Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

Abstract

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.

Two-photon excited-state dynamics of mEGFP-linker-mScarlet-I crowding biosensor in controlled environments

“…Such disagreement could be attributed to the excitation wavelength (488 nm versus 900 nm), the different buffers (NaPi versus PBS), the time window (0-50 ns versus 0-24 ns), and the sample preparation (a cuvette versus a droplet) used in both studies. As a control, Lecinski et al 53 also used mGFP as compared with our mEGFP using the enzymatically cleaved GE2.3, where the fluorescence lifetime of mEGFP is known to be approximately 2.6 ns, [46][47][48][49] in relative agreement with our estimated 2P-fluorescence lifetime of 2.75 ns in enzymatically cleaved GE2.3.…”

“…Our results are also in general agreement with Lecinski et al qualitatively on the same sensor (crGE2.3) in Ficoll-crowded NaPi buffer. 53 We also demonstrated that the outcome of these studies seems independent of the 2Pexcitation mode of GE2.3 (i.e., single-point versus laser scanning FLIM mode). Such control experiments are rather important since 2P-FLIM is compatible with future 2P-FLIM studies in cultured living cells or even thick tissues as a better model for in vivo studies for mapping the correlation between macromolecular crowding and diseased conditions.…”

Editorial: theme issue on complex rheology in biological systems

“…For this theme issue, we gratefully received contributions from across the physics and life sciences with interests in biorheology ranging in length scale from the rheological properties of intracellular biomolecular networks [ 1 , 10 ] to the scale of the direct extracellular environment [ 3 , 7 , 10 , 12 , 13 , 16 ], tissues [ 3 – 5 ] and even entire organs that actively exert forces onto non-Newtonian fluids [ 6 ]. The mechanical properties at the cellular level are discussed in relationship to cancer [ 2 ], as well as in relationship to the transport of red blood cells in disordered porous environments, be it in vascular networks or in microfluidic devices.…”

“…To assess this information, the contributed review by Erlich et al [ 4 ] argues it is necessary to develop novel non-perturbative methodologies to probe the network at a small length scale. Lecinski et al address this using single-bead tracking passive rheology in live S. cerevisiae yeast cells [ 1 ], and Jory et al discuss new methodologies to probe mucus adhesion at the microscopic scale using optical tweezers [ 10 ]. Erlich and co-workers also argue the interpretation of the force–displacement measurements in such experiments relies on the development of suitable theoretical models, which face the challenge of linking the molecular topology of the network to the mechanics at the continuum level; such a modelling contribution is provided by Song and co-workers who discuss hyperelastic continuum models [ 3 ].…”

“…Cell biology in plants, animals and prokaryotes is usually described in terms of components, biochemical networks and signalling. Yet local flows, and deformations of the entire cell as well as its individual parts [ 1 , 2 ], are essential to function. Questions on such mechanical properties and phenomena are rarely addressed.…”