We thank J. Birchler, E. Bach and the Bloomington Drosophila Stock Center for various Drosophila strains; the National Cancer Institute (NCI) Developmental Therapeutics Program for the Oncology Set small-molecule drug library; UCSD undergraduate students including Amy Chang, Taesik You, Jessica Singh-Banga, Rachel Meza, and Alex Chavez. Research reported in this publication was supported by a research grant from American Thoracic Society to J.L. and funding from NIH: R01GM131044 to W.X.L.

1. Drug library preparation
<ol>
	<li>Identify and prepare a drug library to be screened using appropriate solvent at a desired concentration (e.g., 10 mM in DMSO). 
	NOTE: A specific example for a drug library is the Oncology Set III from National Cancer Institute (NCI) Developmental Therapeutics Program (DTP). Compounds in this set are provided as 20 &#181;L at 10 mM in 100% DMSO in two 96-well PP U-bottom plates and stored at -20 &#176;C. The NCI Plate maps, and the basic chemical data of each drug could be found at the following websites: 
	<a href="https://dtp.cancer.gov/dtpstandard/servlet/PlateMap?searchlist=4740&#38;outputformat=html&#38;searchtype=plate&#38;Submit=Submit" target="_blank">NCI plate number 4740</a> 
	<a href="https://dtp.cancer.gov/dtpstandard/servlet/PlateMap?searchlist=4741&#38;outputformat=html&#38;searchtype=plate&#38;Submit=Submit" target="_blank">NCI plate number 4741</a></li>
</ol>
2. Screen for heterochromatin-promoting drugs using Drosophila
<ol>
	<li>Drosophila Breeding strategy (Figure 1A)
	<ol>
		<li>Day 1: Cross three w1118/Y; DX1/CyO male flies and three or more virgin w1118 female flies in a 25 mm x 95 mm food vial with 9 mL of standard Drosophila food media (Bloomington recipe). Set up three replicate vials for each drug and one control for the screen. 
		NOTE: DX1 flies were kindly provided by James Birchler (University of Missouri)9,13.</li>
		<li>Day 2 and Day 3: Allow the flies to lay eggs for 2 days at room temperature (22 &#176;C).</li>
		<li>Day 4: Use a short burst of CO2 gas to anesthetize the parent flies and dispose of the parent flies in a &#34;fly morgue&#34; (a bottle containing 70% alcohol).</li>
	</ol>
	</li>
	<li>Feed drugs to Drosophila for screening.
	<ol>
		<li>Dilute each drug compound from the original stock (20 &#181;L at 10 mM in 100% DMSO in 96-well PP U-bottom plates) to a 10 &#181;M final concentration with 33% DMSO in water. Use 33% DMSO in water without any drug as the control. 
		NOTE: Some drugs are poorly soluble in water and were dissolved in DMSO. 100% DMSO is toxic to flies. 33% is tolerable.</li>
		<li>Day 4: Pipette 60 &#181;L of a 10 &#181;M drug solution or control onto the top of fly food. At this point, ensure that the parent flies have been removed and there are fly eggs and crawling first-instar larvae on the food.</li>
		<li>Day 6: Repeat pipetting 60 &#181;L of the same 10 &#181;M drug solution to the food vial. 
		NOTE: Since drugs were added on to the fly food, the top surface food contains nearly 10 &#181;M concentration and the bottom food could be not be fully penetrated. All the flies lay eggs on the food top and eat the top surface food.</li>
	</ol>
	</li>
	<li>Observe and score eye color changes.
	<ol>
		<li>Two days after the F1 flies emerge (approximately on day 14), examine the flies. Use a short burst of CO2 to anesthetize the F1 flies and remove the flies from their vial onto a porous dissecting pad with CO2 sipping through from underneath a filter paper.</li>
		<li>Inspect the flies on this pad under a dissection microscope. Examine the whole fly and identify all w1118/Y; DX1/+ heterozygous males by their lacking the CyO dominant curly wing phenotype.</li>
		<li>Score the eye color of each of the w1118/Y; DX1/+ heterozygous male flies on a scale of 1 to 5 (Figure 1B). The percentage of white eye color was rated as follows: 
		1. &#60;5% red scattered eye total surface area 
		2. 6% - 25% red spots eye total surface area 
		3. 26% - 50% red spots eye total surface area 
		4. 51% - 75% red spots eye total surface area 
		5. &#62;75% red spots eye total surface area 
		NOTE: Alternatively, homogenize fly heads in 100% methanol and use a spectrometer to measure eye pigmentation at OD 450 nm. Select only males because PEV is more pronouced in DX1 males. Score more than 10 males in each vial.</li>
		<li>Calculate the mean color index of all the males from each vial scored. Perform triplicates for each compound (Figure 1C). 
		NOTE: Since eye color varies with age, this is a time-sensitive step. The eye color should be calculated at the same age &#8211; 2 days old. Usually &#62;10 flies should be scored in each vial.</li>
		<li>To confirm that the compounds indeed promote heterochromatin formation, use a different technique such as western blotting to validate (Figure 1G).</li>
	</ol>
	</li>
</ol>

