Several chemical effectors were used to induce changes in spleen B cell membrane fluidity. Membrane fluidity was monitored by fluorescence polarization analysis of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and cell viability was checked not to be affected by the treatments. Membrane immunoglobulin (Ig) endocytosis by the living B cells with modified or unmodified membranes was quantitatively measured by flow cytometry, using a previously described method (Métézeau et al., 1982, 1984). The kinetics of endocytosis of membrane Ig was not affected by chemical effectors increasing membrane fluidity. On the contrary, increasing membrane microviscosity resulted in the slowing down and eventually the blocking of membrane Ig endocytosis. It is suggested that a step depending on membrane microviscosity is involved in the process of endocytosis; this step may become rate limiting when membranes are artificially rendered or naturally become (i.e. for pathological or particularly differentiated cells) more viscous.
A method is described for directly hybridizing a small number of sorted chromosomes with specific DNA probes. The chromosomes are analyzed by flow cytometry and sorted by deflecting the droplets containing the desired chromosomes onto a nitrocellulose filter. By using probes specific for the human Y chromosome, it has been possible to unambiguously identify the peak corresponding to the Y chromosome in the flow karyotypes of a variety of male cell lines. The position of this peak was found to vary significantly from individual to individual, correlating with the heterochromatin chromosomal polymorphism of the human Y chromosome. The sensitivity of the hybridization was such that, with a probe for a male-specific repetitive sequence, only 2,500 sorted chromosomes were enough to obtain a clear, positive signal; 10,000 were needed with a probe specific for a weakly repeated (maximum, 3-fold) sequence of Y chromosome. With this new method, chromosome sorting may be a rapid and efficient way to assign DNA sequences to chromosomes.
A method is described to study quantitatively and rapidly the kinetics of endocytosis of a receptor-bound ligand by cells of various subclasses within a heterogeneous cell population. The time course of the internalization of the bound ligand is followed by labeling, with a fluorescent antibody, the ligand molecules exposed on the cell surface at various times of the endocytosis process, and by measuring with a flow cytofluorometer the fluorescence of a large number of individual cell within the total cell population. A convenient mathematical treatment of the data is proposed for analyzing the experimental results. This method is applied to the kinetic study of the endocytosis of rabbit antibodies (anti-mouse immunoglobulin) by B-lymphocytes within a mouse spleen cell suspension.
It remains unclear whether polypeptide chains renaturing in vitro from strong denaturants proceed through the same folding pathway as chains released from ribosome within cells. Folding intermediates formed both in vivo and in vitro have been examined using three monoclonal antibodies shown previously to recognize different epitopes of the native P22 tailspike protein (Friguet, B., Djavadi-Ohaniance, L., Haase-Pettingell, C. A., King J., and Goldberg, M. E. (1990) J. Biol. Chem. 265, 10347-10351). The tailspike protein was reconstituted from polypeptide chains unfolded by urea as described by Fuchs et al. (Fuchs, A., Seiderer, C., and Seckler, R. (1991) Biochemistry 30, 6598-6604), and the appearance of immunoreactive forms during the refolding was monitored. The three antibodies discriminated intermediates at different stages in the folding pathway. On the basis of the reconstitution pathway determined from spectroscopic and hydrodynamic measurements by Fuchs et al. (1991), monoclonal antibody (mAb) 236-3 recognized partially folded monomers, mAb 155-3 recognized folded protomers in a protrimer species, and mAb 33-2 recognized the native trimer. The kinetics of appearance of the immunoreactive forms during the in vitro refolding of the protein in crude extracts of phage-infected cells was similar to that observed with the pure tailspike. Thus, the antibodies provided probes for the chain folding and association pathway in vivo. The conformation of the ribosome-bound tailspike polypeptide chains of the infected cells was analyzed with the three antibodies. The antibodies recognizing native trimer and the protrimer did not bind chains associated with the ribosomes. Antibody 236-3, which recognized structured monomers in vitro, bound to the polypeptide chains still associated with ribosomes. This result suggests that steps that take place in solution during in vitro refolding may occur in a ribosome-bound state in vivo.
