Intra-bundle contractions enable extensile properties of active actin networks.

The cellular cortex is a dynamic and contractile actomyosin network modulated by actin-binding proteins. We reconstituted a minimal cortex adhered to a model cell membrane mimicking two processes mediated by the motor protein myosin: contractility and high turnover of actin monomers. Myosin reorganized these networks by extensile intra­bundle contractions leading to an altered growth mechanism. Hereby, stress within tethered bundles induced nicking of filaments followed by repair via incorporation of free monomers. This mechanism was able to break the symmetry of the previously disordered network resulting in the generation of extensile clusters, reminiscent of structures found within cells.

Filopodia formation induced by active mDia2/Drf3.

Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.

Two-step positioning of a cleavage furrow by cortexillin and myosin II.

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.

DdLIM is a cytoskeleton-associated protein involved in the protrusion of lamellipodia in Dictyostelium.

DdLim, a multi-domain member of the cysteine-rich family of LIM domain proteins, was isolated from Dictyostelium cells where it localizes in lamellipodia and at sites of membrane ruffling. The transcription and expression of DdLim are developmentally regulated, and the timing of its increased association with the actin cytoskeleton coincides with the acquisition in starved cells of a motile, chemotactic behavior. Vegetative cells that overexpress DdLim contain large lamella and exhibit ruffling at the cortex. The high frequency of large, multinucleated mutant cells found in suspension culture suggests that excess DdLim interferes with cytokinesis. DdLim was also identified as a protein in a Dictyostelium cell lysate that associated indirectly, but in a guanosine triphosphate-dependent manner, with a GST-rac1 fusion protein. The data presented suggest that DdLim acts as an adapter protein at the cytoskeleton-membrane interface where it is involved in a receptor-mediated rac1-signaling pathway that leads to actin polymerization in lamellipodia and ultimately cell motility.

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum.

A protein accumulated in the cortical region of Dictyostelium discoideum cells proved to be a homologue of GTPase activating proteins that are responsible for the inactivation of ras in yeast and man. Elimination of this protein, DGAP1, by gene replacement resulted in an increased rate of growth of D. discoideum cells on bacterial lawns, and in the formation of aberrant, multi-tipped fruiting bodies. Overexpression of DGAP1 caused the cells to become multi-nucleated since chromosome segregation during mitosis was not reliably followed by cleavage of the cells. These results suggest that in D. discoideum, ras or a related small GTP-binding protein is involved in regulating growth based on the phagocytosis of bacteria, and in coupling activities of the cell cortex to the organization of spindle and asters in mitotic cells.

Cytoskeletal protein mutations and cell motility in Dictyostelium.

Dictyostelium is a suitable experimental system in which to study the effects of mutations in actin-binding proteins on cell motility. Three cytoskeletal mutants that show distinct alterations in cell shape, chemotactic movement and cytokinesis serve to illustrate the diversity of phenotypes. Cells lacking talin, a protein which in many mammalian cell types is a constituent of focal complexes that link the actin cytoskeleton to the plasma membrane, are strongly impaired in adhesion to external surfaces. Coronin is an actin-associated protein that belongs to the WD-repeat family of proteins, which are engaged in protein-protein interactions involved in signalling pathways. Cells lacking coronin build large hyaline protrusions at their leading edge, diagnostic of an imbalance in the actin polymerization/depolymerization cycle. Cells devoid of a pair of cortexillins, which are novel members of the spectrin/alpha-actinin superfamily of actin-binding proteins, form an atypical cleavage furrow on a solid surface and fail to divide in suspension. Other mutants in which one or more actin-binding proteins have been knocked out have weaker phenotypes. With these mutants, cells need to be subjected to special conditions in order to reveal an effect on cell motility. For instance, only on weakly adhesive surfaces is a disturbance in the spatio-temporal co-ordination of protrusion and retraction of the cell body, and of the attachment to and detachment from a substratum, observed in a mutant that lacks three actin-binding proteins: alpha-actinin, 120 kDa F-actin gelation factor and severin.

Maternal thyroid deficiency and pregnancy complications: implications for population screening.

OBJECTIVE: To examine the relation between certain pregnancy complications and thyroid stimulating hormone (TSH) measurements in a cohort of pregnant women. METHODS: TSH was measured in sera obtained from women during the second trimester as part of routine prenatal care. Information was then collected about vaginal bleeding, premature delivery, low birthweight, abruptio placentae, pregnancy induced hypertension, need for cesarean section, low Apgar scores, and fetal and neonatal death. RESULTS: Among 9403 women with singleton pregnancies, TSH measurements were 6 mU/l or greater in 209 (2.2%). The rate of fetal death was significantly higher in those pregnancies (3.8%) than in the women with TSH less than 6 mU/l (0.9%, odds ratio 4.4, 95% confidence interval 1.9-9.5). Other pregnancy complications did not occur more frequently. CONCLUSION: From the second trimester onward, the major adverse obstetrical outcome associated with raised TSH in the general population is an increased rate of fetal death. If thyroid replacement treatment avoided this problem this would be another reason to consider population screening.

