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Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4–5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1–2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues. Here, the authors provide detailed experimental and analytical procedures for Hi-M, a method that enables simultaneous imaging of 3D genome folding and RNA localization in single cells.

International audience; Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulates their function. Genomewide studies revealed a new level of organization at the kilobase-to-megabase scale termed “topological associating domains” (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one “functional unit.” Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review, we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments, and propose an interpretation reconciling population-based findings with direct single-cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes., In most bacteria and archaea, a broadly conserved mitotic-like apparatus assures the inheritance of duplicated genetic material before cell division. Here, the authors use super-resolution microscopies to dissect the activities required for proper DNA segregation through the nucleoid interior.

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA., Instituto de Física de Líquidos y Sistemas Biológicos

With single-molecule localization microscopy (SMLM) it is possible to reveal the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. SMLM remains technically challenging, and frequently its implementation requires tailored experimental conditions that depend on the complexity of the subcellular structure of interest. Here, we describe two simple, robust, and high-throughput protocols to study molecular motors and machineries responsible for chromosome transport and organization in bacteria using 2D- and 3D-SMLM.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome organization while maintaining spatiotemporal information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. We used Hi-M to study chromosome organization during the awakening of the zygotic genome in intact Drosophila embryos. By visualizing chromosome architecture and gene expression simultaneously in single nuclei, we were able to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture in time and space will be key to understand the mechanisms and consequences of the 4D organization of the genome.

With single-molecule localization microscopy (SMLM) it is possible to reveal the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. SMLM remains technically challenging, and frequently its implementation requires tailored experimental conditions that depend on the complexity of the subcellular structure of interest. Here, we describe two simple, robust, and high-throughput protocols to study molecular motors and machineries responsible for chromosome transport and organization in bacteria using 2D- and 3D-SMLM.

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane bound SpoIIIE DNA translocase and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from nonspecific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. We propose a new in vivo model where the match between the orientation of SpoIIIE hexamers in the mother cell respect to the closing septum, defined by the linker connecting the transmembrane and motor domains, and the orientation of the whole chromosome during sporulation, creates a natural directionality pathway that is modulated by sequence-dependent translocation initiation.

DNA intercalators are widely used in cancer therapeutics, to probe protein–DNA interactions and to investigate the statistical–mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43 %, while not affecting its mechanical properties.

Drosophila chromosomes are organized in a series of nanocompartments that correspond to topologically associating domains., Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

The bacterial nucleoid is highly organized, yet it is dynamically remodeled by cellular processes such as transcription, replication, or segregation. Many principles of nucleoid organization have remained obscure due to the inability of conventional microscopy methods to retrieve structural information beyond the diffraction limit of light. Structured illumination microscopy has recently been shown to provide new levels of spatial details on bacterial chromosome organization by surpassing the diffraction limit. Its ease of use and fast 3D multicolor capabilities make it a method of choice for imaging fluorescently labeled specimens at the nanoscale. We describe a simple high-throughput method for imaging bacterial chromosomes using this technique.

At the kilo- to mega-base pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combined high-content super-resolution microscopies with state-of-the-art DNA labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We found that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we were able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs were to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

The bacterial nucleoid is highly organized, yet it is dynamically remodeled by cellular processes such as transcription, replication, or segregation. Many principles of nucleoid organization have remained obscure due to the inability of conventional microscopy methods to retrieve structural information beyond the diffraction limit of light. Structured illumination microscopy has recently been shown to provide new levels of spatial details on bacterial chromosome organization by surpassing the diffraction limit. Its ease of use and fast 3D multicolor capabilities make it a method of choice for imaging fluorescently labeled specimens at the nanoscale. We describe a simple high-throughput method for imaging bacterial chromosomes using this technique.

Fish are often considered a very sensitive indicator of heavy metal contamination in aquatic ecosystems; however, the detailed mechanisms of bioaccumulation remain unclear. Here, we study the bioaccumulation processes of three relevant heavy metals (Cd2+, Cu2+ and Cr6+) in Odontesthes bonariensis using mathematical modelling. We developed a general compartmental kinetic model that describes the transport of heavy metals between the surrounding water and the gills and liver of fish. The general model was reduced to a simple one still capable of reproducing previous experimental data and suggesting a common mechanism for the three metals. The modelling results indicate that bioaccumulation of Cd2+, Cu2+, and Cr6+ is described by a combination of a concentration-independent and saturable uptake kinetics in both organs with a unidirectional path of elimination from gills to liver to waterborne. Finally, the good agreement between the parameter values predicted by the model and previously published data suggests that our modelling approach may shed light on the mechanisms of heavy metal bioaccumulation in other species. Fil: Blanco, Maria Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina Fil: Cattoni, Diego Ignacio. Université de Montpellier; Francia Fil: Carriquiriborde, Pedro. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina Fil: Grigera, Jose Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina Fil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Technische Universität Dresden; Alemania

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing during the awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understand the mechanisms and consequences of the 4D organization of the genome.

