Dietmar J. Manstein

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.

Lighting up the cell surface with evanescent wave microscopy

Evanescent wave microscopy, also termed total internal reflection fluorescence microscopy (TIR-FM), has shed new light on important cellular processes taking place near the plasma membrane. For example, this technique can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. There has been a recent surge in the popularity of this technique with the advent of green-fluorescent protein (GFP) as a fluorescent marker and new technical developments. These technical developments and some of the latest applications of TIR-FM are the subject of this review.

Cloning vectors for the production of proteins in Dictyostelium discoideum

We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products.

Toxoplasma gondii myosin A and its light chain: a fast, single‐headed, plus‐end‐directed motor

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle–laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 μm/s towards the plus end of actin filaments. TgMyoA is the first fast, single‐headed myosin and fulfils all the requirements for power parasite gliding.

A structural model for actin-induced nucleotide release in myosin.

Myosins are molecular motor proteins that harness the chemical energy stored in ATP to produce directed force along actin filaments. Complex communication pathways link the catalytic nucleotide-binding region, the structures responsible for force amplification and the actin-binding domain of myosin. We have crystallized the nucleotide-free motor domain of myosin II in a new conformation in which switch I and switch II, conserved loop structures involved in nucleotide binding, have moved away from the nucleotide-binding pocket. These movements are linked to rearrangements of the actin-binding region, which illuminate a previously unobserved communication pathway between the nucleotide-binding pocket and the actin-binding region, explain the reciprocal relationship between actin and nucleotide affinity and suggest a new mechanism for product release in myosin family motors.

Nanometer targeting of microtubules to focal adhesions.

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP–CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.

Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

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.

Nonmuscle myosin-2: mix and match

Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease.

Single-molecule tracking of myosins with genetically engineered amplifier domains

We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was ∼30°. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.