Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

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.     

Myosin motors with artificial lever arms.

The myosin head consists of a globular catalytic domain and a light chain binding domain (LCBD). The coupling efficiency between ATP hydrolysis and myosin-induced actin movement is known to decline as the LCBD is truncated or destabilized. However, it was not clear whether the observed alteration in the production of force and movement reflects only the mechanical changes to the length of the LCBD or whether these changes also affect the kinetic properties of the catalytic domain. Here we show that replacement of the LCBD with genetically engineered domains of similar rigidity and dimensions produces functional molecular motors with unchanged kinetic properties. The resulting single-chain, single-headed motors were produced in Dictyostelium discoideum and obtained after purification from a standard peptone-based growth medium at levels of up to 12 mg/l. Their actin motility properties are similar or greater than those of native myosin. Rates of 2.5 and 3.3 microm/s were observed for motor domains fused to one or two of these domains, respectively. Their kinetic and functional similarity to the extensively studied myosin subfragment 1 (S1) and their accessibility to molecular genetic approaches makes these simple constructs ideal models for the investigation of chemo-mechanical coupling in the myosin motor.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Loss of functional MYO1C/myosin 1c,a motor protein involved in lipid raft trafficking,disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.     

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.     

Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.     

Molecular engineering of myosin

Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins.     

The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways

Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.