Original Paper

Cell Stress and Chaperones

, Volume 16, Issue 5, pp 549-561

First online:

Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress

  • Kevin C. HadleyAffiliated withDepartment of Medical Biophysics, University of Toronto. Ontario Cancer Institute
  • , Michael J. BorrelliAffiliated withDepartment of Radiology, University of Arkansas for Medical Sciences
  • , James R. LepockAffiliated withDepartment of Medical Biophysics, University of Toronto. Ontario Cancer Institute
  • , JoAnne McLaurinAffiliated withDepartment of Laboratory Medicine and Pathobiology, Centre for Research in Neurodegenerative Diseases, University of Toronto
  • , Sidney E. CroulAffiliated withDepartment of Laboratory Medicine and Pathobiology, University of Toronto, UHN Path 11E426 Toronto General Hospital
  • , Abhijit GuhaAffiliated withArthur and Sonia Labatt Brain Tumour Centre, Hospital for Sick Children’s Research Institute
  • , Avijit ChakrabarttyAffiliated withCampbell Family Institute for Cancer Research, Ontario Cancer Institute, University Health NetworkDepartment of Medical Biophysics, University of TorontoDepartment of Biochemistry, University of Toronto Email author 

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The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer’s disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes—regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.


Protein homeostasis Intracellular protein folding Chaperones Hsp70