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.

In order to study the detailed assembly and regulation mechanisms of complex structures and machineries in the cell, simultaneous in situ observation of all the individual interacting components should be achieved. Multi-color Single-Molecule Localization Microscopy (SMLM) is ideally suited for these quantifications. Here, we build on previous developments and thoroughly discuss a protocol for two-color SMLM combining PALM and STORM, including sample preparation details, image acquisition and data postprocessing analysis. We implement and evaluate a recently proposed colocalization analysis method (aCBC) that allows single-molecule colocalization quantification with the potential of revealing fine, nanometer-scaled, structural details of multicomponent complexes. Finally, using a doubly-labeled nuclear factor (Beaf-32) in Drosophila S2 cells we experimentally validate the colocalization quantification algorithm, highlight its advantages and discuss how using high molecular weight fluorescently labeled tags compromises colocalization precision in two-color SMLM experiments.

The 3D folding of the genome and its relation to fundamental processes such as gene regulation, replication, and segregation remains one of the most puzzling and exciting questions in genetics. In this review, we describe how the use of new technologies is starting to revolutionize the field of chromosome organization, and to shed light on the mechanisms of transcription, replication, and repair. In particular, we concentrate on recent studies using genome-wide methods, single-molecule technologies, and super-resolution microscopy (SRM). We summarize some of the main concerns when employing these techniques, and discuss potential new and exciting perspectives that illuminate the connection between 3D genomic organization and gene regulation.

International audience; Chromosomes of a broad range of species, from bacteria to mammals, are structured by large topological domains whose precise functional roles and regulatory mechanisms remain elusive. Here, we combine super-resolution microscopies and chromosome-capture technologies to unravel the higher-order organization of the Bacillus subtilis chromosome and its dynamic rearrangements during the cell cycle. We decipher the fine 3D architecture of the origin domain, revealing folding motifs regulated by condensin-like complexes. This organization, along with global folding throughout the genome, is present before replication, disrupted by active DNA replication, and re-established thereafter. Single-cell analysis revealed a strict correspondence between sub-cellular localization of origin domains and their condensation state. Our results suggest that the precise 3D folding pattern of the origin domain plays a role in the regulation of replication initiation, chromosome organization, and DNA segregation.

SpoIIIE/FtsK are membrane‐anchored, ATP‐fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence‐recognition modules (SpoIIIE‐γ/FtsK‐γ) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single‐molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single‐molecule and modelling methods that hexameric SpoIIIE binds DNA non‐specifically and finds SRS by an ATP‐independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE‐γ to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS‐directed translocation by SpoIIIE/FtsK. Fil: Cattoni, Diego Ignacio. Universite Montpellier II; Francia Fil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universitat Dresden; Alemania Fil: Godefroy, Cédric. Technische Universität Dresden; Francia Fil: Margeat, Emmanuel. Universite Montpellier II; Francia Fil: Trigueros, Sonia. University of Oxford; Reino Unido Fil: Milhiet, Pierre Emmanuel. Universite Montpellier II; Francia Fil: Nöllmann, Marcelo. Universite Montpellier II; Francia

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.

Bacteria have evolved complex, multi-component cellular machineries to carry out fundamental cellular processes such as cell division/separation, locomotion, protein secretion, DNA transcription/replication, or conjugation/competence. Diffraction of light has so far restricted the use of conventional fluorescence microscopy to reveal the composition, internal architecture and dynamics of these important machineries. This review describes some of the more recent advances on single-molecule super-resolution microscopy methods applied to bacteria and highlights their application to chemotaxis, cell division, DNA segregation, and DNA transcription machineries. Finally, we discuss some of the lessons learned from this approach, and future perspectives.

