Dynamic instability in a DNA-segregating prokaryotic actin homolog

Dynamic instability-the switching of a two-state polymer between phases of steady elongation and rapid shortening-is essential to the cellular function of eukaryotic microtubules, especially during chromosome segregation. Since the discovery of dynamic instability 20 years ago, no other biological polymer has been found to exhibit this behavior. Using total internal reflection fluorescence microscopy and fluorescence resonance energy transfer, we observe that the prokaryotic actin homolog ParM, whose assembly is required for the segregation of large, low-copy number plasmids, displays both dynamic instability and symmetrical, bidirectional polymerization. The dynamic instability of ParM is regulated by adenosine triphosphate (ATP) hydrolysis, and filaments are stabilized by a cap of ATP-bound monomers. ParM is not related to tubulin, so its dynamic instability must have arisen by convergent evolution driven by a set of common constraints on polymer-based segregation of DNA.     

Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations

Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.     

Sustained microtubule treadmilling in Arabidopsis cortical arrays

Plant cells create highly structured microtubule arrays at the cell cortex without a central organizing center to anchor the microtubule ends. In vivo imaging of individual microtubules in Arabidopsis plants revealed that new microtubules are initiated at the cell cortex and exhibit dynamics at both ends. Polymerization-biased dynamic instability at one end and slow depolymerization at the other end result in sustained microtubule migration across the cell cortex by a hybrid treadmilling mechanism. This motility causes widespread microtubule repositioning and contributes to changes in array organization through microtubule reorientation and bundling.     

Actin polymerization and ATP hydrolysis

F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP.Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP.Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.     

Synthetic myosin filaments

A stable preparation of myosin filaments was formed in a medium at pH 8.0. The filament length varied from 0.2 to 0.5 micron. Most of the material sedimented at 21S, but there was a minor peak (due to monomer) at 6.8S. The filaments did not taper and had large bulbous irregularities at the ends.     

Modulation instability and pattern formation in spatially incoherent light beams

We report on the experimental observation of modulation instability of partially spatially incoherent light beams in noninstantaneous nonlinear media and show that in such systems patterns can form spontaneously from noise. Incoherent modulation instability occurs above a specific threshold that depends on the coherence properties (correlation distance) of the wave packet and leads to a periodic train of one-dimensional filaments. At a higher value of nonlinearity, the incoherent one-dimensional filaments display a two-dimensional instability and break up into self-ordered arrays of light spots. This discovery of incoherent pattern formation reflects on many other nonlinear systems beyond optics. It implies that patterns can form spontaneously (from noise) in diverse nonlinear many-body systems involving weakly correlated particles, such as atomic gases at (or near) Bose-Einstein condensation temperatures and electrons in semiconductors at the vicinity of the quantum Hall regime.     

Role of formins in actin assembly: nucleation and barbed-end association

Nucleation of branched actin filaments by the Arp2/3 complex is a conserved process in eukaryotic cells, yet the source of unbranched actin filaments has remained obscure. In yeast, formins stimulate assembly of actin cables independently of Arp2/3. Here, the conserved core of formin homology domains 1 and 2 of Bni1p (Bni1pFH1FH2) was found to nucleate unbranched actin filaments in vitro. Bni1pFH2 provided the minimal region sufficient for nucleation. Unique among actin nucleators, Bni1pFH1FH2 remained associated with the growing barbed ends of filaments. This combination of properties suggests a direct role for formins in regulating nucleation and polarization of unbranched filamentous actin structures.     

光对美洲蛙肌肉纤维衍射强度的分布研究

肌原纤维是由粗丝和细丝重迭而成的Ａ带和只含细丝的Ⅰ带组成，形成了天然光栅，因此可用光学方法探讨肌原纤维分子结构及其动力学问题。试验表明，单色光通过美洲蛙肌原纤维后，衍射光左右两端为非对称性，且左右条纹锋值间隔随肌原纤维节长度增大而减小。肌肉运动有张有弛，于是肌原纤维长度有变异，共变异与非对称有关。当肌原纤维长度增加时，左右两端强度差异变大，而相对应条纹的锋值间隔距离变小。这一现象与布拉格方程和折射     

Dynamic instability of sheared microtubules observed by quasi-elastic light scattering

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.     

不同质粒在大肠杆菌宿主细胞中的不稳定性研究

对几种不同的质粒在相同宿主中的稳定性进行检测,结果表明,在非选择压力下,４种不同的质粒在大肠 杆菌宿主细中的稳定性有明显的差异,其中,ＰＵＣＧ４１８最稳定,该质粒失去的速率仅为０．５％;     

两端固定梁柱失稳问题的尖点突变模型

应用突变理论建立了两端固定梁柱失稳的尖点突变模型.并利用数学方法,揭示了梁柱失稳过程中平衡的特性.     

Distinct properties of the XY pseudoautosomal region crucial for male meiosis

Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform-specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.     

Mitochondrial-satellite and circular DNA filaments in yeast

Mitochondrial DNA of Saccharomyces cerevisiae contains a satellite DNA (density, 1.682) that appears to exist as open-ended filaments at least 5 microns long. DNA from intact cells contains circular filaments whose lengths vary from 0.5 to 7 microns, with a great majority at 1.95 microns. The circular DNA has a density similar to that of the major nuclear peak (1.697). When heat-denatured mitochondrial-satellite DNA is renatured, it cross-links to form a molecule that is larger than the native molecule. The formation of cross-links results in hypersharpening of the density profiles in cesium chloride and also leads to failure to pass Millipore filter paper.     

Assembly mechanism of the contractile ring for cytokinesis by fission yeast

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We used fluorescence microscopy of live fission yeast cells to observe that membrane-bound nodes containing myosin were broadly distributed around the cell equator and assembled into a contractile ring through stochastic motions, after a meshwork of dynamic actin filaments appeared. Analysis of node motions and numerical simulations supported a mechanism whereby transient connections are established when myosins in one node capture and exert force on actin filaments growing from other nodes.     

Chromosome alignment and segregation regulated by ubiquitination of survivin

Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. We report that a de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation.     

Single-molecule speckle analysis of actin filament turnover in lamellipodia

Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip.     

Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation

Meiosis is a specialized cell division in which two chromosome segregation phases follow a single DNA replication phase. The budding yeast Polo-like kinase Cdc5 was found to be instrumental in establishing the meiosis I chromosome segregation program. Cdc5 was required to phosphorylate and remove meiotic cohesin from chromosomes. Furthermore, in the absence of CDC5 kinetochores were bioriented during meiosis I, and Mam1, a protein essential for coorientation, failed to associate with kinetochores. Thus, sister-kinetochore coorientation and chromosome segregation during meiosis I are coupled through their dependence on CDC5.     

The location of DNA in RecA-DNA helical filaments

The helical filament that the RecA protein of Escherichia coli forms around DNA is the active apparatus in protein-catalyzed homologous genetic recombination. The actual position of DNA within this complex has been unknown. Image analysis has been performed on electron micrographs of filaments of RecA on double-stranded DNA and on single-stranded DNA to visualize a difference that is consistent with one strand of the double-stranded DNA. This localization of the DNA gives additional information about the unusual structure of DNA in the complex with RecA protein.     

Pot1, the putative telomere end-binding protein in fission yeast and humans

Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 (protection of telomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeast pot1+ gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.     

Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis

Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.