In vivo amyloid-like fibrils produced under stress

Abstract

The participation of amyloids in neurodegenerative diseases and in functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. We used the autofluorescence from the extended β-sheets of amyloids to follow fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS) and here we suggest the idea of its participation also in heat shock response (HSR). Fluorescence Lifetime Imaging (FLIM) was used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress was further supported by size exclusion chromatography and ultracentrifugation. Our data show the first-time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion

“…ACBP's incorporation within Grh1 droplets intensified over time (up to ~one hour), coinciding with the period when the reversibility of Grh1 droplets started to be significantly impacted (Figure 4C). The isolated non-reversible structure by Grh1 over time under starvation exhibited an amyloid-like signature characterized by an increase in ThT affinity and the development of deep-blue autofluorescence (Figure 4D) [26,52]. These observations agree with the previous in vivo data of S. cerevisiae under starvation for prolonged times [26].…”

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

“…The activation of GRASP fibrillation could be related to a protective mechanism in nonpermissive temperatures. The same profile was also observed in vivo during nutrient starvation, suggesting remarkable plasticity of function for these aggregates [ 31 ]. It is worth pointing that the human GRASPs contain more aggregation hotspots in their GRASP domain compared with the SPR [ 36,55 ].…”

“…Therefore, GRASP could work as tethering factors in UPS and be involved in forming condensates or aggregated structures, recruiting specific proteins for UPS. GRASP fibrillation under stress, both chemically (starvation-triggered) and under heat, was already observed in vivo and shown to be reversible [ 31 ].…”

“…The PDZs are connected in tandem to allow rigid body reorganization, facilitating their interaction with multiple different protein partners [ 25 ]. This promiscuity for interaction with several different protein partners and their unusual structural plasticity [ 26,27 ] might be the convergent features of GRASPs in different cell functional tasks, including as anchoring factors of Golgins [ 12,15,23 ], their direct participation in Golgi dynamics during mitotic and apoptotic times [ [28][29][30] and their self-association during stress conditions triggering UPS [ 4,31 ]. It comes as quite a surprise that there is still a significant gap in understanding how GRASPs can perform those functionalities.…”

Exploring Liquid-Liquid Phase Separation in the organization of Golgi Matrix Proteins

“…GRASPs are collapsed intrinsically disordered proteins (IDPs) with high promiscuity for mediating protein-protein interactions with several different protein partners (13)(14)(15). Their unusual structural plasticity ( 16) might be the converging feature of GRASPs in multiple cell functional tasks, including their direct participation in Golgi dynamics during mitotic and apoptotic times (17)(18)(19) and their self-association during stress conditions triggering unconventional protein secretion pathways (20)(21)(22)(23). GRASPs interact with the membrane via a lipidation of its Gly2 using a myristoyl-switch mechanism (24,25).…”