Distinct architectural requirements for the centromeric sequence of the pSM19035 plasmid partition machinery.

Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like -DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes.

CTP and coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning.

ParABS partition systems, comprising the centromere-like DNA sequence , the -binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParB and move by generating and following a local concentration gradient of nucleoid-bound ParA. However, the process through which ParB activates ParA-ATPase has not been defined.

Probing transient excited states of the bacterial cell division regulator MinE by relaxation dispersion NMR spectroscopy.

Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using N relaxation dispersion NMR: the full-length protein (6-stranded β-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the β1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded β-sheet.

Solidification Microstructures of the Ingots Obtained by Arc Melting and Cold Crucible Levitation Melting in TiNbTaZr Medium-Entropy Alloy and TiNbTaZrX (X = V, Mo, W) High-Entropy Alloys.

The solidification microstructures of the TiNbTaZr medium-entropy alloy and TiNbTaZrX (X = V, Mo, and W) high-entropy alloys (HEAs), including the TiNbTaZrMo bio-HEA, were investigated. Equiaxed dendrite structures were observed in the ingots that were prepared by arc melting, regardless of the position of the ingots and the alloy system. In addition, no significant difference in the solidification microstructure was observed in TiZrNbTaMo bio-HEAs between the arc-melted (AM) ingots and cold crucible levitation melted (CCLM) ingots.

Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranes in vitro.

Bacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the 'Z-ring'. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown.

Mechanistic insights of the Min oscillator via cell-free reconstitution and imaging.

The MinD and MinE proteins of Escherichia coli self-organize into a standing-wave oscillator on the membrane to help align division at mid-cell. When unleashed from cellular confines, MinD and MinE form a spectrum of patterns on artificial bilayers-static amoebas, traveling waves, traveling mushrooms, and bursts with standing-wave dynamics. We recently focused our cell-free studies on bursts because their dynamics recapitulate many features of Min oscillation observed in vivo.

Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids.

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system.

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro.

Reconstituting ParA/ParB-mediated transport of DNA cargo.

Protein gradients play key roles in subcellular spatial organization. In bacteria, ParA adenosine triphosphatases, or ATPases, form dynamic gradients on the nucleoid surface, which imparts positional information for the segregation, transport, and positioning of chromosomes, plasmids, and large protein assemblies. Despite the apparent simplicity of these minimal and self-organizing systems, the mechanism remains unclear.

A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force.

DNA segregation is a critical process for all life, and although there is a relatively good understanding of eukaryotic mitosis, the mechanism in bacteria remains unclear. The small size of a bacterial cell and the number of factors involved in its subcellular organization make it difficult to study individual systems under controlled conditions in vivo. We developed a cell-free technique to reconstitute and visualize bacterial ParA-mediated segregation systems.

Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro.

The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC.

A propagating ATPase gradient drives transport of surface-confined cellular cargo.

The faithful segregation of duplicated genetic material into daughter cells is critical to all organisms. In many bacteria, the segregation of chromosomes involves transport of "centromere-like" loci over the main body of the chromosome, the nucleoid, mediated by a two-protein partition system: a nonspecific DNA-binding ATPase, ParA, and an ATPase stimulator, ParB, which binds to the centromere-like loci. These systems have previously been proposed to function through a filament-based mechanism, analogous to actin- or microtubule-based movement.

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition.

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB.

Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism.

Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC.

ParA-mediated plasmid partition driven by protein pattern self-organization.

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown.

Surfing biological surfaces: exploiting the nucleoid for partition and transport in bacteria.

The ParA family of ATPases is responsible for transporting bacterial chromosomes, plasmids and large protein machineries. ParAs pattern the nucleoid in vivo, but how patterning functions or is exploited in transport is of considerable debate. Here we discuss the process of self-organization into patterns on the bacterial nucleoid and explore how it relates to the molecular mechanism of ParA action.

Endosomal sorting complex required for transport (ESCRT) complexes induce phase-separated microdomains in supported lipid bilayers.

The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals.

DNA requirements for assembly and stability of HIV-1 intasomes.

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly.

ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition.

P1 ParA is a member of the Walker-type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non-specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy.

Phage Mu transposition immunity: protein pattern formation along DNA by a diffusion-ratchet mechanism.

DNA transposons integrate into host chromosomes with limited target sequence specificity. Without mechanisms to avoid insertion into themselves, transposons risk self-destruction. Phage Mu avoids this problem by transposition immunity, involving MuA-transposase and MuB ATP-dependent DNA-binding protein.

Multiple modes of interconverting dynamic pattern formation by bacterial cell division proteins.

Min proteins of the Escherichia coli cell division system oscillate between the cell poles in vivo. In vitro on a solid-surface supported lipid bilayer, these proteins exhibit a number of interconverting modes of collective ATP-driven dynamic pattern formation including not only the previously described propagating waves, but also near uniformity in space surface concentration oscillation, propagating filament like structures with a leading head and decaying tail and moving and dividing amoeba-like structures with sharp edges. We demonstrate that the last behavior most closely resembles in vivo system behavior.

Barrier-to-autointegration factor (BAF) condenses DNA by looping.

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy.

Impact of emission anisotropy on fluorescence spectroscopy and FRET distance measurements.

The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature.