Richard D. Berlin

Surface functions during mitosis I: Phagocytosis, pinocytosis and mobility of surface-bound ConA

Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties. Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is impaired from early prophase through early G1, whereas phagocytosis of nonopsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells. Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37 degrees C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphse. The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization. It is evident that membrane processes which depend upon endocytosis--for example, certain hormone-induced signals--may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.

The Cell Surface

THERE is little doubt that there are important differences in the biologic behavior of tumor cells and normal cells that can be correlated with altered surface properties. Thus, the uncontrolled gr...

Effect of colchicine, colcemid, and vinblastine on the agglutination, by concanavalin A, of transformed cells.

The sensitivity of transformed 3T3 fibroblasts to agglutination by concanavalin A is reduced by alkaloids that bind specifically to protein subunits of microtubules.

Membrane Transport of Purine and Pyrimidine Bases and Nucleosides in Animal Cells

Publisher Summary This chapter discusses the membrane transport of purine and pyrimidine bases and nucleosides in animal cells. In general, these compounds enter animal cells by facilitated diffusion, and transport carriers are clearly separate from the enzymes responsible for subsequent intracellular metabolism. The carriers are proteins which have lateral mobility in the plane of the membrane. The chapter discusses the some general principles about transport processes in animal cells, and then examine: (1) the evidence for specific transport systems for bases and nucleosides, attempting to differentiate between membrane and metabolic events; (2) the detailed characteristics of the systems, with particular attention to implications for carrier structure based on substrate specificity and sensitivity to inhibition by chemical agents; (3) the physiological control of transport; and the evidence that carriers are mobile components of biological membranes and as such may be functionally modified by interactions with other membrane components. Substances that inhibit transport in cells depend on exogenous purines for nucleic acid synthesis may impose selective growth control. Malignancies of bone marrow (leukemia) and of the gastrointestinal tract could fall within this category; such inhibitors should not damage the majority of cells and tissues that have the capacity to synthesize purines de novo. In addition, since carrier properties such as susceptibility to inhibition are highly variable among cells, inhibitors that are particularly effective against specific cell types may be developed.

Mechanisms That Regulate the Structural and Functional Architecture of Cell Surfaces

Publisher Summary This chapter emphasizes on the search for mechanisms controlling cell surface molecular and functional topography. The asymmetric surface topography is a predictable correlate of membrane deformation and microfilament asymmetry, independent of how asymmetry is induced and which cell type is studied. Tthe chapter demonstrates that receptors can be segregated out of and into regions of microfilament accumulation. The topographical heterogeneity is not restricted to membrane molecular determinants, but extends to a range of endocytic functions and to a macromolecular assembly, which is the coated pit. There is also a discussion about the arrest of dynamic surface events during mitosis. The chapter describes a working model to explain that the topographical data depends on initial recruitment of microfilaments to incipient protuberance, uropod, pseudopod, or cleavage furrow membranes. The resulting microfilament-membrane interaction acting inward and along the cell surface leads to the generation of tension that is propagated over the cell surface as a wave. Microfilament polarization is required to establish and maintain membrane structural asymmetry and to eventually relieve the accumulation of membrane components by endocytosis.

Purine transport in polymorphonuclear leukocytes

Abstract The uptake of adenine and xanthine has been studied in rabbit polymorphonuclear neutrophilic leukocytes. Since purines were found to enter neutrophils rapidly a technique was developed for taking initial rate samples as quickly as 10 sec, using cell monolayers on glass coverslips. Adenine was found to enter neutrophils by two saturable transport systems, one with a high affinity and a low maximal velocity, the other with a low affinity and a high maximal velocity. Xanthine enters predominantly by a transport carrier that is distinct from either of the adenine systems. In addition some xanthine entry occurs which cannot be overcome by a competitive inhibitor. Within the cell adenine was metabolized to adenosine nucleotides and xanthine to uric acid. Both products accumulated within the cell. From the absence of free adenine within the cell it was concluded that a low concentrations the limiting step to adenine incorporation into nucleotides was its entrance into the cell.

Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes

The membrane transport of nucleosides in rabbit polymorphonuclear leukocytes has been examined by use of a rapid sampling technique. Both purine and pyrimidine nucleosides are transported by a single saturable system as indicated by the identity of their Kms and Kis against a spectrum of nucleosides. The specificity of the carrier was examined in detail. Adenosine (Km = 0.010 mM, vmax approx. 10 pmoles/106 cells per 45 sec) has the highest affinity for the system. Its fate after uptake is deamination and subsequent conversion to nucleotide. The most critical structural requirements for binding include the pyrimidine base moiety and a 3′-OH configuration on pentose, but other groups make significant contributions to binding. From an analysis of the substrate specificity it is argued that changes in the conformation of the carrier active site are induced by the substrate.

Analysis of transferrin recycling in mitotic and interphase hela cells by quantitative fluorescence microscopy

Recent findings suggest that membrane vesicle transport during mitosis may be generally inhibited. To test this, we examined the kinetics of uptake and exocytosis of RITC-transferrin in mitotic and interphase HeLa cells. We used quantitative image-intensification fluorescence microscopy to analyze the content of ligands in single cells. This technique was validated by comparison of 3H or RITC-transferrin release from interphase cells determined by microscopy or radiometry. Both methods gave a t1/2 of release of 5-6 min. The uptake of RITC-transferrin was depressed in mitotics. More importantly, we monitored the exocytosis of label during mitosis. Labeled mitotics were obtained by the progression of interphase cells into mitosis during a 50 min incubation with RITC-transferrin. After 30 min chase with unlabeled transferrin, the intensities of interphase cells approached background, whereas those of mitotic cells remained nearly constant. Thus both exocytosis and endocytosis of transferrin were exocytosis and endocytosis of transferrin were blocked during mitosis.

A carbamate herbicide causes microtubule and microfilament disruption and nuclear fragmentation in fibroblasts.

Abstract Isopropyl( N -(3-chlorophenyl)carbamate (CIPC) is a widely used herbicide thought to inhibit mitosis by selectively perturbing either the microtubules or microtubule organizing centers of susceptible plant species. Its effects on microtubule and microfilament organization in mouse 3T3 fibroblasts are analyzed here by immunofluorescence techniques using anti-tubulin and anti-actin antibodies. We report that relatively high levels of CIPC (but not higher than the usual levels employed in plant studies) inhibit fibroblast growth, cause the disappearance of cytoplasmic microtubules and also lead to disorientation of microfilament bundles. These responses develop within 24 h of exposure of 3T3 cells to CIPC and persist as long as the herbicide is present in the culture medium. More importantly, cells returned to drug-free medium following 5–7 days exposure to CIPC resume rapid division but show several remarkable morphological responses. In particular they develop multipolar spindles during mitosis and show an unequal partitioning of nuclei between daughter cells. Consequently a high proportion of interphase cells contains multiple and multilobed nuclei. Such fibroblast cultures exposed to CIPC and then returned to drug-free medium may provide chromosome-deficient cells of value for genetic mapping and recombinant studies. On the other hand if similar responses to CIPC exposure and withdrawal occur in vivo then the herbicide may pose a genetic risk to man that is not revealed by usual testing procedures. The mechanism(s) by which CIPC produces these responses remains unclear. Fibroblasts cultured in the continued presence of the known microtubule-specific drugs colchicine and nocodazole (10 −6 M) show microtubule disassembly within 30 min followed by cell rounding and detachment. By contrast CIPC-treated cells lose microtubule integrity more slowly and remain highly elongated and firmly glass adherent even with prolonged treatment. These dissimilar morphological responses, as well as the failure of CIPC to affect microtubule assembly in vitro, indicate that tubulin is not the exclusive target of CIPC in animal cells. The distribution of assembled microtubules into the arms of multipolar mitotic spindles is clearly observed following withdrawal of CIPC. The abnormality could reflect an irreversible effect of CIPC on microtubule organizing centers. However, the redistribution of cytoplasmic microfilaments into dense, discontinuous submembranous bundles in the presence of CIPC and the failure of microfilaments to resume an entirely normal distribution following herbicide withdrawal suggests that microfilaments are additional targets of herbicide action.

[33] Use of horseradish peroxidase and fluorescent dextrans to study fluid pinocytosis in leukocytes

Publisher Summary This chapter discusses the methods to observe and quantify fluid pinocytosis. The procedures have been developed and applied in macrophages and granulocytes. However the same protocols may be used with other leukocytes and in a range of tissue culture cells. Pinocytosis, the uptake of medium and its dissolved or suspended contents by enclosure in small membrane vesicles that bud from the cell surface, occurs in essentially all cells except for mature erythrocytes. The chapter reviews two categories of pinocytosis that have been distinguished. Fluid pinocytosis refers to the internalization of medium constituents by their simple inclusion in droplets of medium engulfed by the cell. Among leukocytes, cells of the monocytic/macrophage lineage show the most active fluid pinocytosis, while lymphocytes and granulocytes are more weakly pinocytic. On the other hand, adsorptive pinocytosis refers to the uptake of extracellular substances, which follow their binding to membrane receptors. Monocytes and macrophages show maximal activity. Granulocytes are also capable of extensive adsorptive pinocytosis and lymphocytes show the lowest activity.