Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.     

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.     

Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion

Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.     

Nup358 integrates nuclear envelope breakdown with kinetochore assembly

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.     

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.     

Abnormal Methylation of Several Tumor Suppressor Genes in Sporadic Breast Cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the R1, p16/CDKN2, p15/CDKN2, p14/ARF, DH1, MGMT, HIC1, and N33 promoter regions in breast cancer (105 tumors). Methylation was often observed for the two major suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, R1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.     

Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis

Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.     

Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes

A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.     

Thoughts on thiolate tethering. Tribute and thanks to a teacher

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.