Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.     

A cytokine-selective defect in interleukin-1 beta-mediated acute phase gene expression in a subclone of the human hepatoma cell line (HEPG2)

Several well-differentiated human hepatoma cell lines (HepG2, Hep3B) have been used to identify factors which regulate hepatic gene expression during the host response to inflammation/tissue injury (acute phase response). Studies in these cell lines, as well as in primary cultures of rat, rabbit, and mouse hepatocytes, have demonstrated that interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interferon-beta 2 (IFN-beta 2) each mediate changes in expression of several hepatic acute phase genes. In this study we identify a subclone of the HepG2 cell line in which there is a selective defect in IL-1 beta-mediated acute phase gene expression. Recombinant human IL-1 beta mediates an increase in synthesis of the positive acute phase complement protein factor B and a decrease in synthesis of negative acute phase protein albumin in the parent uncloned HepG2 cell line (HG2Y), but not in the subclone HG2N. Recombinant human IFN-beta 2 and TNF-alpha, however, regulate acute phase protein synthesis in the subclone HG2N; i.e. IFN-beta 2 and TNF-alpha increase synthesis of factor B and decrease synthesis of albumin in both HG2Y and HG2N cells. Equilibrium binding analysis with 125I-rIL-1 beta at 4 degrees C showed that both HG2N and HG2Y cells bind IL-1 beta specifically and saturably. HG2N and HG2Y possess 3.8 and 4.0 x 10(3) plasma membrane receptors/cell with affinities of 0.96 and 1.07 x 10(-9) M, respectively. Thus, the defect in this subclone of the HepG2 cell line is likely to involve the signal transduction pathway for the biological activity of IL-1 beta and will be useful in elucidation of this signal transduction pathway.     

LvsA, a protein related to the mouse beige protein, is required for cytokinesis in Dictyostelium

We isolated a Dictyostelium cytokinesis mutant with a defect in a novel locus called large volume sphere A (lvsA). lvsA mutants exhibit an unusual phenotype when attempting to undergo cytokinesis in suspension culture. Early in cytokinesis, they initiate furrow formation with concomitant myosin II localization at the cleavage furrow. However, the furrow is later disrupted by a bulge that forms in the middle of the cell. This bulge is bounded by furrows on both sides, which are often enriched in myosin II. The bulge can increase and decrease in size multiple times as the cell attempts to divide. Interestingly, this phenotype is similar to the cytokinesis failure of Dictyostelium clathrin heavy-chain mutants. Furthermore, both cell lines cap ConA receptors but form only a C-shaped loose cap. Unlike clathrin mutants, lvsA mutants are not defective in endocytosis or development. The LvsA protein shares several domains in common with the molecules beige and Chediak-Higashi syndrome proteins that are important for lysosomal membrane traffic. Thus, on the basis of the sequence analysis of the LvsA protein and the phenotype of the lvsA mutants, we postulate that LvsA plays an important role in a membrane-processing pathway that is essential for cytokinesis.     

A two-component histidine kinase gene that functions in Dictyostelium development.

A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration. The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis. In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.     

A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome.

A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.     

An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast

Background: Proteins of the IQGAP family have been identified as candidate effectors for the Rho family of GTPases; however, little is known about their cellular functions. The domain structures of IQGAP family members make them excellent candidates as regulators of the cytoskeleton: their sequences include an actin-binding domain homologous to that found in calponin, IQ motifs for interaction with calmodulin, and a GTPase-binding domain.Results: The genomic sequence of Saccharomyces cerevisiae revealed a single gene encoding an IQGAP family member (denoted IQGAP-related protein: Iqg1). Iqg1 and IQGAPs share similarity along their entire length, with an amino-terminal calponin-homology (CH) domain, IQ repeats, and a conserved carboxyl terminus. In contrast to IQGAPs, Iqg1 lacks an identifiable GAP motif, a WW domain, and IR repeats, although the functions of these domains in IQGAPs are not well defined. Deletion of the IQG1 gene resulted in lethality. Cellular defects included a deficiency in cytokinesis, altered actin organization, aberrant nuclear segregation, and cell lysis. The primary defect appeared to be a cytokinesis defect, and the other problems possibly arose as a consequence of this initial defect. Consistent with a role in cytokinesis, Iqg1 co-localizes with an actin ring encircling the mother–bud neck late in the cell cycle –a putative cytokinetic ring. IQG1 overexpression resulted in premature actin-ring formation, suggesting that Iqg1 activity temporally controls formation of this structure during the cell cycle.Conclusions: Yeast IQGAP-related protein, Iqg1, is an important regulator of cellular morphogenesis, inducing actin-ring formation in association with cytokinesis.     

Mutant Rac1B expression in Dictyostelium: effects on morphology, growth, endocytosis, development, and the actin cytoskeleton

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.     

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.     

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

SadA,a novel adhesion receptor in Dictyostelium

Little is known about cell-substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion-deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA-, completely lacked substrate adhesion in growth medium. The sadA- mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA-GFP in sadA-null cells restored the wild-type phenotype. In sadA-GFP-rescued mutant cells, sadA-GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium.     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Screening of genes involved in cell migration in Dictyostelium

A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration.     

Different extracellular domains of the neural cell adhesion molecule (N- CAM) are involved in different functions

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I- II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig- like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig- like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type III repeats I-II. Intracellular concentrations of Ca2+ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures.     

Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.     

Remi-RFLP Mapping in the Dictyostelium Genome

A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzymemediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.     

连续3次缺口修复构建小鼠乳清酸蛋白-人血清白蛋白杂合基因座

目的:构建人血清白蛋白(hSA)与小鼠乳清酸蛋白(mWAP)调控序列的杂合基因座。方法与结果:在pBR322载体上连入预先无痕连接的3对同源臂,利用基于Red同源重组系统的缺口修复(gap-repair)技术,分别对8kb的mWAP基因座3′调控区、16 kb的hSA基因组编码序列及13 kb的mWAP基因座5′调控区进行连续3次基因抓捕,最终将全长37 kb的乳蛋白杂合基因座构入pBR322载体;通过PCR扩增、限制性内切酶消化和序列测定验证,确定mWAP基因座中的编码序列被精确地置换为hSA的基因组编码序列。结论:构建了整合有mWAP-hSA杂合基因座的pBR322载体,为研究杂合基因座在乳腺中的表达效果及置换型基因座表达的可行性提供了数据。     

肿瘤抑制因子CYLD对细胞功能的调控作用

肿瘤抑制因子（cylindromatosis,CYLD）是一种在体内广泛分布的去泛素酶,其包含去泛素化酶结构域和富含甘氨酸细胞骨架相关蛋白结构域,前者可通过去泛素化信号分子,调控细胞信号传导途径,后者主要通过对微管的调节,改变多聚化和乙酰化过程,进而调控其组装和排列。CYLD主要通过对信号传导和细胞骨架的调节,从而调控细胞增殖、细胞凋亡、细胞运动和细胞分化等细胞功能。该文对近年来肿瘤抑制因子CYLD对细胞功能调控的研究进行概述。     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.