DNA manipulators: caught in the act

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.     

Actin nucleation: cortactin caught in the act

A variety of activators stimulate Arp2/3 complex to nucleate branched actin filament structures. New results provide important biochemical and structural information for activation by the proteins cortactin and N-WASP.     

DNA topoisomerases: single gyrase caught in the act

A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle.     

Double fertilization - caught in the act

In flowering plants, fertilization is unique because it involves two pairs of male and female gametes, a process known as double fertilization. Here, we provide an overview of the field and a detailed review of the outstanding recent advances, including in vivo imaging of double fertilization and the identification of a signaling pathway controlling the release of the male gametes and of a protein involved in gamete membrane fusion. These recent results are stepping stones for further research; our knowledge of double fertilization is expanding as newly discovered molecular pathways are explored and new mutants are characterized. Controlling plant fertilization is essential for seed production, and molecular understanding of double fertilization will provide the tools to improve crops and breeding programs.     

Unfaithful DNA polymerase caught in the act

The 3D structures of all 12 mispairs formed in the active site of a DNA polymerase help explain their differential effects on polymerase stalling and on translocation of the primer terminus to the enzyme's proofreading site.     

MicroRNAs are invading the tumor microenvironment: Fibroblast microRNAs regulate tumor cell motility and invasiveness

Comment on: Aprelikova O, et al. Cell Cycle 2010; 9:4387–98.     

Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.     

The anthers of Senecio vulgaris (Asteraceae): saltatory evolution caught in the act

Anthers of the common annual weed, Senecio vulgaris, show an incomplete development of the two adaxial pollen sacs (microsporangia, MS). One or both adaxial MS can be missing, or they are replaced by sterile lobes. The reduction is stronger in the derived subspecies, S. vulgaris var. vulgaris than in the ancestral subspecies, S. vulgaris ssp. denticulatus. This character in S. vulgaris differs from the usual complete reduction of adaxial MS in other, independent instances of disporangiate anthers in the Asteraceae. It corresponds to the transition phenotypes associated with various recombinant genotypes derived from artificial crosses between tetrasporangiate (4 MS) and disporangiate (2 MS) species in the Asteracean genus Microseris. Senecio vulgaris could be a rare natural instance of homozygosity for a major gene permitting reduction of the adaxial MS in which the expression of the reduced phenotype is determined by different numbers of modifiers in the two subspecies.     

Release of cell fragments by invading melanoma cells

Tumor cell invasion requires coordinated cell adhesion to an extracellular matrix (ECM) substrate at the leading edge and concomitant detachment at the cell rear. Known detachment mechanisms include the slow sliding of focal contacts, the detachment of adhesion receptors by affinity and avidity regulation, as well as the shedding of adhesion receptors, most notably integrins. In highly invasive melanoma cells migrating within 3D collagen matrices, beta1 integrins and CD44 are released upon retraction of the trailing edge, together with ripping-off complete cell fragments to become deposited along the migration trail of remodeled matrix. Cell fragments reach a size up to 12 microm in diameter, contain cytoplasm and occasionally polymerized actin enclosed by intact cell membrane including surface beta1 integrins, but do not include nuclear material. The release of cell fragments was migration dependent, as impairment of motility by a blocking anti-beta1 integrin antibody also blocked cell particle release. Invasion-associated deposition of cell fragments combines the secretory-type release of vesicles with a physical mechanism of rear retraction and migration efficiency. The deposition of cell fragments may further represent a disregulated detachment strategy with implications for neoplastic cell behavior, such as the paracrine effects on neighbor cells or a negative impact on immune effector cells.     

A structural study of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: caught in the act of geminal diamine formation

Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction.     

Wnt signaling and neural stem cells: caught in the Wnt web

Wnt proteins have now been identified as major physiological regulators of multiple aspects of stem cell biology, from self-renewal and pluripotency to precursor cell competence and terminal differentiation. Neural stem cells are the cellular building blocks of the developing nervous system and provide the basis for continued neurogenesis in the adult mammalian central nervous system. Here, we outline the most recent advances in the field about the critical factors and regulatory networks involved in Wnt signaling and discuss recent findings on how this increasingly intricate pathway contributes to the shaping of the developing and adult nervous system on the level of the neural stem cell. Funding: We wish to apologize to those whose work is not included due to the length constraints on the review. Work in the Lie lab is supported by the European Young Investigator Award Program of the European Science Foundation and grants of the Deutsche Forchungsgemeinschaft (LI 858/5-1), the European Union (Marie Curie Excellence Team Award and Marie Curie International Reintegration Grant), and the Bavarian Research Network “Adult Neural Stem Cells” FORNEUROCELL.     

Persistent cell motion in the absence of external signals: a search strategy for eukaryotic cells

Background

Eukaryotic cells are large enough to detect signals and then orient to them by differentiating the signal strength across the length and breadth of the cell. Amoebae, fibroblasts, neutrophils and growth cones all behave in this way. Little is known however about cell motion and searching behavior in the absence of a signal. Is individual cell motion best characterized as a random walk? Do individual cells have a search strategy when they are beyond the range of the signal they would otherwise move toward? Here we ask if single, isolated, Dictyostelium and Polysphondylium amoebae bias their motion in the absence of external cues.

Methodology

We placed single well-isolated Dictyostelium and Polysphondylium cells on a nutrient-free agar surface and followed them at 10 sec intervals for ∼10 hr, then analyzed their motion with respect to velocity, turning angle, persistence length, and persistence time, comparing the results to the expectation for a variety of different types of random motion.

Conclusions

We find that amoeboid behavior is well described by a special kind of random motion: Amoebae show a long persistence time (∼10 min) beyond which they start to lose their direction; they move forward in a zig-zag manner; and they make turns every 1–2 min on average. They bias their motion by remembering the last turn and turning away from it. Interpreting the motion as consisting of runs and turns, the duration of a run and the amplitude of a turn are both found to be exponentially distributed. We show that this behavior greatly improves their chances of finding a target relative to performing a random walk. We believe that other eukaryotic cells may employ a strategy similar to Dictyostelium when seeking conditions or signal sources not yet within range of their detection system.     

Proteins: caught in the trap

Two independent groups have recently devised innovative methods using light to trap and manipulate particles as small as proteins.