48187</td>
			<td>76 x 26 x 1 mm</td>
		</tr>
		<tr>
			<td>Blades for chopper</td>
			<td>TED PELLA</td>
			<td>Model TC752-1</td>
			<td>&#160;Style Razor Blades</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>GIBCO</td>
			<td>41010-026 500ml.&#160;</td>
			<td>No L-Glut</td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Biological Indo.</td>
			<td>02-015-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H-1138-500L</td>
			<td>No Ca+,No Mg+</td>
		</tr>
		<tr>
			<td>HBSS with phenol red</td>
			<td>Sartorius</td>
			<td>02-015-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum&#160;</td>
			<td>GIBCO</td>
			<td>26050088</td>
			<td>heat inactivated</td>
		</tr>
		<tr>
			<td>Iris forceps</td>
			<td>CellPath</td>
			<td>GZX-0100-00A</td>
			<td>130mm blunt serrated</td>
		</tr>
		<tr>
			<td>Iris spatula</td>
			<td>F.S.T</td>
			<td>10092-12</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>41010026</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322412-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium without</td>
			<td>GRIGENE. Ltd, Israel</td>
			<td>K239320A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td>Sigma-Aldrich</td>
			<td>856177-250MG</td>
			<td>Paraquat dichloride (Methyl viologen)</td>
		</tr>
		<tr>
			<td>PARG- Poly(ADP-Ribose) Glycohydrolase inhibitor</td>
			<td>Tocris Bioscience, United Kingdom</td>
			<td>PDD 00017273</td>
			<td>light sensitive&#160;</td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Biological Indo.</td>
			<td>02-018-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, skin-tissue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning incorporated</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Spare Chopping Discs&#160;</td>
			<td>TED PELLA</td>
			<td>Model 10180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue chopper</td>
			<td>McIlwain</td>
			<td>Model TC752</td>
			<td>10180-220</td>
		</tr>
		<tr>
			<td>Tweezers, pointed</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urinary cups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 donkey anti Rabbit IgG</td>
			<td>&#160; 
			Invitrogen</td>
			<td>A21206</td>
			<td>1:500; secondary ab; Secondary dilution buffer;&#160; Incubation for 2 h at room temperature</td>
		</tr>
		<tr>
			<td>Anti glial fibrillary acidic protien</td>
			<td>Milliphore</td>
			<td>MAB3402</td>
			<td>1:1500; primary ab; blocking; Host:mouse</td>
		</tr>
		<tr>
			<td>Anti-calbindin-D-28K</td>
			<td>Swant&#160;</td>
			<td>CB300</td>
			<td>1:2000; primary ab; blocking; host: rabbit&#160;</td>
		</tr>
		<tr>
			<td>Anti-calbindin-D-28K</td>
			<td>Swant</td>
			<td>CB-38a</td>
			<td>1:2000; primary ab; blocking&#160;</td>
		</tr>
		<tr>
			<td>Anti-pan-ADP-ribose reagent</td>
			<td>Milliphore</td>
			<td>MABE1016</td>
			<td>1:800; primary ab; blocking; incubation for 2 h at room temperature</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidine-2&#39;-phenylindole dihydrochloride)</td>
			<td>Cell signaling</td>
			<td>4084</td>
			<td>1:1000; secondary dilution buffer; incubation for 20 min at room temperature</td>
		</tr>
	</tbody>
</table>

visualizing the dna damage response in purkinje cells using cerebellar organotypic cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Figure 1&#160;illustrates the general appearance of the cerebellar organotypic cultures. The top row in Figure 1A shows the cerebellar foliation, which is maintained in culture, while the bottom row shows the PCs stained for Calbindin D-28k (green) and the neuronal nuclei stained for NeuN (red). In Figure 1B, the astrocytes in the PCs (red) are stained for GFAP (green), an intermediate filament type III protein. <strong class="xfig"...

Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional ...

visualizing-dna-damage-response-purkinje-cells-using-cerebellar

Research

Without Section

JoVE Journal

Biology

1.1K Views.  Tel Aviv University. Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

Video: Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...