Michael P. Koonce

Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position.

Cytoplasmic dynein is a microtubule‐based motor protein responsible for vesicle movement and spindle orientation in eukaryotic cells. We show here that dynein also supports microtubule architecture and determines centrosome position in interphase cells. Overexpression of the motor domain in Dictyostelium leads to a collapse of the interphase microtubule array, forming loose bundles that often enwrap the nucleus. Using green fluorescent protein (GFP)–α‐tubulin to visualize microtubules in live cells, we show that the collapsed arrays remain associated with centrosomes and are highly motile, often circulating along the inner surface of the cell cortex. This is strikingly different from wild‐type cells where centrosome movement is constrained by a balance of tension on the microtubule array. Centrosome motility involves force‐generating microtubule interactions at the cortex, with the rate and direction consistent with a dynein‐mediated mechanism. Mapping the overexpression effect to a C‐terminal region of the heavy chain highlights a functional domain within the massive sequence important for regulating motor activity.

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 α-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.

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 vitrodissection of the tail-forming region of the dynein heavy chain fromDictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an ∼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.

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 proteins function. These three approaches taken together indicate that the dynein heavy chain is an essential gene in Dictyostelium.

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.

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ∼10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained β+ registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691–1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil β+ registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the α and β+ registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state.

Centrosome Positioning in Dictyostelium: Moving beyond Microtubule Tip Dynamics

The variability in centrosome size, shape, and activity among different organisms provides an opportunity to understand both conserved and specialized actions of this intriguing organelle. Centrosomes in the model organism Dictyostelium sp. share some features with fungal systems and some with vertebrate cell lines and thus provide a particularly useful context to study their dynamics. We discuss two aspects, centrosome positioning in cells and their interactions with nuclei during division as a means to highlight evolutionary modifications to machinery that provide the most basic of cellular services.