Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum.

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA.

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium.

Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.

Listeria monocytogenes exploits normal host cell processes to spread from cell to cell.

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1-2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.

The coronin family of actin-associated proteins.

Coronin was first isolated from Dictyostelium, but similar proteins have been identified in many species and individual cell types. The coronin-like protein in yeast promotes actin polymerization and also interacts with microtubules. Dictyostelium mutants lacking coronin are impaired in cytokinesis and all actin-mediated processes. Analysis of coronin-GFP (green-fluorescent protein) fusions and knockout mutants shows that coronin participates in the remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and macropinocytosis. Likewise, in mammalian neutrophils, a coronin-like protein is also associated with the phagocytic apparatus. The diversity of function in this family of actin-associated proteins is just beginning to be explored.

Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis.

Coronin is a ubiquitous actin-binding protein representing a member of proteins portraying a WD-repeat sequence, including the beta-subunits of trimeric G-proteins. Coronin has been suggested to participate in multiple, actin-based physiological activities such as cell movement and cell division. Although the slow growth of coronin deletion mutants has been attributed to a defect in the fluid-phase uptake of nutrients, the exact role of coronin in cytoskeletal organization has not been elucidated. In this study, we examined a role of coronin in cytokinesis by analyzing the effect of coronin deletion on the actin cytoskeleton and its dynamic distribution using a green fluorescent protein (GFP)-coronin fusion protein. We show that GFP-coronin works similarly to natural coronin in vivo and in vitro. In live cells, GFP-coronin was found to accumulate into the cleavage furrow during cytokinesis. The fluorescence pattern suggests its association to the contractile ring throughout cytokinesis. Interestingly, a substantial amount of coronin was also bound to F-actin at the prospective posterior cortex of the daughter cells. We also show that the coronin null cells reveal irregularities in organization of actin and myosin II and divide by a process identical to the traction-mediated cytofission reported in myosin II mutants. Overall, this study suggests that coronin is essential for organizing the normal actin cytoskeleton and plays a significant role in cell division.

A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.

In vitro microtubule-based organelle transport in wild-type Dictyostelium and cells overexpressing a truncated dynein heavy chain.

The transport of vesicular organelles along microtubules has been well documented in a variety of systems, but the molecular mechanisms underlying this process are not well understood. We have developed a method for preparing extracts from Dictyostelium discoideum which supports high levels of bidirectional, microtubule-based vesicle transport in vitro. This organelle transport assay was also adapted to observe specifically the motility of vesicles in the endocytic pathway. Vesicle transport can be reconstituted by recombining a high-speed supernatant with KI-washed organelles, which do not move in the absence of supernatant. Furthermore, a microtubule affinity-purified motor fraction supports robust bidirectional movement of the salt-washed organelles. The plus and minus end-directed transport activities can be separated by exploiting differences in their affinities for microtubules in the presence of 0.3 M KCl. We also used our assay to examine organelle transport in a strain of Dictyostelium overexpressing a 380-kDa C-terminal fragment of the cytoplasmic dynein heavy chain, which displays an altered microtubule pattern (380-kDa cells; [Koonce and Samso, Mol. Biol. Cell 7:935-948, 1996]). We have found that the frequency and velocity of minus end-directed membrane organelle movements were significantly reduced in 380-kDa cells relative to wild-type cells, while the frequency and velocity of plus end-directed movements were equivalent in the two cell types. The 380-kDa C-terminal fragment cosedimented with membrane organelles, although its affinity was significantly lower than that of native dynein. An impaired membrane-microtubule interaction may be responsible for the altered microtubule patterns in the 380-kDa cells.

Tdd-3, a tRNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum.

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells.

Coactosin, a 17 kDa F-actin binding protein from Dictyostelium discoideum.

A 17 kDa protein, designated as coactosin, has been purified from an actin-myosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F-actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent-insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA-derived amino-acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin-binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins.

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.

Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii produces a periplasmic arylsulfatase in response to sulfur deprivation. We have isolated and sequenced arylsulfatase cDNAs from a lambda gt11 expression library. The amino acid sequence of the protein, as deduced from the nucleotide sequence, has features characteristic of secreted proteins, including a signal sequence and putative glycosylation sites. The gene has a broad codon usage with seven codons, all having A residues in the third position, not previously observed in C. reinhardtii genes. Arylsulfatase transcription is tightly regulated by sulfur availability. The approximately 2.7 kb arylsulfatase transcript is very susceptible to degradation, disappearing in less than an hour after sulfur starved cells are administered either sulfate or alpha-amanitin. The accumulation of the arylsulfatase transcript is also suppressed by the addition of cycloheximide. Transcription initiation from the arylsulfatase gene occurs approximately 100 bp upstream of the initiation codon, in a region that is 5' to a 43 bp imperfect inverted repeat. Preceding the transcription start site are sequences similar to those present in promoter regions of other genes from C. reinhardtii.

Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii responds to sulfate deprivation by producing an arylsulfatase (Lien, T., and O. Schreiner. 1975. Biochim. Biophys. Acta. 384:168-179; Schreiner, O., 1975. Biochim. Biophys. Acta. 384:180-193) and by developing the capacity to transport sulfate more rapidly (our unpublished data). The arylsulfatase activity, detectable 3 h after the transfer of the cells to low sulfate medium (less than or equal to 10 microM sulfate), is a periplasmic protein released into the culture medium by cw15, a cell wall-less mutant of C. reinhardtii. We have purified the derepressible arylsulfatase to homogeneity and have raised monospecific antibodies to it. The protein monomer (67.6 kD) associates into a dimer, and the enzyme activity shows an alkaline pH optimum and a Km of 0.3 mM for p-nitrophenylsulfate. Studies focused on arylsulfatase biosynthesis demonstrate that it is glycosylated and synthesized as a higher molecular mass precursor. The mature protein contains complex N-linked oligosaccharides and the primary translation product has an apparent molecular mass approximately 5 kD larger than the deglycosylated monomer. Since translatable RNA encoding the arylsulfatase can only be detected in cells after sulfate starvation, it is likely that accumulation of the enzyme is regulated at the level of transcription, although posttranscriptional processes may also be involved.

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize.

A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.