Background

Protein misfolding and aggregation are hallmarks of numerous neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (ALS) [1]. Unraveling how the protein folding and quality control system handles aggregation-prone substrates is key to understanding why protein folding mechanisms fail, leading to age-associated aggregate accumulation in disease [2].

Proteostasis, the intricate network governing protein homeostasis within cells, relies on the coordinated efforts of various organelles and molecular machinery. Each cellular compartment contributes uniquely to the folding, trafficking, and degradation of proteins, with specific proteins and mechanisms orchestrating these processes. The endoplasmic reticulum (ER) stands as a primary site for protein folding and quality control. Within its lumen, chaperone proteins such as BiP/GRP78 and calnexin/calreticulin assist in guiding nascent polypeptides into their native conformations [3]. The ER also houses the unfolded protein response (UPR), a signaling pathway activated in response to ER stress, which coordinates adaptive measures to restore proteostasis. In the cytosol, molecular chaperones such as Hsp70 and Hsp90 safeguard protein folding, preventing misfolding and aggregation [4]. The ubiquitin-proteasome system (UPS) plays a vital role in protein degradation, tagging misfolded or damaged proteins with ubiquitin for proteasomal degradation. Mitochondria harbor molecular chaperones like Hsp60 and Hsp70, which facilitate the folding of mitochondrial proteins [5].

Understanding the specific proteins and mechanisms operating within each cellular compartment is essential for deciphering the molecular underpinnings of proteostasis and its dysregulation in disease. By elucidating these intricate pathways, researchers can identify novel therapeutic targets and develop interventions aimed at restoring protein homeostasis in various pathological conditions. Thus, the ability to probe the performance of the proteostasis machinery through detecting aggregation with organelle-level resolution is essential.

Existing tools for studying protein aggregation offer valuable insights, but they come with limitations. While immunoblots can detect total protein levels, they lack spatial information, preventing precise localization of aggregates within the cell beyond crude fractions. Electron microscopy provides high resolution but requires harsh fixation procedures, hindering live-cell studies and potentially altering the delicate ultrastructure of subcellular organelles. Fluorescent microscopy offers the advantage of live-cell imaging but often struggles to distinguish between misfolded aggregates and native protein structures, particularly within crowded confines of organelles such as the ER.

Our protocol addresses these limitations by introducing a subcellular resolution system combining confocal super-resolution microscopy with fast fluorescence lifetime imaging microscopy (FLIM) [6] by taking advantage of a metastable probe labeled with a solvatochromic fluorophore [7] that changes its lifetime depending on aggregation state [8]. Solvatochromic dyes change fluorescent properties based on their microenvironment, namely by polarity. P1 solvatochromic fluorophore is a modified BODIPY with a chloro-alkane chain ligand designed to covalently bind to Halotag, a modified bacterial haloalkane dehalogenase. HT-aggr^ER is Halotag further modified by the addition of signal peptide and KDEL motif for ER localization and point mutations that make the otherwise metastable protein more aggregate-prone [7]. When P1 binds to and labels HT-aggr^ER in living cells, its fluorescence lifetime is influenced by protein aggregation status, with a longer lifetime observed in aggregated proteins, which can be promoted by heat shock [8]. Monomeric protein can also be visualized by P1 labeling, but with a shorter lifetime, allowing for optical separation of protein species and thus real-time monitoring of protein aggregation status. HT-aggr^ER can also be labeled by fluorescent ligands such as TMR and Janelia Fluor 646, which label non-aggregated HT-aggr more brightly than P1 but are quenched and not fluorescent inside hydrophobic aggregates. This provides another degree of flexibility in the labeling of aggregated and soluble protein species. Resolving protein aggregates with this level of detail in live cells is affordable through the critical improvement in speed of time-correlated single-photon counting (TCSPC) fast FLIM on the Leica SP8 FALCON [9,10], which detects photons at ~80 mega counts per second and plots average arrival time for each pixel in near-real time without exponential fitting [11]. LIGHTNING deconvolution applies the Richardson-Lucy algorithm and the underlying principles of point spread function to remove background and out-of-focus signal from a 3D confocal image voxel by voxel, based on an adaptative decision mask. Implementing fast FLIM alongside LIGHTNING deconvolution of correlated confocal images allows for the localization of the FLIM data to sub-diffraction limit resolution counter images. This combination unlocks new avenues for studying protein aggregation state with subcellular resolution, offering several key advantages over existing methods. Beyond providing unparalleled insights into ER protein aggregation, our protocol is applicable for exploring other areas of cellular proteostasis, using the aggregation probe variants targeted to other cellular compartments. Additionally, this system could be used for live-cell drug screening, allowing researchers to rapidly assess the effectiveness of potential therapeutics in preventing or disassembling protein aggregates. The versatile protocol described here thus fills a gap in existing methodologies by offering high-resolution and quantitative insights into the complex protein aggregation process. Its implementation can help to gain a deeper understanding of protein misfolding-related diseases and the development of therapeutic strategies against them.