<ol><li>Morgan, M. A., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+signatures+of+cancer%5BTitle%5D)+AND+%22Genes+Development%22%5BJournal%5D)">Chromatin signatures of cancer.</a> Genes Development. 29 (3), 238-249 (2015).</li>
<li>Saksouk, N., Simboeck, E., Dejardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constitutive+heterochromatin+formation+and+transcription+in+mammals%5BTitle%5D)+AND+%22Epigenetics+Chromatin%22%5BJournal%5D)">Constitutive heterochromatin formation and transcription in mammals.</a> Epigenetics Chromatin. 8, 3(2015).</li>
<li>Allshire, R. C., Madhani, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ten+principles+of+heterochromatin+formation+and+function%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Ten principles of heterochromatin formation and function.</a> Nature Reviews Molecular Cell Biology. 19 (4), 229-244 (2018).</li>
<li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translating+the+histone+code%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Translating the histone code.</a> Science. 293 (5532), 1074-1080 (2001).</li>
<li>Grewal, S. I., Moazed, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin+and+epigenetic+control+of+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Heterochromatin and epigenetic control of gene expression.</a> Science. 301 (5634), 798-802 (2003).</li>
<li>Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delving+into+the+diversity+of+facultative+heterochromatin%3A+the+epigenetics+of+the+inactive+X+chromosome%5BTitle%5D)+AND+%22Current+Opinion+in+Genetics+%26+Development%22%5BJournal%5D)">Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome.</a> Current Opinion in Genetics & Development. 15 (5), 482-489 (2005).</li>
<li>Ci, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin+Protein+1alpha+Mediates+Development+and+Aggressiveness+of+Neuroendocrine+Prostate+Cancer%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Heterochromatin Protein 1alpha Mediates Development and Aggressiveness of Neuroendocrine Prostate Cancer.</a> Cancer Research. 78 (10), 2691-2704 (2018).</li>
<li>Zhu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin-Encoded+Satellite+RNAs+Induce+Breast+Cancer%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.</a> Molecular Cell. 70 (5), 842-853 (2018).</li>
<li>Dorer, D. R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansions+of+transgene+repeats+cause+heterochromatin+formation+and+gene+silencing+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila.</a> Cell. 77 (7), 993-1002 (1994).</li>
<li>Fanti, L., Dorer, D. R., Berloco, M., Henikoff, S., Pimpinelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin+protein+1+binds+transgene+arrays%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">Heterochromatin protein 1 binds transgene arrays.</a> Chromosoma. 107 (5), 286-292 (1998).</li>
<li>Shi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((JAK+signaling+globally+counteracts+heterochromatic+gene+silencing%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">JAK signaling globally counteracts heterochromatic gene silencing.</a> Nature Genetics. 38 (9), 1071-1076 (2006).</li>
<li>Shi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+STAT+is+required+for+directly+maintaining+HP1+localization+and+heterochromatin+stability%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Drosophila STAT is required for directly maintaining HP1 localization and heterochromatin stability.</a> Nature Cell Biology. 10 (4), 489-496 (2008).</li>
<li>Ronsseray, S., Boivin, A., Anxolabehere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((P-Element+repression+in+Drosophila+melanogaster+by+variegating+clusters+of+P-lacZ-white+transgenes%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.</a> Genetics. 159 (4), 1631-1642 (2001).</li>
<li>Loyola, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+methotrexate+as+a+heterochromatin-promoting+drug%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Identification of methotrexate as a heterochromatin-promoting drug.</a> Scientific Reports. 9 (1), 11673(2019).</li>
<li>Dialynas, G. K., Vitalini, M. W., Wallrath, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linking+Heterochromatin+Protein+1+%28HP1%29+to+cancer+progression%5BTitle%5D)+AND+%22Mutation+Research%22%5BJournal%5D)">Linking Heterochromatin Protein 1 (HP1) to cancer progression.</a> Mutation Research. 647 (1-2), 13-20 (2008).</li>
<li>Janssen, A., Colmenares, S. U., Karpen, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin%3A+Guardian+of+the+Genome%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">Heterochromatin: Guardian of the Genome.</a> Annual Review of Cell and Developmental Biology. 34, 265-288 (2018).</li>
<li>Zhu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BRCA1+tumour+suppression+occurs+via+heterochromatin-mediated+silencing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">BRCA1 tumour suppression occurs via heterochromatin-mediated silencing.</a> Nature. 477 (7363), 179-184 (2011).</li>
<li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unphosphorylated+STAT5A+stabilizes+heterochromatin+and+suppresses+tumor+growth%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Unphosphorylated STAT5A stabilizes heterochromatin and suppresses tumor growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (25), 10213-10218 (2013).</li>
</ol>

