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

Jason M. Beckta; Scott C. Henderson; Kristoffer Valerie

doi:10.3791/4251

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

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

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Full Text

14,695 Views

10:24 min

September 28, 2012

DOI: 10.3791/4251-v

Jason M. Beckta^1,2, Scott C. Henderson³, Kristoffer Valerie^1,2,4

¹Department of Radiation Oncology,Virginia Commonwealth University, ²Department of Biochemistry & Molecular Biology,Virginia Commonwealth University, ³Department of Anatomy & Neurobiology,Virginia Commonwealth University, ⁴Massey Cancer Center,Virginia Commonwealth University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

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. The demonstration utilizes two and 3D live cell imaging to explore the spatial temporal relationships of proteins involved in the DNA damage response.

Key Study Components

Area of Science

Cell Biology
DNA Damage Response
Live Cell Imaging

Background

Understanding DNA damage response is crucial for cell cycle regulation.
Fluorescent markers help visualize protein dynamics in live cells.
Two and 3D imaging techniques enhance spatial and temporal analysis.
53 BP1 is a key protein involved in the DNA damage response.

Purpose of Study

To visualize the activation and localization of DNA damage signaling proteins.
To analyze the effects of DNA damaging agents on cellular dynamics.
To study the behavior of proteins during mitosis.

Methods Used

Transfect or transduce cell lines with fluorescent marker constructs.
Acquire control videos of fluorescently labeled cells under normal conditions.
Apply treatments and capture data on treatment effects.
Analyze video data for spatial temporal relationships of proteins.

Main Results

Live cell fluorescence microscopy reveals dynamics of 53 BP1 foci formation.
Protein behavior during mitosis is influenced by DNA damaging agents.
Two and 3D imaging provides insights into cellular responses to treatments.
Spatial temporal relationships of proteins can be effectively analyzed.

Conclusions

This method allows for detailed observation of DNA damage response proteins.
Live cell imaging is a powerful tool for studying cellular dynamics.
Understanding these processes can inform therapeutic strategies against DNA damage.

Frequently Asked Questions

What is the significance of visualizing DNA damage response proteins?

Visualizing these proteins helps understand their roles in cellular repair mechanisms and mitosis.

How does live cell imaging enhance our understanding of cellular dynamics?

It allows researchers to observe real-time changes and interactions of proteins within living cells.

What are the advantages of using fluorescent markers?

Fluorescent markers enable specific visualization of proteins, facilitating detailed analysis of their behavior.

Can this method be applied to other proteins involved in DNA repair?

Yes, the protocol can be adapted to study various proteins involved in the DNA damage response.

What types of treatments can be applied in this protocol?

Any treatment that induces DNA damage can be applied to study its effects on protein dynamics.

Is this method suitable for high-throughput analysis?

While primarily qualitative, the method can be adapted for quantitative analysis in some cases.

Bu protokol, DNA hasarının yanı sıra, mitoz sırasında lokalizasyonu cevap olarak aktive bir çift iplikli DNA mola sinyal protein görüntülenmesi için bir yöntem açıklanır.

Bu gösteri, DNA hasar tepkisinde yer alan proteinlerin uzamsal zamansal ilişkilerini anlamak için iki ve 3D canlı hücre görüntüleme kullanır. İlk olarak, uygun floresan belirteçleri içeren ifade yapılarıyla seçtiğiniz hücre hattını transfekte edin veya transdüksiyon yapın. Burada, M Cherry ve GFP daha sonra floresan etiketli hücreleri normal koşullar altında gösteren bir dizi kontrol videosu elde ediyor.

Daha sonra uygun bir tedavi uygulayın ve floresan hücreler üzerindeki tedavi etkilerinin verilerini yakalayın, proteinlerin uzamsal zamansal ilişkileri için video verilerini işleyin ve analiz edin. Sonuç olarak, canlı hücre floresan mikroskobu, DNA'ya zarar veren ajanlara yanıt olarak 53 BP bir odak oluşumu veya mitoz sırasında 53 BP kişinin davranışı gibi hücresel dinamikleri detaylandırabilir. İlk olarak, çeşitli tedavilerin belirli hücre dizilerinde mitozu nasıl etkilediğini incelemek istediğimizde iki ve 3D yaşam tarzı görüntülemeye ilgi duymaya başladık.

View the full transcript and gain access to thousands of scientific videos