Biology subjects

Waithe, D.

Publications and source records attributed to Waithe, D..

6 recordsLinked to original sources

bioRxiv2018

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2-1 m) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

biophysics↗

bioRxiv2018

Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging

The diffusion dynamics in the cellular plasma membrane provides crucial insights into the molecular interactions, organization and bioactivity. Fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (STED-FCS) measures such dynamics with high spatial and temporal resolution and reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method; line interleaved excitation scanning STED-FCS (LIESS-FCS) which discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatio-temporal organization of GPI-anchored proteins in the plasma membrane of live cells which interestingly show multiple diffusion modes at different spatial positions.

biophysics↗

bioRxiv2017

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse -globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the -globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

cell biology↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗

bioRxiv2017

Brain Development: Machine Learning Analysis Of Individual Stem Cells In Live 3D Tissue

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D \"point- and-click\" user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on these datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues.\n\nSummaryHailstone et al. develop CytoCensus, a \"point-and-click\" supervised machine-learning image analysis software to quantitatively identify defined cell classes and divisions from large multidimensional data sets of complex tissues. They demonstrate its utility in analysing challenging developmental phenotypes in living explanted Drosophila larval brains, mammalian embryos and zebrafish organoids. They further show, in comparative tests, a significant improvement in performance over existing easy-to-use image analysis software.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC=\"FIGDIR/small/137406v4_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (93K):\norg.highwire.dtl.DTLVardef@68912corg.highwire.dtl.DTLVardef@11300bdorg.highwire.dtl.DTLVardef@95810corg.highwire.dtl.DTLVardef@14b77a6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICytoCensus: machine learning quantitation of cell types in complex 3D tissues\nC_LIO_LISingle cell analysis of division rates from movies of living Drosophila brains in 3D\nC_LIO_LIDiverse applications in the analysis of developing vertebrate tissues and organoids\nC_LIO_LIOutperforms other image analysis software on challenging, low SNR datasets tested\nC_LI

developmental biology↗

bioRxiv2017

Polarity sensitive probes for super resolution STED microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

biophysics↗