Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.

The membrane association sequences of the prostaglandin endoperoxide synthases-1 and -2 isozymes.

Based on the crystal structures of the prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2), four short amphipathic helices near the amino termini of these proteins have been proposed to act as membrane binding domains. We constructed a series of plasmids coding for amino-terminal sequences of the PGHS-1 and PGHS-2 joined to the green fluorescent protein from Aequorea victoria, and we examined the subcellular distribution of the fusion proteins expressed from these plasmids using confocal microscopy of intact cells and Western blot analysis. DNA sequences coding for amino acids 1-139 and 1-136 of PGHS-1 and PGHS-2, respectively, which include the signal peptides, epidermal growth factor homology domains, glycosylation sites, and the putative membrane-binding helices of these two isozymes, were required for targeting the PGHS-green fluorescent protein fusion proteins to the endoplasmic reticulum and nuclear membranes when expressed in NIH 3T3 cells. Chimeric proteins that did not contain the putative membrane binding domains are targeted to the endoplasmic reticulum, but are not associated with membrane structures, and are present only in soluble cell fractions. These are the first experiments to directly confirm that the amphipathic helices present near the amino terminus of the PGHS-1 and PGHS-2 isozymes act as membrane anchors.

Auxins and Cytokinins as Antipodal Modulators of Elasticity within the Actin Network of Plant Cells.

The cytoskeleton of plant and animal cells serves as a transmitter, transducer, and effector of cell signaling mechanisms. In plants, pathways for proliferation, differentiation, intracellular vesicular transport, cell-wall biosynthesis, symbiosis, secretion, and membrane recycling depend on the organization and dynamic properties of actin- and tubulin-based structures that are either associated with the plasma membrane or traverse the cytoplasm. Recently, a new in vivo cytoskeletal assay (cell optical displacement assay) was introduced to measure the tension within subdomains (cortical, transvacuolar, and perinuclear) of the actin network in living plant cells. Cell optical displacement assay measurements within soybean (Glycine max [L.]) root cells previously demonstrated that lipophilic signals, e.g. linoleic acid and arachidonic acid or changes in cytoplasmic pH gradients, could induce significant reductions in the tension within the actin network of transvacuolar strands. In contrast, enhancement of cytoplasmic free Ca2+ resulted in an increase in tension. In the present communication we have used these measurements to show that a similar antipodal pattern of activity exists for auxins and cytokinins (in their ability to modify the tension within the actin network of plant cells). It is suggested that these growth substances exert their effect on the cytoskeleton through the activation of signaling cascades, which result in the production of lipophilic and ionic second messengers, both of which have been demonstrated to directly effect the tension within the actin network of soybean root cells.

Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr).

Alkalinization of normally acidic intracellular compartments or acidification of a mildly alkaline cytoplasm by biochemical or genetic manipulation has been demonstrated to inhibit both endocytosis and secretion (Tartakoff, 1983a; Cosson et al., 1989; Mellman et al., 1986; Davoust et al., 1987; Cosson et al., 1989; van Deurs et al., 1989; Maxfield & Yamashiro, 1991; Hansen et al., 1993). These results provide the basis for the conclusion that the maintenance of pH gradients between acidic vesicular compartments and a mildly alkaline cytoplasm is an essential biochemical requirement for the correct functioning of the endocytotic and secretory machinery. Tumor cells have been shown to have an abnormally acidic cytoplasmic pH (Warburg, 1956; Simon & Schindler, 1994). Here we report that the intracellular vesicular compartments in tumor cells (MCF-7) derived from a human breast cancer fail to acidify. This failure results in a significant decrease in the pH gradient (0.9 pH unit) between the vesicular luminal compartments and the cytoplasm. These defects are correlated with a disruption in the organization and function of the trans-Golgi network (TGN) and the pericentriolar recycling compartment (PRC). In marked distinction, drug-resistant tumor cells (MCF-7adr) derived from the MCF-7 line that are resistant to the most widely employed chemotherapeutic drug, adriamycin, appear normal in both acidification and organization of the PRC and TGN. Treatment of drug-resistant MCF-7adr cells with nigericin and monensin, ionophores demonstrated to disrupt vesicular acidification (Tartakoff, 1983b), leads to a resensitization of these cells to adriamycin. Drug sensitivity is proposed to result from an acidification defect within vesicles of the recycling and secretory pathways. A functional consequence of this defect is the diminished capacity of cells to remove cytotoxic drugs from the cytoplasm by sequestration of protonated drugs within the vesicles, followed by drug secretion through the activity of the secretory and recycling pathways.

Aluminum Induces Rigor within the Actin Network of Soybean Cells.

Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins.

Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements.

An assay has been developed to quantitatively measure the tension and elasticity of the cytoskeleton in living plant cells. The cell optical displacement assay (CODA) uses a focused laser beam to optically trap and displace transvacuolar and cortical strands through a defined distance within the cell. Results from these experiments provide evidence for the classification of at least two rheologically distinct cytoskeletal assemblies, cortical and transvacuolar, that differ in their tension and response to both signaling molecules and reagents that perturb the cytoskeleton. It is further demonstrated that the tension of the transvacuolar strands can be significantly decreased by the addition of either linoleic acid, 1,2 dioctanoyl-sn-glycerol, or 1,3 dioctanoylglycerol. These decreases in tension could also be induced by lowering the cytoplasmic pH. In contrast, addition of Ca2+, Mg2+, or the ionophore A23187 to the cells caused a considerable increase in the tension of the transvacuolar strands. The data provides evidence that: (a) linoleic acid may be a signaling molecule in plant cells; (b) diacylglycerol functions as a signaling molecule through a protein kinase C-independent pathway mediated by PLA2; and (c) Ca2+ and pH have regulatory roles for controlling cytoskeleton tension and organization.

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells.

Intercellular communication between plant cells for low molecular weight hydrophilic molecules occurs through plasmodesmata. These tubular structures are embedded in the plant cell wall in association with the plasmalemma and endoplasmic reticulum (ER). Transmission electron microscopy has provided strong evidence to support the view that both the ER and plasmalemma are structurally continuous across the wall at these sites. In experiments to be described, the technique of fluorescence redistribution after photobleaching was used to examine the lateral mobility and intercellular transport capability of a number of fluorescent lipid and phospholipid analogs. These probes were shown by confocal fluorescence microscopy to partition in either the ER or plasmalemma. Results from these measurements provide evidence for cell communication between contiguous cells for probes localized predominantly in the ER. In contrast, no detectable intercellular communication was observed for probes residing exclusively in the plasmalemma. It was of particular interest to note that when 1-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)aminoacylphosphatidylcholine was utilized as a potential reporter molecule for phospholipids in the plasmalemma, it was quickly degraded to 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminoacyldiglyceride (NBD-DAG), which then appeared predominantly localized to the ER and nuclear envelope. This endogenously synthesized NBD-DAG was found to be capable of transfer between cells, as was exogenously incorporated NBD-DAG. Results from these investigations provide support for the following conclusions: (1) ER, but apparently not the plasmalemma, can form dynamic communication pathways for lipids across the cell wall between connecting plant cells; (2) the plasmodesmata appear to form a barrier for lipid diffusion through the plasmalemma; and (3) lipid signaling molecules such as diacylglycerol are capable of transfer between contiguous plant cells through the ER. These observations speak to issues of plant cell autonomy for lipid synthesis and mechanisms of intercellular signaling and communication.