Cell transport in model extracellular matrices

The rapid transport of cells has been shown to occur by ordered countercurrent convection. This convection can be created by mixtures of macromolecules which make up the extracellular matrix and by the degradation and aggregation products of these macromolecules. The ordered countercurrent convection is manifested in the form of structured flows and arises in isothermal systems with small concentration gradients of solutes. The flows are gravity driven but may rapidly move at angles close to the horizontal axis if they are mechanically constrained to do so. These flows have been shown to rapidly transport cells at rates ranging from 1 to 100 mm h-1, depending on the conditions of the experiment. The transport of cells is nonspecific in that various cell types (chondrocytes, fibroblasts, endothelial cells, and red blood cells) as well as inert particles of similar size (latex beads 6-microns diam) are transported at similar rates. Latex bead transport by structured flow has also been demonstrated to occur in confined spaces in the form of Teflon tubing down to 200 microns in diameter and at angles in the range of 45-90 degrees to the horizontal axis. The flows may also occur over relatively long distances for a prolonged period of time. The conditions for flow formation are simple and widespread. It is suggested that it may contribute to the forces involved in the movement of cells in the extracellular matrix in vivo especially during remodeling and embryogenesis.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Cell wall pH and auxin transport velocity

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.     

Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.     

Cell surface expression of membrane-anchored v-sis gene products: glycosylation is not required for cell surface transport

The v-sis gene is able to transform cells by production of a growth factor that is structurally related to platelet-derived growth factor. This growth factor has been detected in the conditioned media of v-sis transformed cells, and is able to stimulate the autophosphorylation of the platelet-derived growth factor receptor. We have used the v-sis gene product to analyze the role of protein-encoded signals in cell surface transport. We constructed several gene fusions that encode transmembrane forms of the v-sis gene product. These membrane-anchored forms of the v-sis gene product are properly folded into a native structure, as indicated by their dimerization, glycosylation, and NH2- terminal proteolytic processing. Indirect immunofluorescence demonstrated that several of these membrane-anchored gene products are transported to the cell surface. Removal of the N-linked glycosylation site from the v-sis gene product did not prevent cell surface transport. Several of these mutant genes are able to induce focus formation in NIH3T3 cells, providing further evidence that the membrane- anchored proteins are properly folded. These results demonstrate that N- linked glycosylation is not required for the cell surface transport of a protein that is in a native, biologically active conformation. These results provide a correlation between cell surface expression of the membrane-anchored v-sis gene products and transformation.     

Cell cycle related phenothiazine effects on adriamycin transport

In P388 murine leukemic cells, the pheno-thiazine tranquilizer, chlorpromazine, causes a marked increase in intracellular drug retention. Laser excited flow cytometry shows that in log phase cultures this increase in drug retention is not uniform but confined to a sub-population. In cell cycle phase enriched populations of P388 cells obtained by centrifugal elutriation, the phenothiazine enhanced adriamycin retention is seen predominantly in cells in the late S, G2/M part of the cell cycle.