Tracking protein aggregation and mislocalization in cells with flow cytometry

Abstract

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

“…Fig. 6A shows the strategy of PulSA, which we used previously to track inclusion formation of Httex1 and SOD1 (11). In essence, gates can be defined based on pulse width and height parameters that match the localization patterns of GFP fluorescence in cells for dispersed protein (noninclusions, the ni gate) or condensed protein localizations (inclusions; the i gate) (Fig.…”

“…Flow Cytometry and Sorting-Cells were analyzed by PulSA as described (11). For sorting experiments, 2 ϫ 10 7 transfected cells were detached through scraping in 1 ml of PBS supplemented with 10 units/ml DNase I, 5 mM MgCl 2 , and 0.1% w/v bovine serum albumin (BSA).…”

“…Side scatter and forward scatter height and width were also collected. For analysis, cells were gated to remove debris and doublet cells using the forward scatter (FSC-A) and side scatter (SSC-A) plots as described (11). Cells were kept on ice for all steps of the sorting preparation and handling.…”

“…We applied this method successfully to cell lysates to decipher the aggregation patterns of Httex1 and other proteins (10,12,14). We have also developed a strategy to separate cells expressing mutant protein enriched in inclusions from those lacking inclusions using Pulse Shape Analysis (PulSA) by flow cytometry, which enables a second layer of control over the study of protein oligomerization in cells with visible inclusions or not (11). PulSA is based on the principle that the fluorescence width and height parameters of a target protein in a cell vary with its cellular localization.…”

“…We previously showed that PulSA can efficiently distinguish cells with a GFP-tagged protein localized diffusely throughout the cytoplasm from cells with a restricted spatial localization such as an inclusion. In particular, we showed we could separate mutants of superoxide dismutase 1 (SOD1) and Httex1 that had shifted from a dispersed unaggregated state to inclusions (11).…”