In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3∆ cells. This effort yielded three hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.

In eukaryotes, intracellular physical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, meso- and macro- length scale molecular diffusion, and protein folding. However, mapping the molecular crowding and viscoelastic landscapes in living cells remains challenging. One principal tool to measure viscoelasticity is passive rheology, in which the diffusion of exogenous fluorescent particles internalised in living cells is tracked though time and the physical properties inferred from a marker particle's mean square displacement. Recently, the crGE2.3 Förster Resonance Energy Transfer (FRET) based biosensor has been developed to quantify crowding in cells, though it is unclear how this readout depends on viscous and elastic properties and the molecular weight of the crowder. Here, we present correlative multi-technique and multidimensional data to explore the diffusion and molecular crowding characteristics of common molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as candidate in situ viscoelasticity markers in the cytoplasm of live budding yeast cells, Saccharomyces cerevisiae, and we discuss the limitations of this approach.

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of cells lacking RPD3. This effort yielded 3 hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.