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R L Chisholm

Publications and source records attributed to R L Chisholm.

At least 19 recordsLinked to original sources
PubMed2005

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

ATP-Binding Cassette Transporters↗
PubMed2001

During multicellular migration, myosin ii serves a structural role independent of its motor function.

We have shown previously that cells lacking myosin II are impaired in multicellular motility. We now extend these results by determining whether myosin contractile function is necessary for normal multicellular motility and shape control. Myosin from mutants lacking the essential (mlcE(-)) myosin light chain retains the ability to form bipolar filaments that bind actin, but shows no measurable in vitro or in vivo contractile function. The contractile function is necessary for cell shape control since mlcE(-) cells, like myosin heavy-chain null mutants (mhcA(-)), were defective in their ability to control their three-dimensional shape. When mixed with wild-type cells in chimeric aggregation streams, the mlcE(-) cells were able to move normally, unlike mhcA(-) cells which accumulated at the edges of the stream and became distorted by their interactions with wild-type cells. When mhcA(-) cells were mixed with mlcE(-) streams, the mhcA(-) cells were excluded. The normal behavior of the mlcE(-) cells in this assay suggests that myosin II, in the absence of motor function, is sufficient to allow movement in this constrained, multicellular environment. We hypothesize that myosin II is a major contributor to cortical integrity even in the absence of contractile function.

Animals↗
PubMed2001

Molecular motors and membrane traffic in Dictyostelium.

Phagocytosis and membrane traffic in general are largely dependent on the cytoskeleton and their associated molecular motors. The myosin family of motors, especially the unconventional myosins, interact with the actin cortex to facilitate the internalization of external materials during the early steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate transport along microtubules (MTs), facilitate the intracellular processing of the internalized materials and the movement of membrane. Recent studies indicate that some unconventional myosins are also involved in membrane transport, and that the MT- and actin-dependent transport systems might interact with each other. Studies in Dictyostelium have led to the discovery of many motors involved in critical steps of phagocytosis and membrane transport. With the ease of genetic and biochemical approaches, the established functional analysis to test phagocytosis and vesicle transport, and the effort of the Dictyostelium cDNA and Genome Projects, Dictyostelium will continue to be a superb model system to study phagocytosis in particular and cytoskeleton and motors in general.

Actins↗
PubMed2000

Transrectal ultrasound assessment of murine aorta and iliac arteries.

Recent research in arterial aneurysm formation has focused on animal model development. Mice are an ideal experimental organism due to their short life cycle, prolific progeny, and extensively studied genome. Most experiments require the sacrifice of the mice to observe and assess any morphological changes. Noninvasive or minimally invasive imaging is limited due to the relatively small size of the structures. The development of such a technique, therefore, is especially useful for allowing repeated measurement without sacrificing the mice. We introduce a novel technique of imaging and measuring the aorta, the aorta/inferior vena cava complex, and the right and the left common iliac artery/vein complex by the use of an intravascular ultrasound catheter. The catheter is inserted through the anus and rectum and into the sigmoid and left colon, where the aorta can be observed to fluctuate at approximately 500 beats/min. The aortic bifurcation can also be observed. The diameters of the aorta and the inferior vena cava were measured first with the transrectal ultrasound technique and then with direct visualization upon laparotomy for 10 mice. This revealed a percentage error between 13.7 and 14.2% for this novel technique. Fifteen more sets of vessel measurements were also made with 8 male and 7 female mice. The results demonstrated a correlation between vessel size and body weight in male but not female mice and suggested an intersex difference in vessel growth rate. We conclude that transrectal ultrasound is a useful tool in imaging and measuring the murine aorta and its bifurcation.

Animals↗
PubMed2000

Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

Animals↗
PubMed1999

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium.

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

Animals↗
PubMed1999

Interaction mapping of a dynein heavy chain. Identification of dimerization and intermediate-chain binding domains.

Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an approximately 150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed.

Animals↗
PubMed1999

Regulation of cytokinesis.

At the end of mitosis, daughter cells are separated from each other by cytokinesis. This process involves equal partitioning and segregation of cytoplasm between the two cells. Despite years of study, the mechanism driving cytokinesis in animal cells is not fully understood. Actin and myosin are major components of the contractile ring, the structure at the equator between the dividing cells that provides the force necessary to constrict the cytoplasm. Despite this, there are also tantalizing results suggesting that cytokinesis can occur in the absence of myosin. It is unclear what the roles are of the few other contractile ring components identified to date. While it has been difficult to identify important proteins involved in cytokinesis, it has been even more challenging to pinpoint the regulatory mechanisms that govern this vital process. Cytokinesis must be precisely controlled both spatially and temporally; potential regulators of these parameters are just beginning to be identified. This review discusses the recent progress in our understanding of cytokinesis in animal cells and the mechanisms that may regulate it.

Actins↗
PubMed1999

Expression of chicken gizzard RLC complements the cytokinesis and developmental defects of Dictyostelium RLC null cells.

Dictyostelium RLC null cells have defects in cytokinesis and development that can be rescued by expression of either the wild type Dictyostelium RLC or an RLC mutant that cannot be phosphorylated by MLCK (S13A) (Ostrow et al., 1994). The wild type and S13A mutant LCs rescued the cells equally well, despite the fact that RLC phosphorylation increases purified Dictyostelium myosin's activity 5-fold. In this report, we assess the ability of foreign RLCs to rescue the RLC null phenotype. The RLC from smooth muscle myosin, whose activity is tightly controlled by phosphorylation, rescued the null cell phenotype. The purified hybrid myosin had an activity and motility comparable to phosphorylated Dictyostelium myosin. In contrast, cells expressing skeletal muscle RLC were deficient in cytokinesis and development, despite having an activity and motility similar to that of myosin with the unposphorylatable S13A mutant RLC. Neither foreign LC was phosphorylated when expressed in Dictyostelium. These results suggest that the level of actin-activated ATPase activity and motility is not the sole determinant of proper myosin function in vivo. Other heavy chain/light chain interactions, which occur only with the native RLC and smooth muscle RLC, appear to be necessary for optimal function.