Rapid blood separation is superior to fluoride for preventing in vitro reductions in measured blood glucose concentration.

AIMS: To determine whether tubes containing sodium fluoride negatively bias blood glucose concentration by directly comparing glucose concentrations in paired blood samples collected in tubes containing lithium heparin (Li-Heparin) and tubes containing sodium fluoride/potassium oxalate (NaF-KOx). METHODS: Paired blood samples from a group of patients (n = 1040) were collected in tubes containing Li-Heparin and tubes containing NaF-KOx at the same time. All Li-Heparin samples were centrifuged soon after collection and were kept cool in transport along with NaF-KOx samples, which were centrifuged at the receiving location after an average transport time of 4 h, but immediately before analysis. Glucose concentrations in the paired samples were determined simultaneously by an automated oxidase method. RESULTS: The mean glucose concentrations for NaF-KOx samples and Li-Heparin samples were 5.7 mmol/l and 6.1 mmol/l, respectively, with a mean difference of 0.39 mmol/l. CONCLUSION: Rapid separation of heparinised blood is superior to fluoride alone for abrogating glycolytic effects on blood glucose measurements in the clinical laboratory.

Formins and VASPs may co-operate in the formation of filopodia.

Filopodia are finger-like cell protrusions composed of parallel arrays of actin filaments, which elongate through actin polymerization at their tips. These highly dynamic structures seem to be used by many cell types as sensing organs to explore environmental cues and have been implicated in cell motility as well as in cell-substrate adhesion. Formins are highly conserved multidomain proteins that play important roles in the nucleation of actin and the formation of linear actin filaments, yet their role in filopodia formation has remained poorly defined. The Dictyostelium diaphanous-related formin dDia2 is strongly enriched in filopodia tips. Genetic and biochemical analysis revealed that this protein is important for cell migration and cell adhesion, but most importantly for the formation of filopodia. Recently, we have identified the Dictyostelium VASP (vasodilator-stimulated phosphoprotein) orthologue as a binding partner of dDia2 and provide evidence for a co-operative role of both proteins in filopodia formation.

The actin-bundling protein cortexillin is the downstream target of a Rac1-signaling pathway required for cytokinesis.

During the process of cytokinesis by which eukaryotic cells constrict and divide in two, multiple cellular activities have to be precisely coordinated in space and time to guarantee equal distribution of chromosomes and cytoplasm to the emerging daughter cells. Eventually, constriction of the cleavage furrow leads to the complete separation of the daughter cells. Since the basic observation of cell division some 100 years ago, the principal challenge has been to unravel the detailed molecular mechanisms and signaling events leading to cytokinesis. Regulation of this fundamental cellular process is still poorly understood yet a central issue in modern cell biology. In the recent past it became evident that small GTPases of the Ras super family play a major role during this process. This review is focused on a Rho family GTPase-mediated signaling pathway that is required for cleavage furrow assembly and cytokinesis by the actin-bundling protein cortexillin of D. discoideum cells.

Constitutive overexpression of the contact site A glycoprotein enables growth-phase cells of Dictyostelium discoideum to aggregate.

The contact site A (csA) glycoprotein is a developmentally regulated cell adhesion molecule which mediates EDTA-stable cell contacts during the aggregation stage of Dictyostelium discoideum. A transformation vector was constructed which allows overexpression of the csA protein during the growth phase. In that stage the csA protein is normally not expressed; in the transformants it was transported to the cell surface and carried all modifications investigated, including a phospholipid anchor and two types of oligosaccharide chain. csA expression enabled the normal non-aggregative growth-phase cells to form EDTA-stable contacts in suspension and to assemble into three-dimensional aggregates when moving on a substratum. After prolonged cultivation of csA overexpressing transformants in nutrient medium the developmental program was found to be turned on, as it normally occurs only in starving cells. During later development of transformed cells, the csA glycoprotein remained present on the cell surface, while it is down-regulated in the wild type. It was detected in both the prestalk and prespore regions of the multicellular slugs made from transformed cells.

Overexpression of the csA cell adhesion molecule under its own cAMP-regulated promoter impairs morphogenesis in Dictyostelium.

