Expression‐level dependent perturbation of cell proteostasis and nuclear morphology by aggregation‐prone polyglutamine proteins

Abstract

We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance, and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to ∼80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs.

Long Term Aggresome Accumulation Leads to DNA Damage, p53-dependent Cell Cycle Arrest, and Steric Interference in Mitosis

“…To investigate this phenomenon with EGFP-polyQ proteins, we established stable cell lines expressing inducible, single copy constructs of either EGFP-HDQ72 or EGFP-HDQ23 in a Flp-In T-REx293 cell background; the former protein is highly prone to aggregation, whereas the latter should not form aggregates and acts as a control. Both cell lines are constructed with the same promoter, and their respective genes are expressed at comparable levels (19). Using EGFP-HDQ72-expressing cells as an example, we verified the consistency of gene expression by flow cytometry over a 5-week period (Fig.…”

“…The GeneJammer/ DMEM mixture was added dropwise to each plate, and cells were incubated for 3 h at 37°C in the incubator before an additional 1 ml of complete medium containing the appropriate selection antibiotics was added. Stable cell line construction has been described previously (19). Histone 2B-DsRed and pericentrin-mCherry plasmids were gifts from Dr. Viji Draviam, Cambridge (61).…”

“…2C). Our previous data (19) showed that the rate of formation, penetrance, and morphology of aggresomes within the cell population are dependent on polyQ expression level. We therefore investigated whether a similar correlation pertains between polyQ expression level and the presence of DSBs using three independent, singlecopy cell lines where SNAP-HDQ72 expression is driven by low, intermediate-strength, or strong promoters.…”

“…shown that aggresomes can have a detrimental effect on nuclear morphology (7,8,18,19). To investigate this phenomenon with EGFP-polyQ proteins, we established stable cell lines expressing inducible, single copy constructs of either EGFP-HDQ72 or EGFP-HDQ23 in a Flp-In T-REx293 cell background; the former protein is highly prone to aggregation, whereas the latter should not form aggregates and acts as a control.…”

Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells

“…SNAP-HDQ72-expressing HEK 293T cells were labeled with SNAP-Cell 505-Star and fixed on gridded slides 9,10 (see Supporting Information); they were then imaged using both TCSPC-FLIM and SROS-SIM as described previously. Note that we fixed the cells only for the purpose of enabling correlative FLIM/SIM measurements; in principle, the lifetime sensor can equally be applied in living cells.…”