Amino Acid Sequence↗
PubMed1999

Regulatory light chain mutations affect myosin motor function and kinetics.

The actin-based motor protein myosin II plays a critical role in many cellular processes in both muscle and non-muscle cells. Targeted disruption of the Dictyostelium regulatory light chain (RLC) caused defects in cytokinesis and multicellular morphogenesis. In contrast, a myosin heavy chain mutant lacking the RLC binding site, and therefore bound RLC, showed normal cytokinesis and development. One interpretation of these apparently contradictory results is that the phenotypic defects in the RLC null mutant results from mislocalization of myosin caused by aggregation of RLC null myosin. To distinguish this from the alternative explanation that the RLC can directly influence myosin activity, we expressed three RLC point mutations (E12T, G18K and N94A) in a Dictyostelium RLC null mutant. The position of these mutations corresponds to the position of mutations that have been shown to result in familial hypertrophic cardiomyopathy in humans. Analysis of purified Dictyostelium myosin showed that while these mutations did not affect binding of the RLC to the MHC, its phosphorylation by myosin light chain kinase or regulation of its activity by phosphorylation, they resulted in decreased myosin function. All three mutants showed impaired cytokinesis in suspension, and one produced defective fruiting bodies with short stalks and decreased spore formation. The abnormal myosin localization seen in the RLC null mutant was restored to wild-type localization by expression of all three RLC mutants. Although two of the mutant myosins had wild-type actin-activated ATPase, they produced in vitro motility rates half that of wild type. N94A myosin showed a fivefold decrease in actin-ATPase and a similar decrease in the rate at which it moved actin in vitro. These results indicate that the RLC can play a direct role in determining the force transmission and kinetic properties of myosin.

Animals↗
PubMed1998

Cell-type-specific rescue of myosin function during Dictyostelium development defines two distinct cell movements required for culmination.

Mutant Dictyostelium cells lacking any of the component polypeptides of myosin II exhibit developmental defects. To define myosin's role in establishing Dictyostelium's developmental pattern, we have rescued myosin function in a myosin regulatory light chain null mutant (mlcR-) using cell-type-specific promoters. While mlcR- cells fail to progress beyond the mound stage, expression of RLC from the prestalk promoter, ecmA, produces culminants with normal stalks but with defects in spore cell localization. When GFP-marked prestalk and prespore cells expressing ecmA-RLC are mixed with wild-type cells, the mislocalization of prestalk cells, but not prespore cells, is rescued. Time-lapse video recording of ecmA-RLC cells showed that the posterior prespore zone failed to undergo a contraction important for the upward movement of prespore cells. Prespore cells marked with green fluorescent protein (GFP) failed to move toward the tip with the spiral motion typical of wild type. In contrast, expression of RLC in prespore cells using the psA promoter produced balloon-like structures reminiscent of sorocarps but lacking stalks. GFP-labeled prespore cells showed a spiral movement toward the top of the structures. Expression of RLC from the psA promoter restores the normal localization of psA-GFP cells, but not ecmA-GFP cells. These results define two distinct, myosin-dependent movements that are required for establishing a Dictyostelium fruiting body: stalk extension and active movement of the prespore zone that ensures proper placement of the spores atop the stalk. The approach used in these studies provides a direct means of testing the role of cell motility in distinct cell types during a morphogenetic program.

Animals↗
PubMed1997

Substitution mutations in the myosin essential light chain lead to reduced actin-activated ATPase activity despite stoichiometric binding to the heavy chain.

Myosin essential light chain (ELC) wraps around an alpha-helix that extends from the myosin head, where it is believed to play a structural support role. To identify other role(s) of the ELC in myosin function, we have used an alanine scanning mutagenesis approach to convert charged residues in loops I, II, III, and helix G of the Dictyostelium ELC into uncharged alanines. Dictyostelium was used as a host system to study the phenotypic and biochemical consequences associated with the mutations. The ELC carrying loop mutations bound with normal stoichiometry to the myosin heavy chain when expressed in ELC-minus cells. When expressed in wild type cells these mutants competed efficiently with the endogenous ELC for binding, suggesting that the affinity of their interaction with the heavy chain is comparable to that of wild type. However, despite apparently normal association of ELC the cells still exhibited a reduced efficiency to undergo cytokinesis in suspension. Myosin purified from these cells exhibited 4-5-fold reduction in actin-activated ATPase activity and a decrease in motor function as assessed by an in vitro motility assay. These results suggest that the ELC contributes to myosin's enzymatic activity in addition to providing structural support for the alpha-helical neck region of myosin heavy chain.

Actins↗
PubMed1995

Both the amino and carboxyl termini of Dictyostelium myosin essential light chain are required for binding to myosin heavy chain.

Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951-962). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH2-terminal 11 or 28 amino acid residues (delta N11 or delta N28) or the COOH-terminal 15 amino acid residues (delta C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE-) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, delta N28 and delta N11 bound to the MHC at 15% of WT levels and delta C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE- cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.

Animals↗
PubMed1995

Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis.

We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7-11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE- mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE- mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7-11 cells. This phenotype was rescued by introducing a construct that expressed the wild-type Dictyostelium ELC cDNA. Myosin purified from the mlcE- cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE- mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotactic defect of mlcE- cells. We have shown that while mlcE- cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE- cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin.

Animals↗
PubMed1994

Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment et al., 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987; Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild-type myosin, but the filaments then exhibit abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.

Actins↗