PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules

Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-PAINT

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments

Associations of Partner Support and Acculturation With Physical Activity in Mexican American Women.

IntroductionInsufficient physical activity (PA) and obesity-related health conditions have reached epidemic proportions worldwide. Mexican American women (MAW) report low leisure time physical activity. Few studies examine activities beyond leisure time. Qualitative research suggests that partner support influence provides a cultural approach relevant to PA among MAW.MethodThis cross-sectional study used an ecological model to investigate community (the physical environment), interpersonal (partner support, attitudinal familism), and intrapersonal (age, health conditions, acculturation, employment, and body mass index) factors associated with PA among 112 MAW. Community-based participatory research recommendations guided the preparatory phase of the study and the face-to-face interviews. Frequencies and descriptive statistics were computed. Multivariable linear regression analyses were used to examine associations between study variables.ResultsModerate to high PA levels were found based on combined activities performed during leisure time, transportation, household tasks, and occupational duties. Women with greater partner support reported higher PA levels. Although acculturation levels were low among women, those with higher acculturation were found to be more physically active.ConclusionsFuture studies should examine strategies to increase partner support and address acculturation within intervention programs to enhance overall PA among MAW

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method to quantify clustering in super-resolution localization images of static surfaces in two dimensions. The method also describes how over-counting of labeled molecules contributes to apparent self-clustering and how the effective lateral resolution of an image can be determined. This treatment applies to clustering of proteins and lipids in membranes, where there is significant interest in using super-resolution localization techniques to probe membrane heterogeneity. When images are quantified using pair correlation functions, the magnitude of apparent clustering due to over-counting will vary inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. Over-counting does not yield apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (Fc{\epsilon}RI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (SEM). We find that apparent clustering of labeled IgE bound to Fc{\epsilon}RI detected with both methods arises from over-counting of individual complexes. Thus our results indicate that these receptors are randomly distributed within the resolution and sensitivity limits of these experiments.Comment: 22 pages, 5 figure

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle

Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy

Reporting on novel complex intervention development for adults with social communication impairments after acquired brain injury

Purpose: Interventions are often poorly described in published controlled trials, with relatively little information regarding intervention development, content and fidelity. This makes it difficult to conduct replication studies, interpret and compare findings across studies and for therapists to deliver the intervention in clinical practice. Complete reporting of interventions (including fidelity) is now recommended for treatment studies, and this standardised approach is achieved using the Template for Intervention Description and Replication (TIDieR). The aim of this paper is to describe the multi-phase process of developing a novel intervention for adults with acquired brain injury (ABI), and report on the findings from involving practicing therapists in this process. Methods: Phase 1 involved a review of relevant literature and specifying the intervention as a prototype intervention manual. Phase 2 comprised a focus group with eight practicing therapists exploring their experiences and perceptions of the intervention, potential active components, and essential elements; it also included review of the prototype manual. Data from the focus group discussion was transcribed and analysed thematically. Phase 3 investigated actual fidelity of the intervention undertaken, achieved by observers viewing videoed sessions and appraising against the fidelity checklist, which was then analysed using Cohen’s kappa. Results: Project-based intervention was defined as having six essential elements: a project or tangible end product focus; group-based intervention; individualised communication-based goals; communication partner involvement; acknowledgement and support of participants’ cognitive ability; and consideration and plan to address impaired awareness. Analysis of focus group data revealed four themes of essential elements; group context; therapeutic skills; and manual core components and informed the development of a fidelity checklist with 13 essential and 6 desirable criteria. Fidelity assessed using percent agreement was acceptable for almost all rater pairs; where significant, Kappa coefficients had values ranging from poor to excellent (k=0.34 – 1.0) depending on rater pair and session. Discussion: The TIDieR framework provided a clear systematic approach for the complete description and reporting of a complex communication intervention for people with ABI. This paper comprehensively described the development and manualisation of an intervention in collaboration with practicing therapists which can be used for future testing. In addition, the process undertaken has the potential to inform rehabilitation researchers in other fields on the development of complex interventions

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.We thank the Wellcome Trust for the PhD studentship of MP (093756/B/10/Z), and the Royal Society for the University Research Fellowship of SFL (UF120277). The work by SB and DL was also funded by the Wellcome Trust (082010/Z/07/Z). UE and MH acknowledge funding by the German Science Foundation (grants EXC 115 and SFB 902). SB is funded by a BBSRC grant (BB/K013726/1). AMC acknowledges ERC Award 268788-SMI-DDR. We also thank the European Commision for support through the 4DCellFate project (EC FP7 CP 277899).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.012543

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds