The movement of bound ligands over cell surfaces

The long range movements of membrane-bound ligands into surface caps and into the pseudopods of phagocytizing cells, the uropods of motile cells and the cleavage furrow of dividing cells appear to be analogous processes. A common mechanism to explain these movements must take into account several new and central observations: ligand-receptor complexes can migrate to regions of existing microfilament accumulation; laser photobleaching studies with fluorescent Con A indicate that ligand-receptor movement occurs unidirectionally; video computer analyses of Con A redistribution show that movement may exceed the maximum rates measured for protein diffusion in membranes. These observations are not consistent with models in which ligand-receptor movement occurs by diffusion or by direct interaction with contractile microfilaments. However, they can be satisfied by a new model that proposes the entrainment of selected membrane determinants on membrane waves directed towards regions such as caps, pseudopodia, uropods or cleavage furrow. These oriented waves are initiated by tension due to asymmetric microfilamentmembrane interaction.     

Distribution of receptors and functions on cell surfaces: quantitation of ligand-receptor mobility and a new model for the control of plasma membrane topography

The long-range movements of membrane ligand-receptor complexes into surface caps and into the pseudopods of cells performing phagocytosis, the uropods of motile cells and the cleavage furrows of dividing cells appear to be analogous processes. A common mechanism to explain these movements must take into account several recent observations. First, laser photobleaching studies have indicated that Concanavalin A-receptor movement occurs unidirectionally; and analyses of Con A redistribution by quantitative video intensification microscopy (QUAVIM) have shown that movement may exceed the maximum rates measured for protein diffusion in membranes. These are the results predicted for a process of directed migration but not for a process of diffusion with entrapment. In addition it has been found that membrane receptors may segregate out of as well as into cap, pseudopod, uropod and cleavage furrow regions and that topographical heterogeneity on asymmetric cells is not restricted to membrane molecular determinants but extends to a range of endocytic functions and to a macromolecular complex, the coated pit. All dynamic surface events are arrested during mitosis. A new model for the regulation of plasma membrane topography has been developed from these diverse quantitative, functional and morphological data. Its essence is the entrainment of selected membrane determinants on membrane waves directed towards regions such as caps, pseudopods, uropods and cleavage furrows. The waves are initiated by tension due to asymmetric microfilament-membrane interaction.     

Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

Isolation of concanavalin a caps during various stages of formation and their association with actin and myosin

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell’s actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.     

人胃癌不同周期细胞膜表面ConA受体的分布与侧向运动

本文研究了人胃低分化粘液性腺癌细胞MGC 80-3不同周期时相中ConA受体的分布与侧向运动。MGc 80-3细胞经同步化培养,用F-ConA标记。被标记细胞中G_1、S和G_2期呈不连续的分布,但它们之间又存在显著的差异。M期呈较均匀的强荧光分布(与其它时相细胞比较)。荧光漂白恢复方法测定ConA受体复合物侧向运动表明:各个周期时相之间不仅运动方式不同,而且运动速率也有显著差异。M期与G_1期主要表现出扩散型运动;而S期与G_2期表现为流动型运动。G_1期的扩散系数大干M期的;S期的流动速率大于G_2期的。但可动分子百分比以G_2期最高。这些结果表明了ConA受体的动力学性质。它受到细胞周期的调节。     

Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.     

A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis

Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP(2)) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.     

Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.

We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.     

Cell lineage-specific inhibition of cytokinesis by concanavalin A in a molluscan embryo (Nassarius reticulatus,Gastropoda)

Summary The effects of the lectin concanavalin A (Con A) on cleavage were studied in early embryos of the gastropodNassarius reticulatus. Progression of the first cleavage furrow is inhibited by incubating eggs before the first cleavage with 0.3–20 μg/ml Con A. Treatment with 1.0–20 μg/ml Con A during first cleavage causes regression of the cleavage furrow. Treatment with low concentrations (0.3–1.0 μg/ml) during the same period does not affect first cleavage. However, when further development of such eggs is followed, one finds that second cleavage is inhibited typically in only one of the two blastomeres of the 2-cell stage, i.e. the CD-blastomere. As a result, a 3-cell embryo is formed. At third cleavage of such embryos, the CD-blastomere forms either one double-sized micromere (1cd-micromere) or two normal-sized micromeres (1c and 1d) simultaneously. Sometimes micromere formation in the CD-blastomere is inhibited. Con A binding does not affect karyokinesis, nor does it affect the division asynchronies typical for normal development. On the basis of these and other results it is argued that binding of Con A to sites located at the vegetal pole of the egg is responsible for the cell lineage-specific inhibition of cleavage by Con A. This effect is most probably mediated by changes in the organization of the egg cortex.     

