<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 and in vitro Studies of Adaptor-clathrin Interaction

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Biology

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In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vivo and in vitro Studies of Adaptor-clathrin Interaction

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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

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.

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

During clathrin mediated endocytosis, a polyhedral lattice of clathrin is linked to membrane proteins or lipids via clathrin binding adapta proteins such as SL one, SL one binds clathrin through its variant clathrin box or VCB. In this video, three methods are used for analysis of clathrin SLA one interactions. First yeast cells expressing GFP tagged native and VCB mutant mutants SLA one are imaged using spinning disc confocal microscopy.

Time-lapse movies are then analyzed to determine the effect of mutation on the kinetics of endocytosis. Second yeast cytosolic extracts are prepared from wild type cells as a source of clathrin and G-S-T-V-C-B fusion protein pull down assays are performed to examine clathrin VCB interaction in vitro. Third total extracts are prepared from yeast cells expressing native and VCB mutants.

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