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes. Fil: Dodes Traian, Martín Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Cattoni, Diego Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Inserm; Francia Fil: Levi, Valeria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Background: Enterocutaneous fistulae, pathological communications between the intestinal lumen and the abdominal skin, can arise as serious complication of gastrointestinal surgery. A current non-surgical treatment for this pathology involves topical application of sub-atmospheric pressure, also known as vacuum assisted closure (VAC). While this technique appears to be promising, surgeons report a number of cases in which its application fails to achieve fistula closure. Here, we evaluate the fistula's physical properties during the vacuum assisted closure process in a computational approach exploring the relevance of intraluminal intestinal pressure. Methods: A mathematical model formulated by differential equations based on tissue elasticity properties and principles of fluid mechanics was created and forcing functions were integrated to mimic intestinal pressure dynamics. A software to solve equations and to fit the model to experimentally obtained data was developed. This enabled simulations of vacuum assisted fistula closure under different intestinal pressure. Results: The simulation output indicates conditions, in which fistula closure can or cannot be expected suggesting favoured or impeded healing, respectively. When modifications of intestinal pressure, as observed in fistula accompanying pathologies, are integrated, the outcome of fistula closure changes considerably. Rise of intestinal pressure is associated with delay of fistula closure and temporary fistula radius augmentation, while reduction of intestinal pressure during sub-atmospheric pressure treatment contributes to a faster and direct fistula closure. Conclusion: From the model predictions, we conclude that administration of intestinal pressure decreasing compounds (e.g. butylscopolamine, glucagon) may improve VAC treatment, while intestinal pressure increasing drugs should be avoided., Facultad de Ciencias Exactas, Instituto de Física de Líquidos y Sistemas Biológicos

Although 1-anilino-naphthalene-8-sulfonate (ANS) has been widely used in protein folding and binding studies, the detailed mechanism of this interaction is not fully understood. In this work the binding of ANS was analyzed at pre-equilibrium and equilibrium conditions using bovine serum albumin (BSA) as model. We employed a combined approach including the analysis of fluorescence, near-UV circular dichroism and isothermal titration calorimetric data. Experiments at equilibrium with these techniques identify three ANS molecules bound at hydrophobic cavities in BSA. Pre-equilibrium fluorescence analysis unambiguously indicated that the binding of ANS at hydrophobic cavities of BSA occurs at two different and independent classes of sites with similar affinities and quantum yields, two features that are undetectable by the equilibrium analysis. The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (DeltaH = -22 kJ/mol) and entropic terms (-TDeltaS = -17 kJ/mol), while the binding to the second site is enthalpically driven (DeltaH = -31 kJ/mol; -TDeltaS = -0.6 kJ/mol). Complementary information from molecular docking showed three ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally and three additional ANS molecules bound at water exposed sites.

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites., Instituto de Física de Líquidos y Sistemas Biológicos, Facultad de Ciencias Exactas

Super-resolution and fluctuation microscopy in a model DNA-segregation system reveal the architecture and assembly mechanism of the motor responsible for DNA translocation during bacterial cell division., ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes., Author Summary Molecular motors are implicated in myriad cellular processes, notably in the transcription, replication, and segregation of DNA. Segregation or packaging of DNA is essential for production of viable viral particles, proper division of bacterial cells, and production of spores. A dramatic example of this process occurs during sporulation in Bacillus subtilis, in which a large proportion of the chromosome is actively transferred across a division septum by the SpoIIIE motor protein. Here, we use advanced microscopy methods to study the mechanism of recruitment and assembly of the SpoIIIE pump and the architecture of its complex with DNA. We found that SpoIIIE complexes are recruited before the beginning of cell division, and are subsequently escorted by the constriction machinery to the center of the septum. We show that the directionality of DNA transport by SpoIIIE results in the establishment of an asymmetric complex that exports DNA into the nascent spore. Our data are inconsistent with previous models that predicted the formation of a membrane-spanning DNA-conducting channel. Instead, the greater resolution afforded by our approach supports a model in which DNA movement occurs through an open pore structurally maintained by the division apparatus, with SpoIIIE working as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed model may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

SpoIIIE/FtsK are membrane-anchored, ATP-fueled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence-recognition modules (SpoIIIE-γ/FtsK-γ) and highly-skewed chromosomal sequences (SRS/KOPS). using a novel combination of bulk and single-molecule methods we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence-independent with binding specificity being uniquely determined by dissociation. Next, we show by single-molecule and modeling methods that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE-γ to SRS triggering allosteric motor activation. We propose a new model that provides an entirely novel interpretation of previous observations for the origin of SRS/KOPS-directed translocation by SpoIIIE/FtsK.

