A hidden Markov model for detecting confinement in single particle tracking trajectories

State-of-the-art single-particle tracking (SPT) techniques can generate long trajectories with high temporal and spatial resolution. This offers the possibility of mechanistically interpreting particle movements and behavior in membranes. To this end, a number of statistical techniques have been developed that partition SPT trajectories into states with distinct diffusion signatures, allowing a statistical analysis of diffusion state dynamics and switching behavior. Here, we develop a confinement model, within a hidden Markov framework, that switches between phases of free diffusion and confinement in a harmonic potential well. By using a Markov chain Monte Carlo algorithm to fit this model, automated partitioning of individual SPT trajectories into these two phases is achieved, which allows us to analyze confinement events. We demonstrate the utility of this algorithm on a previously published interferometric scattering microscopy data set, in which gold-nanoparticle-tagged ganglioside GM1 lipids were tracked in model membranes. We performed a comprehensive analysis of confinement events, demonstrating that there is heterogeneity in the lifetime, shape, and size of events, with confinement size and shape being highly conserved within trajectories. Our observations suggest that heterogeneity in confinement events is caused by both individual nanoparticle characteristics and the binding-site environment. The individual nanoparticle heterogeneity ultimately limits the ability of interferometric scattering microscopy to resolve molecule dynamics to the order of the tag size; homogeneous tags could potentially allow the resolution to be taken below this limit by deconvolution methods. In a wider context, the presented harmonic potential well confinement model has the potential to detect and characterize a wide variety of biological phenomena, such as hop diffusion, receptor clustering, and lipid rafts

Actin turnover ensures uniform tension distribution during cytokinetic actomyosin ring contraction

In many eukaryotes, cytokinesis is facilitated by the contraction of an actomyosin ring (AMR). The exact mechanisms that lead to this contractility are unknown, although some models posit that actin turnover in the AMR is essential. The effect of reduced actin dynamics during AMR formation has been well studied in Schizosaccharomyces pombe; however, the corresponding effects on AMR contraction are not well understood. By using mutants of the fission yeast actin severing protein Adf1, we observed that contracting AMRs display a “peeling” phenotype, where bundles of actin and myosin peel off from one side of the AMR, and are pulled across to the opposite side. This occurs multiple times during cytokinesis and is dependent on the activity of myosins Myo2, Myp2, and Myo51. We found that the distribution of Myo2 in the AMR anticorrelates with the location of peeling events, suggesting that peeling is caused by a nonuniform tension distribution around the AMR, and that one of the roles of actin turnover is to maintain a uniform tension distribution around the AMR

Human kinetochores are swivel joints that mediate microtubule attachments.

Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle - a dynamic microtubule-based force-generating machine. Connections to this spindle are mediated by sister kinetochore pairs, that form dynamic end-on attachments to microtubules emanating from opposite spindle poles. This bi-orientation generates forces that have been reported to stretch the kinetochore itself, which has been suggested to stabilise attachment and silence the spindle checkpoint. We reveal using three dimensional tracking that the outer kinetochore domain can swivel around the inner kinetochore/centromere, which results in large reductions in intra-kinetochore distance (delta) when viewed in lower dimensions. We show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, suggesting an organisational change linked to checkpoint satisfaction and/or obligatory changes in kinetochore mechanochemistry may occur before dissolution of sister chromatid cohesion

KiT : a MATLAB package for kinetochore tracking

Summary: During mitosis, chromosomes are attached to the mitotic spindle via large protein complexes called kinetochores. The motion of kinetochores throughout mitosis is intricate and automated quantitative tracking of their motion has already revealed many surprising facets of their behaviour. Here, we present ‘KiT’ (Kinetochore Tracking)—an easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies. KiT supports 2D, 3D and multi-colour movies, quantification of fluorescence, integrated deconvolution, parallel execution and multiple algorithms for particle localization

