<em>In vivo</em> and <em>in vitro</em> Studies of Adaptor-clathrin Interaction

Daniel Feliciano; Jarred J. Bultema; Andrea L. Ambrosio; Santiago M. Di Pietro

doi:10.3791/2352

In vivo e in vitro de Estudos Adaptor-clathrin Interação

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Biology

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JoVE Journal Biology

In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vivo e in vitro de Estudos Adaptor-clathrin Interação

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17:14 min

January 26, 2011

DOI: 10.3791/2352-v

Daniel Feliciano¹, Jarred J. Bultema¹, Andrea L. Ambrosio¹, Santiago M. Di Pietro¹

¹Department of Biochemistry and Molecular Biology,Colorado State University

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Overview

This study focuses on clathrin-mediated endocytosis, emphasizing the role of adaptor proteins in cargo selection and clathrin coat assembly. Using the yeast endocytic adaptor protein Sla1p, various methods are employed to analyze adaptor-clathrin interactions and live cell imaging.

Key Study Components

Area of Science

Cell Biology
Neuroscience
Biochemistry

Background

Clathrin-mediated endocytosis is crucial for cellular processes.
Adaptor proteins link clathrin to membrane proteins or lipids.
Sla1p is a key adaptor protein in yeast.
Understanding these interactions can reveal insights into endocytosis mechanisms.

Purpose of Study

To investigate the physical interactions between Sla1p and clathrin.
To analyze the effects of mutations on endocytosis kinetics.
To utilize live cell imaging techniques for real-time observation.

Methods Used

Spinning disc confocal microscopy for imaging yeast cells expressing GFP-tagged Sla1p.
Time-lapse movies to assess the impact of VCB mutations on endocytosis.
Pull-down assays using yeast cytosolic extracts to study clathrin interaction in vitro.
Preparation of total extracts from yeast cells for further analysis.

Main Results

Live imaging revealed differences in endocytosis kinetics due to mutations.
In vitro assays confirmed the interaction between clathrin and Sla1p.
GFP-tagging allowed for visualization of Sla1p dynamics in live cells.
Results contribute to understanding the role of adaptor proteins in endocytosis.

Conclusions

Adaptor proteins like Sla1p are essential for clathrin-mediated endocytosis.
Mutations can significantly affect the kinetics of endocytosis.
Live cell imaging is a powerful tool for studying protein interactions.

Frequently Asked Questions

What is clathrin-mediated endocytosis?

Clathrin-mediated endocytosis is a process by which cells internalize molecules by engulfing them in vesicles formed from clathrin-coated pits.

What role do adaptor proteins play in this process?

Adaptor proteins facilitate the binding of clathrin to membrane proteins or lipids, coordinating cargo selection and coat assembly.

How was Sla1p studied in this research?

Sla1p was studied using live cell imaging and in vitro assays to analyze its interactions with clathrin.

What techniques were used for imaging?

Spinning disc confocal microscopy was used to visualize yeast cells expressing GFP-tagged Sla1p.

What were the main findings of the study?

The study found that mutations in Sla1p affect endocytosis kinetics and confirmed the interaction between Sla1p and clathrin.

Why is this research important?

Understanding the mechanisms of endocytosis can provide insights into cellular processes and potential therapeutic targets.

Clatrina-endocitose mediada depende de proteínas adaptadoras que coordenam a seleção de carga e montagem casaco de clatrina. Aqui nós descrevemos os procedimentos para estudar adaptador clathrin interação física e imagens de células vivas abordagens utilizando como modelo a levedura Sla1p proteína endocítica adaptador.

Durante a endocitose mediada por clatrina, uma rede poliédrica de clatrina está ligada a proteínas de membrana ou lipídios por meio de proteínas adapta de ligação à clatrina, como SL one, SL one liga-se à clatrina através de sua caixa de clatrina variante ou VCB. Neste vídeo, três métodos são usados para análise das interações SLA um da clatrina. As primeiras células de levedura que expressam os mutantes nativos e mutantes VCB marcados com GFP SLA um são fotografadas usando microscopia confocal de disco giratório.

Os filmes de lapso de tempo são então analisados para determinar o efeito da mutação na cinética da endocitose. Em segundo lugar, extratos citosólicos de levedura são preparados a partir de células do tipo selvagem como fonte de clatrina e ensaios de extração de proteína de fusão G-S-T-V-C-B são realizados para examinar a interação VCB da clatrina in vitro. Em terceiro lugar, os extratos totais são preparados a partir de células de levedura que expressam mutantes nativos e VCB.

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