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Biomedical subjects

M P Koonce

Publications and source records attributed to M P Koonce.

At least 19 recordsLinked to original sources
PubMed1998

Structural characterization of a dynein motor domain.

Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule's minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of seven to eight globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin.

Adenosine Triphosphate↗
PubMed1998

Cytoplasmic dynein heavy chain is an essential gene product in Dictyostelium.

We describe here three different approaches to perturb cytoplasmic dynein heavy chain (DHC) gene function in Dictyostelium: integration of a marker into the heavy chain coding sequence by homologous recombination to disrupt transcription, expression of antisense RNA to inhibit translation, and expression of a 158 kDa amino-terminal coding region to perturb the native protein organization. By homologous recombination, we fail to obtain cells that lack an intact DHC gene product. Cells containing antisense orientation plasmids (but not sense) appear to die 4 to 6 days following transformation. Plasmids designed to overexpress an amino-terminal region of the DHC result in substantially reduced transformation efficiency. When expressed at low levels, the truncated amino-terminal product appears capable of dimerizing with an intact heavy chain or with itself, essentially producing a cargo-binding domain lacking mechanochemical activity. This, in turn, likely competes with the native protein's function. These three approaches taken together indicate that the dynein heavy chain is an essential gene in Dictyostelium.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

A specific light chain of kinesin associates with mitochondria in cultured cells.

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Amino Acid Sequence↗
PubMed1997

Identification of a microtubule-binding domain in a cytoplasmic dynein heavy chain.

As a molecular motor, dynein must coordinate ATP hydrolysis with conformational changes that lead to processive interactions with a microtubule and generate force. To understand how these processes occur, we have begun to map functional domains of a dynein heavy chain from Dictyostelium. The carboxyl-terminal 10-kilobase region of the heavy chain encodes a 380-kDa polypeptide that approximates the globular head domain. Attempts to further truncate this region fail to produce polypeptides that either bind microtubules or UV-vanadate cleave, indicating that the entire 10-kilobase fragment is necessary to produce a properly folded functional dynein head. We have further identified a region just downstream from the fourth P-loop that appears to constitute at least part of the microtubule-binding domain (amino acids 3182-3818). When deleted, the resulting head domain polypeptide no longer binds microtubules; when the excised region is expressed in vitro, it cosediments with added tubulin polymer. This microtubule-binding domain falls within an area of the molecule predicted to form extended alpha-helices. At least four discrete sites appear to coordinate activities required to bind the tubulin polymer, indicating that the interaction of dynein with microtubules is complex.

Adenosine Triphosphate↗
PubMed1997

SSP1, a gene necessary for proper completion of meiotic divisions and spore formation in Saccharomyces cerevisiae.

During meiosis, a diploid cell undergoes two rounds of nuclear division following one round of DNA replication to produce four haploid gametes. In yeast, haploid meiotic products are packaged into spores. To gain new insights into meiotic development and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Our results indicate that there are at least five different classes of transcripts representing genes expressed at different stages of the sporulation program. Here we describe one of these differentially expressed genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is expressed midway through meiosis, and homozygous ssp1 diploid cells fail to sporulate. In the ssp1 mutant, meiotic recombination is normal but viability declines rapidly. Both meiotic divisions occur at the normal time; however, the fraction of cells completing meiosis is significantly reduced, and nuclei become fragmented soon after meiosis II. The ssp1 defect does not appear to be related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of chromosome segregation. The data suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation.

Amino Acid Sequence↗
PubMed1996

Overexpression of cytoplasmic dynein's globular head causes a collapse of the interphase microtubule network in Dictyostelium.

Cytoplasmic dynein is a minus-end directed microtubule-based motor. Using a molecular genetic approach, we have begun to dissect structure-function relationships of dynein in the cellular slime mold Dictyostelium. Expression of a carboxy-terminal 380-kDa fragment of the heavy chain produces a protein that approximates the size and shape of the globular, mechanochemical head of dynein. This polypeptide cosediments with microtubules in an ATP-sensitive fashion and undergoes a UV-vanadate cleavage reaction. The deleted amino-terminal region appears to participate in dimerization of the native protein and in binding the intermediate and light chains. Overexpression of the 380-kDa carboxy-terminal construct in Dictyostelium produces a distinct phenotype in which the interphase radial microtubule array appears collapsed. In many cells, the microtubules form loose bundles that are whorled around the nucleus. Similar expression of a central 107-kDa fragment of the heavy chain does not produce this result. The data presented here suggest that dynein may participate in maintaining the spatial pattern of the interphase microtubule network.

