Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings.Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochete's alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

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The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20-40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.

BackgroundLive-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.ResultsPresented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.ConclusionsThis method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.Electronic supplementary materialThe online version of this article (10.1186/s13008-018-0039-z) contains supplementary material, which is available to authorized users.

SUMMARY While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo . Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

Neutrophils are innate immune effector cells that migrate from the blood to resolve bacterial and fungal infections. Understanding how neutrophils migrate is critical for regulating excessive inflammation and subsequent collateral injury. β2 integrins are essential to classical neutrophil recruitment from the blood, and the activation of β2 integrins has been well defined in previous studies. Adhesion stabilization of neutrophils on the endothelial surface as they crawl into a favorable position for transmigration is not as well defined. Neutrophils do not make mature focal adhesions, but do express the focal adhesion protein vinculin. Vinculin associates with integrins by binding to talin‐1 and stabilizes integrin adhesions by recruiting various actin‐associated proteins or by associating with actin directly. This study characterizes the role of vinculin in neutrophil β2 integrin‐dependent adhesion, motility and anti‐bacterial function. Intrinsic activation of β2 integrins is unaffected by vinculin knockout after CXCL1 activation. Vinculin knockout attenuates neutrophil adhesion, spreading, and motility on glass coated with β2 integrin ligand, ICAM‐1, and activating CXCL1. Vinculin knockout also reduces neutrophil spreading in response to ICAM‐1/CXCL1 on polyacrylamide gels of high stiffness but not lower stiffness. Vinculin knockout reduces traction stresses of neutrophils and the actin stiffening response after stimulation. Unlike static conditions, vinculin knockout does not affect neutrophil motility under flow conditions. Vinculin knockout attenuates respiratory burst, but does not affect phagocytosis. In mixed chimeric mice given intraperitoneal thioglycollate, we find comparable migration of vinculin‐knockout and vinculin‐sufficient neutrophils into the peritoneum. Altogether, while vinculin enhances neutrophil β2 integrin adhesion strength, vinculin knockout does not affect neutrophil motility and trafficking under physiological conditions. Support or Funding Information American Heart Association (12SDG12080281), Scientist Development Grant CL Department of Surgery, Rhode Island Hospital This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.