Dependence of the redistribution of ligand-receptor complexes in cultured cells on the type of growth and on the cell cycle phase

The process of active redistribution of ligand-receptor complexes on the surface of both suspension (LS) and adapted for growing in monolayer (LMS) sublines of mouse fibroblasts L was studied. The binding of ligands to its specific receptors on the surface of the LS cells induced an accumulation of ligand-receptor complexes on one pole of a cell with the formation of a typical cap. Under the same conditions in the LSM cells the cleaning of processes and lamello-plasma surfaces from the ligand-receptor complexes was registered. The binding of ligands to the surface of the LSM cells, detached with EDTA, induced the same capping process, as in the case of the LS cells. No differences were found in the redistribution capacity of the ligand-receptor complexes in synchronized cultures of the LS and LSM cells in the G1 and S phase of the cycle. But such a redistribution was not registered on the surface of cells in metaphase and anaphase. The accumulation of ligand-receptor complexes was found in the region of cleavage between daughter cells in telophase. These results are in a good agreement with the well-known data on the changes in the cytoskeleton organization during transition from a monolayer to a suspension state and during mitosis.     

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.     

Mitochondria Localize to the Cleavage Furrow in Mammalian Cytokinesis

Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.     

Chromosomal Proteins and Cytokinesis: Patterns of Cleavage Furrow Formation and Inner Centromere Protein Positioning in Mitotic Heterokaryons and Mid-anaphase Cells

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.     

Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining

We have studied the distribution of myosin and tubulin molecules inside the same tissue culture cells by using two antibodies labeled with contrasting fluorochromes. Antimyosin raised against human platelet myosin was labeled with rhodamine. Antitubulin raised against sea urchin vinblastine-induced tubulin crystals was labeled with fluorescein. The two antibodies stained entirely different structures inside the same flat interphase cell: antimyosin bound to stress fibers and antitubulin bound to thin, wavy fibers thought to be individual microtubules. Compact interphase cells stained diffusely with both antibodies. From prophase through early anaphase both antibodies stained the mitotic spindle, although the fluorescence contrast between the spindle and the cytoplasm was much higher with antitubulin than with antimyosin. From anaphase through telophase, strong antimyosin staining occurred in the cleavage furrow, while antitubulin stained the region between the separated chromosomes. This study established the feasibility of high-resolution fluorescent antibody localization of pairs of motility proteins in the cytoplasm of single cells, an approach which will make it possible to map out the sites of the various contractile protein interactions in situ.     

Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells

Thin-section electron microscope analysis of rat and rabbit-cultured granulosa cells treated with concanavalin A (Con A) at 37 degrees C revealed coordinated changes in the cytoplasmic disposition of microfilaments, thick filaments, and microtubules during cap formation and internalization of lectin-receptor complexes. Con A-receptor clustering is accompanied by an accumulation of subplasmalemmal microfilaments which assemble into a loosely woven ring as patches of receptor move centrally on the cell surface. Periodic densities appear in the microfilament ring which becomes reduced in diameter as patches coalesce to form a single central cap. Microtubules and thick filaments emerge associated with the capped membrane. Capping is followed by endocytosis of the con A-receptor complexes. During this process, the microfilament ring is displaced basally into the cytoplasm and endocytic vesicles are transported to the paranuclear Golgi complex along microtubules and thick filaments. Eventually, these vesicles aggregate near the cell center where they are embedded in a dense meshwork of thick filaments. Freeze-fracture analysis of Con A-capped granulosa cells revealed no alteration in the arrangement of peripheral intramembrane particles but large, smooth domains were conspicuous in the capped region of the plasma membrane. The data are discussed with reference to the participation of microtubules and microfilaments in the capping process.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

Abstract. A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 μg/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary colium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 microgram/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary cilium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Cleavage furrow positioning

To complete the cell cycle, the cleavage furrow draws the plasma membrane toward the cell center, pinching the cytoplasm into two lobes that are subsequently separated into two cells. The position of the cleavage furrow is induced by the mitotic spindle during early anaphase. Although the mechanism of cleavage furrow positioning is not understood at a molecular level, recent results suggest that it might be mediated by local relief from the inhibitory effects of microtubules.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of calcium and phosphorus during the first cell division of the sea urchin egg

The intracellular distribution of calcium and phosphorus during metaphase and anaphase of the first cleavage in sea urchin eggs was studied with the electron-probe microanalyzer. This study allowed a comparison of the relative concentrations of both elements on the polar and cleavage furrow regions of the membrane and on the mitotic asters and cytoplasm. The results show that in most eggs, both calcium and phosphorus are more highly concentrated in the mitotic asters than in surrounding cytoplasm during both anaphase and metaphase. Calcium is more concentrated at the furrow region than at the polar region during metaphase but not anaphase. The role of calcium during mitosis was reviewed with special reference to the theories on the formation of the cleavage furrow along the equatorial zone between two mitotic centers.