The authors have no conflicts of interest to disclose.

Heterochromatin is a condensed form of DNA that plays a central role in regulating gene expression. It is becoming increasingly more recognized in cancer and may serve as a potential target for cancer therapy15,16,17,18. Small-molecule compounds are commonly used in drug development due to advantages in manufacture, preservation, and metabolism in human bodies. To identify heterochromatin-promo...

Heterochromatin is a condensed form of DNA that plays a central role in gene expression, in regulating chromosome segregation during cell division, and in protecting against genome instability1. Heterochromatin has been considered to be a gene repression regulator and to protect chromosome integrity during cell mitosis2,3. It is associated with the di- and tri-methylation of histone H3 lysine 9 (H3K9me) during lineage commitment4,5. Moreover, recruitment of Heterochromatin Protein 1 (HP1) chromodomain proteins is also considered to be associated with heterochromatin and epigenetic repression of gene expression6. These proteins are essential components and markers of heterochromatin formation.
Since genomic instability enables cells to acquire genetic alterations that promote carcinogenesis, heterochromatin is becoming more recognized in cancer development and may be targeted for cancer treatment7,8. Currently, there are no drugs that are well-established in assisting heterochromatin formation. Here, we present a simple and quick yet efficient method for screening small-molecule compounds that promote heterochromatin formation. The screening is done by treating Drosophila with a library of small-molecule drugs. This method takes advantage of a variegated eye color phenotype in the DX1Drosophila strain that is influenced by heterochromatin levels. DX1 flies contain a tandem array of seven P[lac-w] transgenes, which have the variegated expression/depression depending on heterochromatinization, therefore, the extent of variegation in the eye color reflect the heterochromatin level. Specifically, increasing heterochromatin could be detected by the rising proportion of variegated eye color (white eye). On the contrary, decreasing heterochromatin would be detected by the rising proportion of the P[lac-w] transgene expression (red eye)9,10,11,12.
Therefore, we take advantage of this Drosophila transgene system that produces a variegated eye color phenotype since its expression is directly correlated to the amount of heterochromatin present. Upon discovery of compounds that are suspected to promote heterochromatin formation, we may confirm this suspicion using other methods such as western blot. These heterochromatin-promoting substances may be further developed for clinical trials in patients in the future.

<table><tbody><tr><td>Drosophila</td><td></td><td>&lt;em&gt;DX1&lt;/em&gt; strain</td><td>&lt;em&gt;DX1&lt;/em&gt; flies were kindly provided by James Birchler (University of Missouri)</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; food media</td><td>UCSD fly kitchen</td><td></td><td></td></tr><tr><td>Methotrexate</td><td>NCI drug library</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma</td><td>E7023-500ML</td><td></td></tr></tbody></table>

a screening method for identification of heterochromatin-promoting drugs using drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila&#39;s DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.

This protocol was successfully used to screen compounds that promote heterochromatin formation in Drosophila, which is an efficient and low-cost in vivo system for drug development (Figure 1). We screened a small-molecule drug library, Oncology Set III, which is composed of 97 FDA authorized oncology drugs, using the DX1 strain of Drosophila melanogaster (Figure 1B). The results for a significant drug were represented ...

Watch this Scientific Journal Video about A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila at JoVE.com

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

a-screening-method-for-identification-heterochromatin-promoting-drugs

Research

JoVE Journal

Genetics

6.0K Views.  University of California San Diego. Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

Video: A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Behavior

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Biology

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

Neuroscience

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Summary

Explore More Videos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells