Cell Poles Research Articles

Meta-Substituted benzenesulfonamide: a potent scaffold for the development of metallo-β-lactamase ImiS inhibitors.

Metallo-β-lactamase (MβL) ImiS contributes to the emergence of carbapenem resistance. A potent scaffold, meta-substituted benzenesulfonamide, was constructed and assayed against MβLs. The twenty-one obtained molecules specifically inhibited ImiS (IC50 = 0.11-9.3 μM); 2g was found to be the best inhibitor (IC50 = 0.11 μM), and 1g and 2g exhibited partially mixed inhibition with K i of 8.0 and 0.55 μM. The analysis of the structure-activity relationship revealed that the meta-substitutes improved the inhibitory activity of the inhibitors. Isothermal titration calorimetry (ITC) assays showed that 2g reversibly inhibited ImiS. The benzenesulfonamides exhibited synergistic antibacterial effects against E. coli BL21 (DE3) cells with ImiS, resulting in a 2-4-fold reduction in the MIC of imipenem and meropenem. Also, mouse experiments showed that 2g had synergistic efficacy with meropenem and significantly reduced the bacterial load in the spleen and liver after a single intraperitoneal dose. Tracing the ImiS in living E. coli cells by RS at a super-resolution level (3D-SIM) showed that the target was initially associated on the surface of the cells, then there was a high density of uniform localization distributed in the cytosol of cells, and it finally accumulated in the formation of inclusion bodies at the cell poles. Docking studies suggested that the sulfonamide group acted as a zinc-binding group to coordinate with Zn(ii) and the residual amino acid within the CphA active center, tightly anchoring the inhibitor at the active site. This study provides a highly promising scaffold for the development of inhibitors of ImiS, even the B2 subclasses of MβLs.

Live Imaging Reveals <i>Listeria</i> Hijacking of E-Cadherin Recycling as It Crosses the Intestinal Barrier

Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, a severe systemic infection. Its translocation across the intestinal epithelium is mediated by the interaction of internalin (InlA), a Listeria surface protein, with its host species-specific receptor E-cadherin (Ecad). It occurs through goblet cells, on which Ecad is luminally accessible, via an unknown mechanism. In the absence of cell lines recapitulating this phenotype in vitro, we developed an ex vivo experimental system, based on the intraluminal microinjection of Listeria in untreated, pharmacologically-treated and genetically modified intestinal organoids. Using both live light sheet microscopy and confocal imaging, we show that Listeria translocates through goblet cells within a vacuole in an InlA- and microtubule-dependent manner. As Ecad undergoes constant apical-basal recycling, we hypothesized that Lm may transit through goblet cells by hijacking Ecad recycling pathway. Indeed, Listeria is stuck at goblet cell apex when Ecad endocytosis is blocked, and remains trapped intracellularly at the basolateral pole of goblet cells when Rab11-dependent Ecad recycling is compromised. Together, these results show that Listeria, upon docking onto its luminally accessible receptor Ecad, hijacks its recycling pathway to be transferred by transcytosis across goblet cells. Live imaging of host-pathogen interactions in organoids is a promising approach to dissect their underlying cell and molecular biology.

Peptidoglycan layer and disruption processes in Bacillus subtilis cells visualized using quick-freeze, deep-etch electron microscopy.

Peptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands cross-linked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface in Bacillus subtilis at the nanometer resolution, using quick-freeze, deep-etch electron microscopy (EM). Filamentous structures were observed on the entire surface of the cell, where filaments about 11 nm wide formed concentric circles on cell poles, filaments about 13 nm wide formed a circumferential mesh-like structure on the cylindrical part and a 'piecrust' structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during EM. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using EM suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

The phenanthrene derivative PJ34 exclusively eradicates human pancreatic cancer cells in xenografts.

Recent reports demonstrate an exclusive eradication of a variety of human cancer cells by the modified phenanthridine PJ34. Their eradication during mitosis is attributed to PJ34 preventing NuMA clustering in the mitotic spindle poles of human malignant cells, which is crucial for their normal mitosis. Here, the effect of PJ34 is tested in cell cultures and xenografts of human pancreas ductal adenocarcinoma. Evidence is presented for a substantial reduction (80–90%) of PANC1 cancer cells in xenografts, measured 30 days after the treatment with PJ34 has been terminated. Benign cells infiltrated into the PANC1 tumors (stroma) were not affected. Growth, weight gain and behavior of the treated nude mice were not impaired during, and 30 days after the treatment with PJ34. The efficient eradication of malignant cells in human pancreas cancer xenografts presents a new model of pancreas cancer treatment.

Cell Width Dictates Type VI Secretion Tail Length.

The type VI secretion system (T6SS) is a multiprotein apparatus that injects protein effectors into target cells, hence playing a critical role in pathogenesis and in microbial communities [1-4]. The T6SS belongs to the broad family of contractile injection systems (CISs), such as Myoviridae bacteriophages and R-pyocins, that use a spring-like tail to propel a needle loaded with effectors [5, 6]. The T6SS tail comprises an assembly baseplate on which polymerizes a needle, made of stacked Hcp hexamers, tipped by the VgrG-PAAR spike complex and wrapped by the contractile sheath made of TssB and TssC [7-13]. The T6SS tail is anchored to the cell envelope by a membrane complex that also serves as channel for the passage of the needle upon sheath contraction [14-16]. In most CISs, the length of the tail sheath is invariable and is usually ensured by a dedicated protein called tape measure protein (TMP) [17-22]. Here, we show that the length of the T6SS tail is constant in enteroaggregative Escherichia coli cells, suggesting that it is strictly controlled. By overproducing T6SS tail subunits, we demonstrate that component stoichiometry does not participate to the regulation of tail length. The observation of longer T6SS tails when the apparatus is relocalized at the cell pole further shows that tail length is not controlled by a TMP. Finally, we show that tail stops its elongation when in contact with the opposite membrane and thus that T6SS tail length is determined by the cell width.

A PopZ-linked apical recruitment assay for studying protein-protein interactions in the bacterial cell envelope.

Most bacteria are surrounded by a complex cell envelope. As with many biological processes, studies of envelope assembly have benefited from cell-based assays for detecting protein-protein interactions. These assays use simple readouts and lack a protein purification requirement, making them ideal for early stage investigations. The most widely used two-hybrid interaction assay for proteins involved in envelope biogenesis is based on the reconstitution of adenylate cyclase activity from a split enzyme. Because adenylate cyclase is only functional in the cytoplasm, both protein fusions used in the assay must have a terminus located in this compartment. However, many envelope assembly factors are wholly extracytoplasmic. Detecting interactions involving such proteins using two-hybrid systems has therefore been problematic. To address this issue, we developed a cytological assay in Escherichia coli based on PopZ from Caulobacter crescentus. Here, we demonstrate the utility of this PopZ-Linked Apical Recruitment (POLAR) method for detecting interactions between proteins located in different cellular compartments. Additionally, we report that recruitment of an active peptidoglycan synthase to the cell pole is detrimental for E. coli and that interactions between proteins in the inner and outer membranes of the Gram-negative envelope may provide a mechanism for recruiting protein complexes to subpolar sites.

Cadmium toxicity and its relationship with disturbances in the cytoskeleton, cell cycle and chromosome stability.

This study aimed to investigate the mode of action of cadmium (Cd) toxicity at cell level, especially at early stages of plant exposure. Tomato seedlings were cultivated in growth media containing from 0.1 to 70 µM CdCl2 for 24 h. Mitotic index, chromosome abnormality, DNA integrity and organization of tubulin-based structures were assessed in root cells. As higher the Cd concentration in the growth media, higher was the DNA damage intensity and the occurrence of chromosomal abnormalities that included chromosome lost, bridges, stickiness, C-metaphase and polyploidy. The profile of chromosomal aberrations also varied with elevated Cd concentration, being observed increases in the frequency of chromosome stickiness. The mitotic index was reduced at the lowest Cd concentration, but such reduction was statistically similar to that detected at the highest concentration, suggesting that mitotic depression is a rapid outcome and, at same time, a Cd-induced effect that is limited at the first 24 h of direct root exposure to this metal. Under exposure to 20 µM CdCl2, heterogenous distribution of the spindle fibers, formation of two spindle complexes in both of the cell poles, absence of centrosome center, polarization of the spindle fibers during cell division, and non-uniform tubulin deposition in microtubule and phragmoplast were noticed. The results indicate that the tubulin-dependent components of cytoskeleton are Cd targets, and the sensitivity of tubulin-based structures to Cd exposure depends on cell cycle phase. Moreover, DNA damage intensity and chromosomal abnormality profile can be employed as markers of Cd toxicity level.

Air-liquid interface cell culture: From airway epithelium to the female reproductive tract.

The air-liquid interface (ALI) approach is primarily used to mimic respiratory tract epithelia in vitro. It is also known to support excellent differentiation of 3D multilayered skin models. To establish an ALI culture, epithelial cells are seeded into compartmentalized culture systems on porous filter supports or gel substrata. After an initial propagation period, the culture medium is removed from the apical side of the epithelium, exposing the cells to the surrounding air. Therefore, nutritive supply to the cells is warranted only by the basolateral cell pole. Under these conditions, the epithelial cells differentiate and regain full baso-apical polarity. Some types of epithelia even generate in vivo-like apical fluid or mucus. Interestingly, the ALI culture approach has also been shown to support morphological and functional differentiation of epithelial cells that are not normally exposed to ambient air in vivo. This review aims at giving a brief overview on the characteristics of ALI cultures in general and ALI models of female reproductive tract epithelia in particular. We discuss the applicability of ALI models for the investigation of the early embryonic microenvironment and for its implications in assisted reproductive technologies.

Microtubule‑severing protein Katanin p60 ATPase‑containing subunit A‑like 1 is involved in pole‑based spindle organization during mouse oocyte meiosis.

Microtubule‑severing proteins (MTSPs) are a group of microtubule‑associated proteins essential for multiple microtubule‑related processes, including mitosis and meiosis. Katanin p60 ATPase‑containing subunit A‑like 1 (p60 katanin‑like1) is an MTSP that maintains the density of spindle microtubules at the poles in mitotic cells; however, to date, there have been no studies about its role in female meiosis. Using invitro‑matured(IVM) oocytes as a model, it was first revealed that p60 katanin‑like 1 was predominant in the ovaries and oocytes, indicating its essential roles in oocyte meiosis. It was also revealed that p60 katanin‑like 1 was concentrated at the spindle poles and co‑localized and interacted with γ‑tubulin, indicating that it may be involved in pole organization. Next, specific siRNA was used to deplete p60 katanin‑like 1; the spindle organization was severely disrupted and characterized by an abnormal width:length ratio, multipolarity and extra aster microtubules out of the main spindles. Finally, it was determined that p60 katanin‑like 1 knockdown retarded oocyte meiosis, reduced fertilization, and caused abnormal mitochondrial distribution. Collectively, these results indicated that p60 katanin‑like 1 is essential for oocyte meiosis by ensuring the integrity of the spindle poles.

A Multiprotein Complex Anchors Adhesive Holdfast at the Outer Membrane of Caulobacter crescentus.

Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium Caulobacter crescentus to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series of C. crescentus mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations.IMPORTANCE Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. Caulobacter crescentus biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.

Inducible asymmetric cell division and cell differentiation in a bacterium.

Multicellular organisms achieve greater complexity through cell divisions that generate different cell types. We engineered a simple genetic circuit that induces asymmetric cell division and subsequent cell differentiation in Escherichia coli. The circuit involves a scaffolding protein, PopZ, that is stably maintained at a single cell pole over multiple asymmetric cell divisions. PopZ was functionalized to degrade the signaling molecule, c-di-GMP. By regulating synthesis of functionalized PopZ via small molecules or light, we can chemically or optogenetically control the relative abundance of two distinct cell types, characterized by either low or high c-di-GMP levels. Differences in c-di-GMP levels can be transformed into genetically programmable differences in protein complex assembly or gene expression, which in turn produce differential behavior or biosynthetic activities. This study shows emergence of complex biological phenomena from a simple genetic circuit and adds programmable bacterial cell differentiation to the genetic toolbox of synthetic biology and biotechnology.

Does the Nucleoid Determine Cell Dimensions in Escherichia coli?

Bacillary, Gram-negative bacteria grow by elongation with no discernible change in width, but during faster growth in richer media the cells are also wider. The mechanism regulating the change in cell width W during transitions from slow to fast growth is a fundamental, unanswered question in molecular biology. The value of W that changes in the divisome and during the division process only, is related to the nucleoid complexity, determined by the rates of growth and of chromosome replication; the former is manipulated by nutritional conditions and the latter—by thymine limitation of thyA mutants. Such spatio-temporal regulation is supported by existence of a minimal possible distance between successive replisomes, so-called eclipse that limits the number of replisomes to a maximum. Breaching this limit by slowing replication in fast growing cells results in maximal nucleoid complexity that is associated with maximum cell width, supporting the notion of Nucleoid-to-Divisome signal transmission. Physical signal(s) may be delivered from the nucleoid to assemble the divisome and to fix the value of W in the nascent cell pole.

Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction.

Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell.IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

Two-step chromosome segregation in the stalked budding bacterium Hyphomonas neptunium

Chromosome segregation typically occurs after replication has finished in eukaryotes but during replication in bacteria. Here, we show that the alphaproteobacterium Hyphomonas neptunium, which proliferates by bud formation at the tip of a stalk-like cellular extension, segregates its chromosomes in a unique two-step process. First, the two sister origin regions are targeted to opposite poles of the mother cell, driven by the ParABS partitioning system. Subsequently, once the bulk of chromosomal DNA has been replicated and the bud exceeds a certain threshold size, the cell initiates a second segregation step during which it transfers the stalk-proximal origin region through the stalk into the nascent bud compartment. Thus, while chromosome replication and segregation usually proceed concurrently in bacteria, the two processes are largely uncoupled in H. neptunium, reminiscent of eukaryotic mitosis. These results indicate that stalked budding bacteria have evolved specific mechanisms to adjust chromosome segregation to their unusual life cycle.

Staphylococcal Protein Secretion and Envelope Assembly.

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of Staphylococcus aureus including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division. Peptidoglycan, WTA and capsular polysaccharide are directly synthesized onto undecaprenol. Surface proteins are anchored by Sortase A, a membrane-embedded transpeptidase that scans secreted polypeptides for the C-terminal LPXTG motif of sorting signals. The resulting acyl enzyme intermediate is resolved by lipid II, the undecaprenol-bound peptidoglycan precursor. While these pathways share membrane diffusible undecaprenol, assembly of these molecules occurs either at the cross-walls or the cell poles. In S. aureus, the cross-wall represents the site of de novo peptidoglycan synthesis which is eventually split to complete the cell cycle yielding newly divided daughter cells. Peptidoglycan synthesized at the cross-wall is initially devoid of WTA. Conversely, lipoteichoic acid (LTA) synthesis which does not require bactoprenol is seemingly restricted to septal membranes. Similarly, S. aureus distinguishes two types of surface protein precursors. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with conserved YSIRK-GXXS motif signal peptides traffic to the cross-wall. A model for protein trafficking in the envelope and uneven distribution of teichoic acids is discussed.

DNA segregation under Par protein control.

The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion by interacting with ParA bound to the nucleoid. In the case of plasmids, a single ParB bound plasmid is observed to execute oscillations between cell poles while multiple plasmids eventually settle at equal distances from each other along the cell’s length. While the potential mechanism underlying the ParA-ParB interaction has been discussed, it remains unclear whether ParB-complex oscillations are stable limit cycles or merely decaying transients to a fixed point. How are dynamics affected by substrate length and the number of complexes? We present a deterministic model for ParA-ParB driven DNA segregation where the transition between stable arrangements and oscillatory behaviour depends only on five parameters: ParB-complex number, substrate length, ParA concentration, ParA hydrolysis rate and the ratio of the lengthscale over which the ParB complex stimulates ParA hydrolysis to the lengthscale over which ParA interacts with the ParB complex. When the system is buffered and the ParA rebinding rate is constant we find that ParB-complex dynamics is independent of substrate length and complex number above a minimum system size. Conversely, when ParA resources are limited, we find that changing substrate length and increasing complex number leads to counteracting mechanisms that can both generate or subdue oscillatory dynamics. We argue that cells may be poised near a critical level of ParA so that they can transition from oscillatory to fixed point dynamics as the cell cycle progresses so that they can both measure their size and faithfully partition their genetic material. Lastly, we show that by modifying the availability of ParA or depletion zone size, we can capture some of the observed differences in ParB-complex positioning between replicating chromosomes in B. subtilis cells and low-copy plasmids in E. coli cells.

Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

Abstract 430: Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer

Abstract Since several decades, prostate specific antigen (PSA) is regarded as a specific marker for healthy and neoplastic prostate tissues, and PSA level changes in blood and tissue are discussed as prognostic markers of prostate cancer. Using a custom-made mouse monoclonal anti-PSA antibody, we studied expression of PSA in a tissue microarray (TMAs) comprising 17,456 prostate cancers with long-term follow-up data. PSA staining was typically present in the cytoplasm and pronounced at the apical cell pole. PSA expression was often reduced (15%) or even completely lost (1%). Reduction of PSA expression was significantly linked to adverse features of prostate cancer, including advanced stage, high classical and quantitative Gleason grade, presence of lymph node metastasis, a positive surgical margin, increased cell proliferation, and reduced time to recurrence (p<0.0001 each). Complete lack identified a small subset of patients with the worst prognosis. There was also in impact of the subcellular localization of PSA expression: Loss of the apical staining pattern worsened the prognosis in cancers with otherwise identical levels of cytoplasmic staining. In a multivariable analysis including pT stage, Gleason grade, nodal stage, and resection margin status, loss of PSA expression proved to predict outcome independently from these parameters. Of note, PSA even stratified the prognosis of 971 cancers harboring PTEN deletions in our cancer set. In conclusion, gradual reduction of PSA staining, including a loss of the apical staining pattern, is a strong and independent prognostic marker in prostate cancer. Citation Format: Ronald Simon, Martina Kluth, Kristine Fischer, Sarah Bonk, Claudia Hube-Magg, Doris Höflmayer, Maximilian Lennartz, Florian Lutz, Guido Sauter, Franziska Büscheck, Ria Uhlig, David Dum, Andreas M. Luebke, Corinna Wittmer, Frank Jacobsen, Eike Burandt, Stefan Steurer, Waldemar Wilczak, Sarah Minner. Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 430.

DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance in Bacillus subtilis.

Chromosome segregation in sporulating Bacillus subtilis involves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation. Here we demonstrate that, in addition to positioning and condensing the chromosome, RacA contributes to efficient transport of DNA by the chromosome segregation motor SpoIIIE. When RacA is deleted, one-quarter of cells fail to capture DNA in the nascent spore, yet 70% of cells fail to form viable spores without RacA. This discrepancy indicates that RacA possesses a role in sporulation beyond DNA capture and condensation. We observed that the mutant cells had reduced chromosome translocation into the forespore across the entire length of the chromosome, requiring nearly twice as much time to move a given DNA locus. Additionally, functional abolition of the RacA-DivIVA interaction reduced translocation to a similar degree as in a racA deletion strain, demonstrating the importance of the RacA-mediated tether in translocation and chromosome packaging during sporulation. We propose that the DNA-membrane anchor facilitates efficient translocation by SpoIIIE, not through direct protein-protein contacts but by virtue of physical effects on the chromosome that arise from anchoring DNA at a distance.IMPORTANCE To properly segregate their chromosomes, organisms tightly regulate the organization and dynamics of their DNA. Aspects of the process by which DNA is translocated during sporulation are not yet fully understood, such as what factors indirectly influence the activity of the motor protein SpoIIIE. In this work, we have shown that a DNA-membrane tether mediated by RacA contributes to the activity of SpoIIIE. Loss of RacA nearly doubles the time of translocation, despite the physically distinct locations these proteins and their activities occupy within the cell. This is a rare example of an explicit effect that DNA-membrane connections can have on cell physiology and demonstrates that distant changes to the state of the chromosome can influence motor proteins which act upon it.

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.