mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

[Laminin-binding protein as a cellular receptor for the equine Venezuelan encephalomyelitis virus: Report 2. Inhibition of replication of equine Venezuelan encephalomyelitis virus by blocking laminin-binding protein on the surface of Vero cells]. / Lamininsviazyvaiushchii belok kak kletochnyi retseptor dlia virusa venesuél'skogo éntsefalomielita loshadei: Soobshchenie. 2. Ingibiriovanie replikatsii virusa venesuél'skogo éntsefalomielita loshchadei putem blokirovaniia lamininsviazyvaiushchego belka na poverkhnosti kletok Vero.

A possible inhibition of the Venezuelan equine encephalomyelitis (VEE) virus replication in Vero cells through the laminin-binding protein (LBP) blocking in the surface of such cells was investigated in order to verify the LBP value. It was demonstrated, on the basis of the flow scanning cytometry and FITC-labeled antibodies to LBP, that there are at least 263 thousand LBP molecules in the Vero cells' surface. Blocking of the molecules by rabbit polyclonal antibodies to 43 kD of the recombinant LBP (rLBP) was shown to inhibit the VEE virus replication in Vero cells by more than 300,000 times, which made them virtually resistant to the possibility of VEE virus infection. This also confirmed that LBP is a target-molecule for VEE virus in Vero cells with the interplay between VEE virus and LBP in the cells' surface being the initial stage of virions' penetration into the cell and of their replication inside the cell.

[Laminin-binding protein (LBP) as a cellular receptor for the virus of Venezuelan equine encephalomyelitis (VEE): Part 1. A study of the interaction between VEE virus virions and the human recombinant LBP]. / Laminosviazyvaiushchii belok (LSB) kak kletochnyi retseptor dlia virusa venesuél'skogo éntsefalomielita loshadei (VEL): Soobshchenie 1. Issledovanie vzaimodeistviia virionov virusa VEL s rekombinantnym LSB cheloveka.

The interaction of the VEE virus virions with human LBP was investigated. The affinely purified 43 kDa recombinant LBP (rLBP) of man was found to interact effectively with the VEE virus virions purified in immune enzyme assay. The affinity constant of 43 kDa rLBP with virions was equal to 4.3-4.8 x 10(7) M-1. The rabbit antiviral polyclonal antibodies blocked the interaction of rLBP with the VEE virus virions. According to Western blot, rLBP is capable of interacting with the E1 glycoprotein of the VEE virus, which suggests the presence of a specific epitope of binding with LBP in the surface of the E1-E2 heterodimer of the VEE virus. The results confirm that human LBP could be a receptor for the VEE virus.

[Inhibition of replication of Venezuelan equine encephalomyelitis with polyclonal antibodies to laminin-binding protein]. / Ingibirovanie replikatsii virusa venesuél'skogo éntsefalomielita loshadei poliklonal'nymi antitelami k lamininsviazyvaiushchemu belku.

A study of temporal and quantitative characteristics of inhibition of replication of Venezuelan equine encephalomyelitis (VEE) virus, strain TC-83, in Vero and CPE on PK cells showed purified polyclonal rabbit antibodies to human recombinant laminin-binding protein (LBP) to be able to block completely the development of cytopathic effect (CPE) in such cells, when infected with 10(7) CPE60. The extent of VEE infection inhibition in Vero was in direct proportion to a concentration of specific antibodies within a range of 0.44-3 microg/100 microl. When antibodies were added to Vero cells after they were infected, there was a gradual attenuation of the inhibition effect, which stopped almost completely 9 hours after the antibodies were placed. Inhibition was effective at 4 degrees C and 37 degrees C. A lack of synthesis of viral glycoprotein E2 in Vero cells infected in the presence of antibodies to LBP is an extra argument proving that the VEE replication is inhibited at early infection stages. The data obtained demonstrated the general LBP significance for the penetration of VEE into mammalian cells and the related importance of designing new antiviral drugs against alpha-viral infection, which are based on blocking the mechanism of receptor penetration of the virus into the cell.

Particle classification from light scattering with the scanning flow cytometer.

BACKGROUND: The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. METHODS: The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. RESULTS: The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm. CONCLUSIONS: The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.

Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer.

BACKGROUND: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding.

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.