International audience; Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS.This assembly model and approach to characterizing large-scale, dynamic interactions between macro-molecules may be generalizable to many unrelated machineries that self-assemble in superstructures.

Summary Condensin—an SMC-kleisin complex—is essential for efficient segregation of sister chromatids in eukaryotes [1–4]. In Escherichia coli and Bacillus subtilis, deletion of condensin subunits results in severe growth phenotypes and the accumulation of cells lacking nucleoids [5, 6]. In many other bacteria and under slow growth conditions, however, the reported phenotypes are much milder or virtually absent [7–10]. This raises the question of what role prokaryotic condensin might play during chromosome segregation under various growth conditions. In B. subtilis and Streptococcus pneumoniae, condensin complexes are enriched on the circular chromosome near the single origin of replication by ParB proteins bound to parS sequences [11, 12]. Using conditional alleles of condensin in B. subtilis, we demonstrate that depletion of its activity results in an immediate and severe defect in the partitioning of replication origins. Multiple copies of the chromosome remain unsegregated at or near the origin of replication. Surprisingly, the growth and chromosome segregation defects in rich medium are suppressed by a reduction of replication fork velocity but not by partial inhibition of translation or transcription. Prokaryotic condensin likely prevents the formation of sister DNA interconnections at the replication fork or promotes their resolution behind the fork., Highlights • Smc-ScpAB inactivation causes a severe chromosome segregation defect in B. subtilis • Replication origins remain interconnected in the absence of prokaryotic condensin • Defects in chromosome segregation are highly dependent on growth conditions • Reduction of replication fork velocity rescues segregation of replication origins, Gruber et al. show that conditional inactivation of prokaryotic condensin in B. subtilis results in immediate and severe defects in chromosome segregation under conditions promoting fast growth. The separation of replication origins is blocked in the absence of Smc-ScpAB but can be rescued by artificial reduction of replication fork speed.

1-anilino-naphtalene-8-sulfonate (ANS) is a fluorescent probe widely used in protein folding and conformational transitions studies. The fluorescent features of ANS, a blue shift of the emission maximum and the increase of quantum yield and lifetime, are generally attributed to the binding at hydrophobic sites. Despite the interaction of ANS with proteins has been extensively studied since the early works of Gregorio Weber, few high-resolution structures of proteins complexed with ANS have been resolved. In this work the binding of ANS to BSA was analyzed at equilibrium and pre-equilibrium conditions. The combined analysis of fluorescence, near UV circular dichroism and isothermal titration calorimetric data provided a detailed description of the binding mechanism. Three ANS molecules bound to BSA in 100 mM phosphate pH 7 at 25°C. Pre-equilibrium experiments allowed to determine the affinity and the relative quantum yield at each binding site by fitting a microscopic model to the fluorescence time-course data. This analysis unambiguously indicated that the binding of ANS to BSA occurs at two different and independent binding sites with similar quantum yields and affinities (ΔG° @ -35 kJ/mol). The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (ΔH = -16.3 kJ/mol) and entropic terms (-TΔS = -19.4 kJ/mol), while the binding to the second site is enthalpically driven (ΔH = -36.6 kJ/mol; -TΔS = 4.3 kJ/mol). Complementary information from molecular docking showed 3 ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally. The sulfonate group of ANS was oriented towards clusters of polar residues, a common feature in the reported crystal structures of other ANS-protein complexes.Supported with grants from UBA, CONICET and ANPCyT.

Although the importance of lipid-protein interactions in determining the biological function of integral membrane proteins is well recognized, the underlying molecular mechanisms remain unclear. In this work we explore the modulation of the enzymatic activity of an integral membrane protein by phospholipids, using the Plasma Membrane Calcium Pump (PMCA) reconstituted in mixed detergent C12E10-phospholipid micelles as model system. Increasing amounts of phospholipids were added to purified PMCA preparations reconstituted in detergent micelles producing a reversible increase in activity reaching a maximum value. No major structural changes occur during this process neither in the protein as assessed by far UV circular dichroism (CD) and tryptophan fluorescence nor in the micellar system as determined by fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). In addition the relative affinities of phospholipids for the PMCA transmembrane region were evaluated by a FRET method using a pyrene labelled PC as fluorescent probe. These results were analyzed in terms of a macroscopic model that includes the affinities of the phospholipids covering the PMCA transmembrane region and a transduction parameter that correlates the composition of the boundary monolayer with the enzyme activity. The model predictions show good agreement with the experimental data, linking amphiphile/protein interactions with enzymatic activity.