Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments.

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Protein motion in the nucleus: from anomalous diffusion to weak interactions.

Understanding how transcription factors (TFs) regulate mammalian gene expression in space and time is a central topic in biology. To activate a gene, a TF has first to diffuse in the available space of the nucleus until it reaches a target DNA sequence or protein (target site). This eventually results in the recruitment of the whole transcriptional machinery. All these processes take place in the mammalian nucleoplasm, a highly organized and dynamic environment, in which some complexes transiently assemble and break apart, whereas others appear more stable. This diversity of dynamic behaviors arises from the number of biomolecules that make up the nucleoplasm and their pairwise interactions. Indeed, interactions energies that span several orders of magnitude, from covalent bounds to transient and dynamic interactions, can shape nuclear landscapes. Thus, the nuclear environment determines how frequently and how fast a TF contacts its target site, and it indirectly regulates gene expression. How exactly transient interactions are involved in the regulation of TF diffusion is unclear, but are reflected by live cell imaging techniques, including single-particle tracking (SPT). Overall, the macroscopic result of these microscopic interactions is almost always anomalous diffusion, a phenomenon widely studied and modeled. Here, we review the connections between the anomalous diffusion of a TF observed by SPT and the microscopic organization of the nucleus, including recently described topologically associated domains and dynamic phase-separated compartments. We propose that anomalous diffusion found in SPT data result from weak and transient interactions with dynamic nuclear substructures, and that SPT data analysis would benefit from a better description of such structures.

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme.

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.

High-spatial resolution epidemic surveillance of bacterial meningitis in the African meningitis belt in Burkina Faso.

Despite improved surveillance capacities and WHO recommendations for subdistrict analysis, routine epidemic surveillance of acute bacterial meningitis in the African meningitis belt remains largely limited to the district level. We evaluated the appropriateness and performance of analyses at higher spatial resolution. We used suspected meningitis surveillance data at health centre (HC) resolution from Burkina Faso from 14 health districts spanning years 2004-2014 and analysed them using spatio-temporal statistics and generative models. An operational analysis compared epidemic signals at district and HC-level using weekly incidence thresholds. Eighty-four percent (N = 98/116) of epidemic clusters spanned only one HC-week. Spatial propagation of epidemic clusters was mostly limited to 10-30 km. During the 2004-2009 (with serogroup A meningitis) and 2010-2014 (after serogroup A elimination) period, using weekly HC-level incidence thresholds of 100 and 50 per 100,000 respectively, we found a gain in epidemic detection and timeliness in 9 (41% of total) and 10 (67%), respectively, district years with at least one HC signal. Individual meningitis epidemics expanded little in space, suggesting that a health centre level analysis is most appropriate for epidemic surveillance. Epidemic surveillance could gain in precision and timeliness by higher spatial resolution. The optimal threshold should be defined depending on the current background incidence of bacterial meningitis.

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing minor roles for cross-links and topological effects and challenging the view that interphase chromatin is a gel-like material. Our technology opens avenues for future research in areas from chromosome mechanics to genome functions.

How the Genome Folds: The Biophysics of Four-Dimensional Chromatin Organization.

The genetic information that instructs transcription and other cellular functions is carried by the chromosomes, polymers of DNA in complex with histones and other proteins. These polymers are folded inside nuclei five orders of magnitude smaller than their linear length, and many facets of this folding correlate with or are causally related to transcription and other cellular functions. Recent advances in sequencing and imaging-based techniques have enabled new views into several layers of chromatin organization. These experimental findings are accompanied by computational modeling efforts based on polymer physics that can provide mechanistic insights and quantitative predictions. Here, we review current knowledge of the main levels of chromatin organization, from the scale of nucleosomes to the entire nucleus, our current understanding of their underlying biophysical and molecular mechanisms, and some of their functional implications.

Atmospheric Dust, Early Cases, and Localized Meningitis Epidemics in the African Meningitis Belt: An Analysis Using High Spatial Resolution Data.