Size matters for single-cell C4 photosynthesis in Bienertia

Bienertia cycloptera belongs to a diverse set of plants, recently discovered to perform C4 photosynthesis within individual mesophyll cells. How these plants accomplish high photosynthetic efficiency without adopting Kranz anatomy remains unanswered. By modelling the processes of diffusion, capture, and release of carbon dioxide and oxygen inside a typical Bienertia mesophyll cell geometry, we show that a spatial separation as low as 10 μm between the primary and the secondary carboxylases, can, on its own, provide enough diffusive resistance to sustain a viable C4 pathway at 20 °C, with a CO2 leakage <35%. This critical separation corresponds to a cell diameter of 50 μm, consistent with the observed range where Bienertia’s mesophyll cells start to develop their characteristic mature anatomy. Our results are robust to significant alterations in model assumptions and environmental conditions, their applicability extending even to aquatic plants

Evidence for the intense exchange of MazG in marine cyanophages by horizontal gene transfer

Background: S-PM2 is a phage capable of infecting strains of unicellular cyanobacteria belonging to the genus Synechococcus. S-PM2, like other myoviruses infecting marine cyanobacteria, encodes a number of bacterial-like genes. Amongst these genes is one encoding a MazG homologue that is hypothesized to be involved in the adaption of the infected host for production of progeny phage. Methodology/Principal Findings: This study focuses on establishing the occurrence of mazG homologues in other cyanophages isolated from different oceanic locations. Degenerate PCR primers were designed using the mazG gene of S-PM2. The mazG gene was found to be widely distributed and highly conserved among Synechococcus myoviruses and podoviruses from diverse oceanic provinces. Conclusions/Significance: This study provides evidence of a globally connected cyanophage gene pool, the cyanophage mazG gene having a small effective population size indicative of rapid lateral gene transfer despite being present in a substantial fraction of cyanophage. The Prochlorococcus and Synechococcus phage mazG genes do not cluster with the host mazG gene, suggesting that their primary hosts are not the source of the mazG gene

Probing microtubule polymerisation state at single kinetochores during metaphase chromosome motion

Kinetochores regulate the dynamics of attached microtubule bundles (kinetochore-fibres, K-fibres) to generate the forces necessary for chromosome movements in mitosis. Current models suggest that poleward-moving kinetochores are attached to depolymerising K-fibres and anti-poleward-moving kinetochores to polymerising K-fibres. How the dynamics of individual microtubules within the K-fibre relate to poleward and anti-poleward movements is poorly understood. To investigate this, we developed a live-cell imaging assay combined with computational image analysis that allows eGFP-tagged EB3 (also known as MAPRE3) to be quantified at thousands of individual metaphase kinetochores as they undergo poleward and anti-poleward motion. Surprisingly, we found that K-fibres are incoherent, containing both polymerising and depolymerising microtubules – with a small polymerisation bias for anti-poleward-moving kinetochores. K-fibres also display bursts of EB3 intensity, predominantly on anti-poleward-moving kinetochores, equivalent to more coherent polymerisation, and this was associated with more regular oscillations. The frequency of bursts and the polymerisation bias decreased upon loss of kinesin-13, whereas loss of kinesin-8 elevated polymerisation bias. Thus, kinetochores actively set the balance of microtubule polymerisation dynamics in the K-fibre while remaining largely robust to fluctuations in microtubule polymerisation

Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase

Chromosome mis-segregation during mitosis leads to aneuploidy, which is a hallmark of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although their mechanistic origins and likelihood of correction are incompletely understood. Here, we combine lattice light-sheet microscopy, endogenous protein labeling, and computational analysis to define the life history of >10 kinetochores. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable risk of mis-segregation. We show that the majority of lazy kinetochores are corrected rapidly in anaphase by Aurora B; if uncorrected, they result in a higher rate of micronuclei formation. Quantitative analyses of the kinetochore life histories reveal a dynamic signature of metaphase kinetochore oscillations that forecasts their anaphase fate. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with an additional layer of anaphase error correction required for stable karyotype propagation

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells

This protocol measures the 3D Euclidean distance (Δ3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ3D is corrected through a Bayesian algorithm, with ΔEC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure ΔEC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells)

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing “HURP-gap”. The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps