In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Niels Alberts; Yasith Mathangasinghe; Nadinath  B. Nillegoda

doi:10.3791/60172

Monitoraggio in Situ di gruppi di chaperone molecolari formati in farmaci, lieviti e cellule umane

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Biology

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JoVE Journal Biology

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Monitoraggio in Situ di gruppi di chaperone molecolari formati in farmaci, lieviti e cellule umane

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08:58 min

September 2, 2019

DOI: 10.3791/60172-v

Niels Alberts*¹, Yasith Mathangasinghe*², Nadinath B. Nillegoda³

¹Department of Biomedical Sciences of Cells & Systems,University of Groningen, ²Department of Anatomy, Faculty of Medicine,University of Colombo, ³Australian Regenerative Medicine Institute (ARMI),Monash University

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Overview

This study investigates the role of cognate J-domain proteins in association with the Hsp70 chaperone to facilitate various biological processes. Utilizing an in situ proximity ligation assay, the research monitors transient chaperone complexes in bacteria, yeast, and human cells, shedding light on cellular proteostasis.

Key Study Components

Research Area

Cellular proteostasis
Protein interactions and complexes
Biological implications in microbial infections

Background

Importance of chaperone machineries in protein dynamics
Relevance of transient interactions in cell biology
Potential applications in disease research

Methods Used

In situ proximity ligation assay (PLA)
Prokaryotic (E. coli) and eukaryotic (HeLa, S. cerevisiae) cell models
Immunofluorescence and specific antibody techniques

Main Results

Successful monitoring of J-domain and Hsp70 chaperone interactions
Visualization of sub-cellular localization of protein assemblies
Adaptability of the method for various organisms

Conclusions

The study demonstrates the critical involvement of J-domain proteins in protein folding and degradation.
This method can significantly enhance understanding of protein dynamics in a variety of biological systems.

Frequently Asked Questions

What are J-domain proteins?

J-domain proteins are essential co-chaperones that assist the Hsp70 chaperone in processes like protein folding and degradation.

How does the in situ proximity ligation assay work?

The assay detects transient protein interactions by utilizing specific antibodies and a series of biochemical reactions that amplify the signal.

In which organisms can this method be applied?

The method can be applied to a range of organisms, including bacteria, yeast, and human cells.

What is the significance of studying protein interactions?

Understanding protein interactions is crucial for elucidating cellular mechanisms and can provide insights into various diseases.

How can this approach help in disease research?

This technique can reveal insights into molecular interactions involved in diseases, particularly those related to microbial infections.

What kind of antibodies should be used?

It is important to select high-quality antibodies that have been validated for techniques like immunohistochemistry or immunofluorescence.

What are the main advantages of this assay?

The assay provides real-time insights into protein dynamics and localization, which are vital for understanding cellular function.

Le proteine Cognate J-domain cooperano con l'accompagnatore Hsp70 per assistere in una miriade di processi biologici che vanno dal ripiegamento proteico alla degradazione. Qui, descriviamo un analisi di legatura di prossimità in situ, che consente il monitoraggio di questi macchinari chaperone tradotti transitoriamente in cellule batteriche, lieviti e umane.

Questo metodo cattura le interazioni proteiche transitori in organismi come batteri e lieviti e in diversi tipi di cellule. Qui monitoriamo complessi di chaperone specifici formati transitoriamente che sono coinvolti nella proteostasi cellulare. Il principale vantaggio di questa tecnica è la capacità di generare informazioni sulla dinamica e localizzazione subcellulare degli assemblaggi proteici nelle cellule procariotiche ed eucariotiche.

Adattamenti di questa tecnica in organismi unicellulari come batteri e lieviti, consente ai ricercatori di utilizzarla come un potente strumento per indagare malattie legate alle infezioni microbiche. Questa tecnica potrebbe potenzialmente essere utilizzata per analizzare le interazioni proteiche in quasi tutti gli organismi semplicemente cambiando le condizioni nella fase di preparazione del campione del protocollo. È importante selezionare anticorpi di buona qualità specifici e testati in immunoistochimica, immunofluorescenza, ELISA o immunoprecipitazioni.

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