Membrane Hsp70-supported cell-to-cell connections via tunneling nanotubes revealed by live-cell STED nanoscopy

Judith Reindl,Stefan Stangl,Gabriele Multhoff,Günther Dollinger,Maxim Shevtsov

doi:10.1007/s12192-018-00958-w

Abstract

Heat shock protein Hsp70 (Hsp70) is found on the cell surface of a large variety of human and mouse tumor cell types including U87, GL261 glioblastoma, and 4T1 mammary carcinoma cells. We studied the role of membrane-bound Hsp70 (mHsp70) in the formation of cell-to-cell connections via tunneling nanotubes (TNTs) using live-cell STED nanoscopy. This technique allows the visualization of microstructures in the 100-nm range in the living cells. We could show that the presence of tumor-derived mHsp70 in TNTs with a diameter ranging from 120 to 140 nm predominantly originates from cholesterol-rich-microdomains containing the lipid compound globoyltriaosylceramide (Gb3). Under non-stress conditions, Hsp70 and Gb3 are structurally clustered in the membrane of TNTs of tumor cells that showed tumor type specific variations in the amount of cell-to-cell connection networks. Furthermore depletion of cholesterol and ionizing radiation as a stress factor results in a complete loss of Hsp70-containing TNTs.

Highlights

Heat shock proteins (HSPs) with a molecular weight of approximately 70 kDa constitute to a large family of highly conserved proteins that are involved in protein homeostasis, cell proliferation, differentiation, and carcinogenesis (Shevtsov and Multhoff 2016; Hartl and Hayer-Hartl 2002; Mayer and Bukau 2005)
The formation of tunneling membrane nanotubes (TNTs) that enable connections between living neighboring tumor cells under non-stress conditions could be determined as a novel function for membrane-bound Hsp70 (mHsp70) by live-cell STED nanoscopy
The expression of the membrane-bound Hsp70 on the tumor cells was determined by flow cytometry using either the fluorescein isothiocyanate (FITC)-conjugated cmHsp70.1 monoclonal antibody (mAb) (IgG1; multimmune GmbH), which is directed against the extracellular-exposed sequence of membrane Hsp70

Summary

Introduction

Heat shock proteins (HSPs) with a molecular weight of approximately 70 kDa constitute to a large family of highly conserved proteins that are involved in protein homeostasis, cell proliferation, differentiation, and carcinogenesis (Shevtsov and Multhoff 2016; Hartl and Hayer-Hartl 2002; Mayer and Bukau 2005). Apart from its cytosolic overexpression, the highly stress-inducible member Hsp (HSPA1A) is frequently found on the cell membrane in a large variety of different tumor types (Thorsteinsdottir et al 2017; Multhoff et al 1995). The presence of Hsp on the plasma membrane of tumor cells was determined by global profiling of membrane proteins (Shin et al 2003) and flow cytometry using cmHsp70.1 monoclonal antibody (Stangl et al 2011; Multhoff et al 1995) that detects a conformational epitope of Hsp in the context with lipids. Apart from its documented immunological relevance as a tumor-specific biomarker for antibody- (Stangl et al 2001) and NK cell-based-targeted therapies (Specht et al 2015), the role of mHsp is poorly understood. The formation of tunneling membrane nanotubes (TNTs) that enable connections between living neighboring tumor cells under non-stress conditions could be determined as a novel function for mHsp by live-cell STED nanoscopy

Methods

Results

Conclusion