Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid-liquid phase separation.

Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.

Biochemical characterization of G64W mutant of acidic beta-crystallin 4.

Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic ß-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic ß-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.

[The validation of the effect of correcting spectral background changes based on floating reference method by simulation].

There are some challenges in near-infrared non-invasive blood glucose measurement, such as the low signal to noise ratio of instrument, the unstable measurement conditions, the unpredictable and irregular changes of the measured object, and etc. Therefore, it is difficult to extract the information of blood glucose concentrations from the complicated signals accurately. Reference measurement method is usually considered to be used to eliminate the effect of background changes. But there is no reference substance which changes synchronously with the anylate. After many years of research, our research group has proposed the floating reference method, which is succeeded in eliminating the spectral effects induced by the instrument drifts and the measured object's background variations. But our studies indicate that the reference-point will changes following the changing of measurement location and wavelength. Therefore, the effects of floating reference method should be verified comprehensively. In this paper, keeping things simple, the Monte Carlo simulation employing Intralipid solution with the concentrations of 5% and 10% is performed to verify the effect of floating reference method used into eliminating the consequences of the light source drift. And the light source drift is introduced through varying the incident photon number. The effectiveness of the floating reference method with corresponding reference-points at different wavelengths in eliminating the variations of the light source drift is estimated. The comparison of the prediction abilities of the calibration models with and without using this method shows that the RMSEPs of the method are decreased by about 98.57% (5%Intralipid)and 99.36% (10% Intralipid)for different Intralipid. The results indicate that the floating reference method has obvious effect in eliminating the background changes.