Animals↗
PubMed1993

Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

Amino Acid Sequence↗
PubMed1992

Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.

Adenosine Triphosphate↗
PubMed1990

Identification and immunolocalization of cytoplasmic dynein in Dictyostelium.

A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules, UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium. These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.

Adenosine Triphosphatases↗
PubMed1989

Mitosis.

Data that describe both the structure and the physiology of the mitotic spindle are reviewed. Some of the molecules that have been shown to play a role in mitosis are tabulated, and how mitosis might work is considered.

Animals↗
PubMed1988

An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa.

The rapid, vectorial, microtubule-associated transport of organelles is believed to be mediated by specific mechanochemical transducers. Recent studies of various metazoan cells have allowed the identification of novel microtubule-dependent translocator molecules capable of promoting microtubule gliding across glass surfaces and translocation of inert beads along microtubules. These translocators could be involved in force generation for directional organelle movements in vivo. Here we report the identification of a microtubule-binding protein with characteristics expected for an organelle translocator in the giant freshwater amoeba Reticulomyxa. This factor has an apparent relative molecular mass (Mr) of 440,000 (440K) and sediments at 20-22S in sucrose-density gradients. It binds to microtubules under conditions of ATP depletion, possesses an ATPase activity and is sensitive to ultraviolet-induced, vanadate-dependent cleavage. Although its pharmacological properties differ from those of axonemal dynein, it can be considered to be a variant of cytoplasmic dynein. The Reticulomyxa high-molecular-weight protein (HMWP) promotes rapid, bidirectional movement of latex beads along Reticulomyxa microtubules in vitro at an average speed of 3.6 micron s-1. This protein, therefore, is a likely candidate for a microtubule-dependent motor.

Adenosine Triphosphatases↗
PubMed1986

Cytoskeletal architecture and motility in a giant freshwater amoeba, Reticulomyxa.

Reticulomyxa is a large, multinucleated freshwater protozoan with striking intracellular transport. Cytoplasmic streaming and saltatory movements of individual organelles (at rates of up to 25 micron/sec) are observed within the naked cell body and the extensive reticulate peripheral network of fine cytoplasmic strands. As demonstrated by video-enhanced light microscopy, individual organelles move only when associated with cytoskeletal linear elements. The linear elements are composed of mixed colinear bundles of microtubules and actin filaments, which form the backbone of the reticulopodial network. The constant branching, sprouting, and fusion of network strands suggest unique membrane properties and an unusually dynamic cytoskeleton. The electrophoretic mobility of Reticulomyxa tubulins and the lack of crossreactivity with several antibodies known to react with many plant and animal tubulins suggest that they may differ from other tubulins more widely than might be expected. Reticulomyxa's large size, the rapidity and pervasiveness of the two forms of transport, and the simple and ordered cytoskeleton make the organism well suited for future studies on the mechanisms of intracellular transport.

Actin Cytoskeleton↗
PubMed1986

Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba.

The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.

Actin Cytoskeleton↗
PubMed1986

Reticulomyxa: a new model system of intracellular transport.

Reticulomyxa is a large multinucleated freshwater protozoan that provides a new model system in which to study intracellular transport and cytoskeletal dynamics. Within the cell body and reticulopodial network, rapid, visually striking saltatory organelle motility as well as bulk cytoplasmic streaming can be readily observed. In addition, the cytoskeletal elements within these strands undergo dynamic splaying and fusing rearrangements, which can be visualized by video-enhanced light microscopy. A reactivatable lysed cell model has been developed that appears to preserve, and therefore permits examination of, these three forms of motility in a more controlled environment. Individual organelle movements are microtubule-based and have similarities to, but also differences from, the recently described kinesin-based transport. This lysed cell model can be further manipulated to provide native, ordered, completely exposed networks of either microtubules or microfilaments, or a combination of both, and thus may serve as a versatile motility assay system in which to examine the movement of exogenously added isolated organelles or latex beads.

Actin Cytoskeleton↗
PubMed1985

Bidirectional organelle transport can occur in cell processes that contain single microtubules.

Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.

Amoeba↗