Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells.
Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.
Department of Physics, McGill University, Montréal, Québec, Canada; Institut Universitaire en Santé Mentale de Québec, Québec, Canada.
Antoine G
AG
Godin
Department of Physics, McGill University, Montréal, Québec, Canada; Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montréal, Québec, Canada; Program in NeuroEngineering, McGill University, Montréal, Québec, Canada.
Benjamin
B
Rappaz
Department of Physics, McGill University, Montréal, Québec, Canada.
Laurent
L
Potvin-Trottier
Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montréal, Québec, Canada; Program in NeuroEngineering, McGill University, Montréal, Québec, Canada.
Timothy E
TE
Kennedy
Institut Universitaire en Santé Mentale de Québec, Québec, Canada; Département de Psychiatrie et Neurosciences, Université Laval, Québec, Canada; Department of Pharmacology & Therapeutics, McGill University, Montréal, Québec, Canada; Alan Edwards Center for Research of Pain, McGill University, Montréal, Québec, Canada.
Yves
Y
De Koninck
Department of Physics, McGill University, Montréal, Québec, Canada; Program in NeuroEngineering, McGill University, Montréal, Québec, Canada; Department of Chemistry, McGill University, Montréal, Québec, Canada. Electronic address: paul.wiseman@mcgill.ca.
Paul W
PW
Wiseman
D000818:Animals; D016477:Artifacts; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D009154:Mutation; D018991:Myelin Proteolipid Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D055503:Protein Multimerization; D017470:Receptors, Glutamate / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis
D018991:Myelin Proteolipid Protein; C472930:PLP1 protein, human; D017470:Receptors, Glutamate