Do not touch! The surface of a living cell is soft and responsive and therefore high‐resolution imaging of the cell membrane has not been possible to date. Now noncontact imaging of protein complexes in the plasma membrane of living cells has been demonstrated (see picture) and has been used to follow the cells' structural reorganization. This breakthrough opens up a wealth of new experiments in membrane and cell biology.

The Drosophila HP1 gene contains a highly conserved sequence, the chromobox, which can be used to isolate HP1-like genes from both mouse (M31 and M32) and man (HSM1) (Singh et al., 1991). Here we report that a monoclonal antibody (MoAb) raised against the M31 protein recognises a 26-kDa protein in murine and human nuclear extracts and localises to large masses of condensed chromatin within murine interphase nuclei, some of which are associated with the nucleoli. At metaphase, the MoAb binds to the centromeres of both human and murine chromosomes. The evolutionary conservation of this chromosomal localisation indicates that the M31 protein is likely to be important in the packaging of mammalian chromosomal DNA into constitutive heterochromatin.

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.

A comprehensive study of how age and weaning affect intestinal structure and enterocyte ability to digest and absorb nutrients has been carried out in 4-and 6-weekold piglets.Villus length, which did not change significantly in unweaned piglets 4-6 weeks after birth, was halved 5 days after weaning. Crypt depth, which increased normally in unweaned piglets, is further increased by weaning in both 4-and 6-week-old animals.Lactase activity, which decreased normally with age, was inhibited more than a-glucosidase by weaning. Weaning of 6-week-old piglets also caused a significant increase in maltase II and III activities. Alkaline phosphatase activity was unaffected by age or weaning in 4-and 6-week-old piglet intestine.Na-dependent alanine transport was reduced in 6-compared with 4-week-old unweaned piglet intestine. Weaning inhibited Na-dependent alanine transport in 4-but not 6-week-old pigs. Na-independent alanine transport, which was considerably less than that found in the presence of Na, was not noticeably affected by age or weaning.Weaning-induced problems in intestinal function appear from the present results to be caused more by changes in intestinal structure and specific loss of digestive enzymes rather than by any gross change in absorptive function. The possible role of immune as well as nutritional factors in causing these weaning-dependent changes in intestinal function is discussed.

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 +/- 0.04 microm(2)/s in the acrosomal region and 0.10 +/- 0.02 microm(2)/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.