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A De Lozanne

Publications and source records attributed to A De Lozanne.

At least 19 recordsLinked to original sources
PubMed2001

Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability.

The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions, the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow ingression.

Animals↗
PubMed2000

Molecular analysis of racE function in Dictyostelium.

Dictyostelium has long proven to be a valuable system for studying various aspects of the cytoskeleton and cell motility. In this review we describe the isolation of a novel gene, racE, and how we have used multiple approaches to learn how the product of this gene is involved in cytokinesis. The racEgene was isolated in a screen designed to identify genes specifically required for cytokinesis. The use of GFP fusion proteins, coupled with mutational analysis, allowed us to determine that racE exerts its function at the plasma membrane throughout the entire cell cycle. Measurements of cortical tension and observations of live cells in suspension culture revealed that racE is involved in the regulation of cortical tension and actin organization at the cortex. We postulate that in the absence of proper cortical tension, cytokinesis cannot proceed normally.

Actins↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

A role for Dictyostelium racE in cortical tension and cleavage furrow progression.

The small GTPase racE is essential for cytokinesis in Dictyostelium. We found that this requirement is restricted to cells grown in suspension. When attached to a substrate, racE null cells form an actomyosin contractile ring and complete cytokinesis normally. Nonetheless, racE null cells fail completely in cytokinesis when in suspension. To understand this conditional requirement for racE, we developed a method to observe cytokinesis in suspension. Using this approach, we found that racE null cells attempt cytokinesis in suspension by forming a contractile ring and cleavage furrow. However, the cells form multiple blebs and fail in cytokinesis by regression of the cleavage furrow. We believe this phenotype is caused by the extremely low level of cortical tension found in racE null cells compared to wild-type cells. The reduced cortical tension of racE null cells is not caused by a decrease in their content of F-actin. Instead, mitotic racE null cells contain abnormal F-actin aggregates. These results suggest that racE is essential for the organization of the cortical cytoskeleton to maintain proper cortical integrity. This function of racE is independent of attachment to a substrate, but can be bypassed by other signaling pathways induced by adhesion to a substrate.

Actins↗
PubMed1998

Identification of darlin, a Dictyostelium protein with Armadillo-like repeats that binds to small GTPases and is important for the proper aggregation of developing cells.

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.

Amino Acid Sequence↗
PubMed1997

Role of Dictyostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein.

The small GTPase racE is essential for cytokinesis in Dictyostelium but its precise role in cell division is not known. To determine the molecular mechanism of racE function, we undertook a mutational analysis of racE. The exogenous expression of either wild-type racE or a constitutively active V20racE mutant effectively rescues the cytokinesis deficiency of racE null cells. In contrast, a constitutively inactive N25racE mutant fails to rescue the cytokinesis deficiency. Thus, cytokinesis requires only the activation of racE by GTP and not the inactivation of racE by hydrolysis of GTP. To determine the spatial distribution of racE, we created a fusion protein with GFP at the amino terminus of racE. Remarkably, GFP-racE fusion protein was fully competent to rescue the phenotype of racE null cells and, therefore, must reside in the same location as native racE. We found that GFP-racE localized to the plasma membrane of the cell throughout the entire cell cycle. Furthermore, constitutively active and inactive GFP-racE fusion proteins also localized to the plasma membrane. We mapped the domain required for plasma membrane localization to the carboxyl-terminal 40 amino acids of racE. This domain, however, is not sufficient to confer racE function onto a closely related GTPase. Taken together, these results suggest that racE functions at the cell cortex but it is not involved in determining the timing or placement of the contractile ring.

Animals↗
PubMed1996

Sequences in the myosin II tail required for self-association.

The assembly of myosin II molecules into a filament requires the electrostatic interaction of a domain localized toward the carboxyl-terminus of the myosin II tail. However, the precise sequences involved in this interaction are not known. Here we show that the smallest Dictyostelium myosin II fragment that is necessary and sufficient for self-association is a fragment of 294 amino acids that contains four clusters of positively charged and negatively charged residues. Fragments of the same length but which lack one of these positively or negatively charged clusters are incapable of self-assembly. We postulate that this assembly domain is also found in myosin II from other species. Such charged clusters are found in a similar location in rabbit myosin II and are also essential for filament formation.

Animals↗
PubMed1996

Isolation and characterization of a novel cytokinesis-deficient mutant in Dictyostelium discoideum.

Cytokinesis is a dramatic event in the life of any cell during which numerous mechanisms must coordinate the legitimate and complete mechanical separation into two daughter cells. We have used Dictyostelium discoideum as a model system to study this highly orchestrated event through genetic analysis. Transformants were generated using a method of insertional mutagenesis known as restriction enzyme-mediated integration (REMI) and subsequently screened for defects in cytokinesis. Mutants isolated in a similar screen suffered a disruption in the myosin II heavy chain gene, a protein known to be essential for cytokinesis and in a novel gene encoding a rho-like protein termed racE [Larochelle et al., 1996]. In the screen reported here we isolated a third type of mutant, called 10BH2, which also had a complete defect in cytokinesis. 10BH2 mutant cells are able to propagate on tissue culture plates by fragmenting into smaller cells by a process known as traction-mediated cytofission. However, when grown in suspension culture, 10BH2 cells fail to divide and become large and multinucleate. Phenotypic characterization of the mutant cells showed that other cytoskeletal functions are preserved. The distribution of myosin and actin is identical to wild type cells. The cells can chemotax, phagocytose, cap crosslinked receptors, and contract normally. However, the 10BH2 mutants are unable to complete the Dictyostelium developmental program beyond the finger stage. The mutant cells contain functional genes for myosin II heavy and light chains and the racE gene. Thus, based on these findings, we conclude that 10BH2 represents a novel cytokinesis-deficient mutant.

Actins↗
PubMed1996

A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis.

Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein, specifically in cytokinesis, and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle.

Actins↗
PubMed1995

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Amino Acid Sequence↗
PubMed1995

Expression of light meromyosin in Dictyostelium blocks normal myosin II function.

The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.

Amino Acid Sequence↗
PubMed1990

Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.

To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.

Animals↗
PubMed1989

Gene replacement in Dictyostelium: generation of myosin null mutants.

The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5' coding sequence and 0.9 kb of 3' coding sequence or 1.5 kb of 5' flanking sequence and 1.1 kb of 3' flanking sequence. Myosin null mutants (mhcA- cells) were obtained after transformation with either of these plasmids. The mhcA- cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.

Dictyostelium↗
PubMed1989

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination.

A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL. couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.

Dictyostelium↗
PubMed1988

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain.

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.

Actins↗
PubMed1988

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain.

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.

Actins↗
PubMed1987

Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.

The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.

Cell Division↗