Abstract
Hsp70-1A—the major stress-inducible member of the HSP70 chaperone family—is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.
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Abbreviations
- AFM :
-
atomic force microscopy
- chol :
-
cholesterol
- DOPC :
-
1,2-dioleoyl-sn-glycero-3-phosphocholine
- DOPS :
-
1,2-dioleoyl-sn-glycero-3-phospho-L-serine
- DPPC :
-
1,2-dipalmitoylphosphatidylcholine
- DPPS :
-
1,2-dioleoyl-sn-glycero-3-phospho-L-serine
- eSM :
-
sphingomyelin from chicken egg yolk
- GUV :
-
giant unilamellar vesicle
- Hsp70-1A :
-
heat shock protein 70-1A
- Lα :
-
liquid crystalline phase
- Lβ :
-
solid or gel phase
- Ld :
-
liquid disordered phase
- Lo :
-
liquid ordered phase
- PC :
-
phosphatidylcholine
- SLB :
-
supported lipid bilayer
- SUV :
-
small unilamellar vesicle
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Acknowledgments
This project received funding from the European Union’S Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Sklodowska-Curie Grant Agreement No. 656842 and the Carl-Zeiss-Stiftung (Carl Zeiss Foundation) (Az. 0563-2.8/685/4). The work has been supported in part by the German Federal Ministry of Education and Research (BMBF) in the framework of the EU ERASynBio project SynGlycTis (031A464), by the Ministry of Science, Research and the Arts of Baden-Württemberg (Az: 33-7532.20) and by the Excellence Initiative of the German Research Foundation (EXC 294).
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CL and MG designed the experiments. CL and JM conducted the experiments. CL analyzed the data. AE, WR, GM, and MG helped to conceive the project and discuss results. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
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Lamprecht, C., Gehrmann, M., Madl, J. et al. Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol. Cell Stress and Chaperones 23, 673–683 (2018). https://doi.org/10.1007/s12192-018-0879-0
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DOI: https://doi.org/10.1007/s12192-018-0879-0