The contact site A (csA) glycoprotein is a strictly developmentally regulated plasma membrane component responsible for the EDTA-stable (Ca(2+)-independent) form of intercellular adhesion in Dictyostelium discoideum. Using inverse polymerase chain reaction and a terminator vector we have isolated a 1.6 kb genomic fragment carrying a 1.1 kb upstream region of the csA gene. This fragment had promoter activity in D. discoideum cells, giving rise to a 3'-truncated csA RNA that was regulated like the mRNA of the endogenous gene. Cyclic AMP pulses strongly enhanced transcription from the cloned csA promoter. These findings provide evidence that the cloned region of the csA gene comprises the complete promoter. It contains a G/C-rich octamer motif similar to other cAMP-regulated D. discoideum promoters. When the csA protein was strongly overexpressed under the developmental control of the csA promoter, morphogenesis was substantially altered. Aggregation was delayed, and secondary centres were formed along aggregation streams that led to fragmentation of the aggregates and multiple slug formation. At high cell density a substantial portion of aggregated cells was left behind on the substratum when slugs and fruiting bodies were built. The transformation vector was also employed to rescue a csA-negative mutant, HG1287, from its cell adhesion defect.

Antibody enhances lymphocyte proliferation in guinea pigs immunized to express cutaneous basophil hypersensitivity.

Lymphocytes from HSA-sensitized guinea pigs expressing cutaneous basophil hypersensitivity (CBH) proliferate in the presence of specific antigen. We report that this proliferative response is enhanced by the addition of anti-HSA antibody, either in the form of whole immune serum or as purified antibody. The enhancement was characterized as a marked shift of the antigen dose-response curve such that significant [3H]thymidine incorporation was observed at antigen concentrations much lower than those eliciting a comparable response in the absence of immune serum. Enhancement was antigen specific and required an intact immunoglobulin molecule. Antibodies capable of enhancing antigen-specific lymphocyte proliferation could be isolated from serum by affinity chromatography as early as 7 days after sensitization and were also evident in sera obtained at later intervals. It is unlikely that such antibodies account for the progressive decline of CBH reactivity and they may actually influence its initial expression.

Detection of subtle phenotypes: the case of the cell adhesion molecule csA in Dictyostelium.

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.

Indirect estimation of thyroid hormone-binding proteins to calculate free thyroxine index: comparison of nonisotopic methods that use labeled thyroxine ("T-uptake")

There are many alternative ways of estimating free thyroxine (T4) when thyrotropin screening results are abnormal. In addition to free T4 immunoassays, the menu of most automated immunoassay instruments includes a nonisotopic version of the original triiodothyronine (T3)-uptake assay called "T-uptake." We evaluated the ability of five such assays (Access, ES-300, IMx, Magnum Opus, and Stratus) to accurately estimate the free thyroxine index (FTI) in euthyroid, hyperthyroid, and hypothyroid patients with abnormal concentrations of thyroid hormone-binding proteins, and in patients with nonthyroidal illness. For comparison, we calculated a similar FTI, using either T3-uptake or direct measurement of thyroxine-binding globulin (TBG). Euthyroid reference ranges were comparable. Of euthyroid patients with increased TBG, 12-32% and 5-20% had increased or suppressed FTI, respectively, depending on the T-uptake method used. Except for IMx, 6-35% of hypothyroid patients with increased TBG had inappropriately increased FTI. Patients with nonthyroidal illness had comparable results regardless of the method used, and T-uptake methods were variably affected by known inhibitors of thyroid hormone binding. The most reliable T-uptake method appeared to be the IMx, which, despite claims that it measures all thyroid hormone-binding proteins, correlated best with TBG concentrations.

Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.

A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. The homophilic cell adhesion molecule contact site A, a lipid-anchored glycoprotein from cells of the slime mold Dictyostelium discoideum, is used as receptor. The success of the reconstitution, the structure and the dynamics of the model membranes are studied by various techniques including film balance techniques, micro fluorescence, fluorescence recovery after photobleaching, electron microscopy, and phase contrast microscopy. The interaction of the functionalized giant vesicles with the supported bilayer is studied by reflection interference contrast microscopy, and the adhesion strength is evaluated quantitatively by a recently developed technique. At low receptor concentrations adhesion-induced receptor segregation in the membranes leads to decomposition of the contact zone between membranes into domains of strong (receptor-mediated) adhesion and regions of weak adhesion while continuous zones of strong adhesion form at high receptor densities. The adhesion strengths (measured in terms of the spreading pressure S) of the various states of adhesion are obtained locally by analysis of the vesicle contour near the contact line in terms of elastic boundary conditions of adhesion: the balance of tensions and moments. The spreading pressure of the weak adhesion zones is S approximately 10(-9) J/m(2) and is determined by the interplay of gravitation and undulation forces whereas the spreading pressure of the tight adhesion domains is of the order S approximately 10(-6) J/m(2).

Epithelioid sarcoma:radiologic and pathologic manifestations.

The radiologic and pathologic manifestations of epithelioid sarcoma are presented based on an analysis of five cases, and the literature is reviewed. This rare entity tends to orginate in the extremities and metastasizes primarily via the lymphatics. Although the lesion grows slowly, it recurs with high frequency. Males are affected three times as often as females. The differential diagnosis includes both malignant and benign entities. The natural history and methods of treatment are reported.