Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation

Zakaria Ezzoukhry,Sylvaine Di-Tommaso,Jean-William Dupuy,Marlène Maître,Marion Peter,Luc Mercier,Fabrice P Cordelières,Frédéric Saltel,Jacky G Goetz,Nathan Gay,Frédéric Bard,Anne-Aurélie Raymond,Violaine Moreau,Elodie Henriet

doi:10.1038/s41467-018-04461-9

Abstract

Invadosomes are F-actin-based structures involved in extracellular matrix degradation, cell invasion, and metastasis formation. Analyzing their proteome is crucial to decipher their molecular composition, to understand their mechanisms, and to find specific elements to target them. However, the specific analysis of invadosomes is challenging, because it is difficult to maintain their integrity during isolation. In addition, classical purification methods often suffer from contaminations, which may impair data validation. To ensure the specific identification of invadosome components, we here develop a method that combines laser microdissection and mass spectrometry, enabling the analysis of subcellular structures in their native state based on low amounts of input material. Using this combinatorial method, we show that invadosomes contain specific components of the translational machinery, in addition to known marker proteins. Moreover, functional validation reveals that protein translation activity is an inherent property of invadosomes, which is required to maintain invadosome structure and activity.

Highlights

Invadosomes are F-actin-based structures involved in extracellular matrix degradation, cell invasion, and metastasis formation
The combination of mass spectrometry (MS)-based proteomics with biochemical fractionation or immunoprecipitation is the classical approach for the characterization of protein interactions in subcellular complexes[14,15]
Previous studies used a combination of laser capture microdissection and MS analysis for the molecular characterization of isolated cells or tissue sections but these approaches were not applied at the subcellular level[19,20,21]

Summary

Introduction

Invadosomes are F-actin-based structures involved in extracellular matrix degradation, cell invasion, and metastasis formation Analyzing their proteome is crucial to decipher their molecular composition, to understand their mechanisms, and to find specific elements to target them. To ensure the specific identification of invadosome components, we here develop a method that combines laser microdissection and mass spectrometry, enabling the analysis of subcellular structures in their native state based on low amounts of input material. Using this combinatorial method, we show that invadosomes contain specific components of the translational machinery, in addition to known marker proteins. To guarantee the specificity of the identified proteins, we combine the proteomics analysis with isotopic labeling, accounting for the fact that the high sensitivity mass spectrometric analysis may otherwise result in the identification of undesirable contaminating proteins[25]

Methods

Results

Conclusion