BACKGROUND: Bacterial meningitis causes a high burden of disease in the African meningitis belt, with regular seasonal hyperendemicity and sporadic short, but intense, localized epidemics during the late dry season occurring at a small spatial scale [i.e., below the district level, in individual health centers (HCs)]. In addition, epidemic waves with larger geographic extent occur every 7-10 y. Although atmospheric dust load is thought to be an essential factor for hyperendemicity, its role for localized epidemics remains hypothetic. OBJECTIVES: Our goal was to evaluate the association of localized meningitis epidemics in HC catchment areas with the dust load and the occurrence of cases in the same population early in the dry season. METHODS: We compiled weekly reported cases of suspected bacterial meningitis at the HC resolution for 14 districts of Burkina Faso for the period 2004-2014. Using logistic regression, we evaluated the association of epidemic HC-weeks with atmospheric dust [approximated by the aerosol optical thickness (AOT) satellite product] and with the observation of early meningitis cases during October-December. RESULTS: Although AOT was strongly associated with epidemic HC-weeks in crude analyses across all HC-weeks during the meningitis season [odds ratio (OR) [Formula: see text]; 95% CI: 4.90, 9.50], the association was no longer apparent when controlling for calendar week (OR [Formula: see text]; 95% CI: 0.60, 1.50). The number of early meningitis cases reported during October-December was associated with epidemic HC-weeks in the same HC catchment area during January-May of the following year (OR for each additional early case [Formula: see text]; 95% CI: 1.06, 1.21). CONCLUSIONS: Spatial variations of atmospheric dust load do not seem to be a factor in the occurrence of localized meningitis epidemics, and the factor triggering them remains to be identified. The pathophysiological mechanism linking early cases to localized epidemics is not understood, but their occurrence and number of early cases could be an indicator for epidemic risk. https://doi.org/10.1289/EHP2752.

Robust model-based analysis of single-particle tracking experiments with Spot-On.

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

The association between respiratory tract infection incidence and localised meningitis epidemics: an analysis of high-resolution surveillance data from Burkina Faso.

Meningococcal meningitis epidemics in the African meningitis belt consist of localised meningitis epidemics (LME) that reach attack proportions of 1% within a few weeks. A meningococcal serogroup A conjugate vaccine was introduced in meningitis belt countries from 2010 on, but LME due to other serogroups continue to occur. The mechanisms underlying LME are poorly understood, but an association with respiratory pathogens has been hypothesised. We analysed national routine surveillance data in high spatial resolution (health centre level) from 13 districts in Burkina Faso, 2004-2014. We defined LME as a weekly incidence rate of suspected meningitis ≥75 per 100,000 during ≥2 weeks; and high incidence episodes of respiratory tract infections (RTI) as the 5th quintile of monthly incidences. We included 10,334 health centre month observations during the meningitis season (January-May), including 85 with LME, and 1891 (1820) high-incidence episodes of upper (lower) RTI. In mixed effects logistic regression accounting for spatial structure, and controlling for dust conditions, relative air humidity and month, the occurrence of LME was strongly associated with high incidence episodes of upper (odds ratio 23.9, 95%-confidence interval 3.1-185.3), but not lower RTI. In the African meningitis belt, meningitis epidemics may be triggered by outbreaks of upper RTI.

Geometry of the nucleus: a perspective on gene expression regulation.

Gene expression control results from the combined interactions of the nearly hundred proteins forming the pre-initiation complex, thousands of transcription regulators, and genomic DNA. In the recent years, new technologies have revealed several key aspects of nuclear spatial organization that showed a fine interplay between the function of nuclear proteins, their 3D organization, and their dynamics. Here we review several concepts that link biochemical reactivity in the nucleus to its 3D spatial organization. We present the analogies between the emerging understanding of nuclear organization in the field of cell biology, and the more established disciplines of heterogeneous catalysis and the physics of random walks. We provide several recent examples showing how nuclear geometry affects protein reactivity in the nucleus.