17 research outputs found
Spatial Point Pattern Analysis of Neurons Using Ripley's K-Function in 3D
The aim of this paper is to apply a non-parametric statistical tool, Ripley's K-function, to analyze the 3-dimensional distribution of pyramidal neurons. Ripley's K-function is a widely used tool in spatial point pattern analysis. There are several approaches in 2D domains in which this function is executed and analyzed. Drawing consistent inferences on the underlying 3D point pattern distributions in various applications is of great importance as the acquisition of 3D biological data now poses lesser of a challenge due to technological progress. As of now, most of the applications of Ripley's K-function in 3D domains do not focus on the phenomenon of edge correction, which is discussed thoroughly in this paper. The main goal is to extend the theoretical and practical utilization of Ripley's K-function and corresponding tests based on bootstrap resampling from 2D to 3D domains
Three-Dimensional Spatial Analyses of Cholinergic Neuronal Distributions Across The Mouse Septum, Nucleus Basalis, Globus Pallidus, Nucleus Accumbens, and Caudate-Putamen
Neuronal networks are regulated by three-dimensional spatial and structural properties. Despite robust evidence of functional implications in the modulation of cognition, little is known about the three-dimensional internal organization of cholinergic networks in the forebrain. Cholinergic networks in the forebrain primarily occur in subcortical nuclei, specifically the septum, nucleus basalis, globus pallidus, nucleus accumbens, and the caudate-putamen. Therefore, the present investigation analyzed the three-dimensional spatial organization of 14,000 cholinergic neurons that expressed choline acetyltransferase (ChAT) in these subcortical nuclei of the mouse forebrain. Point process theory and graph signal processing techniques identified three topological principles of organization. First, cholinergic interneuronal distance is not uniform across brain regions. Specifically, in the septum, globus pallidus, nucleus accumbens, and the caudate-putamen, the cholinergic neurons were clustered compared with a uniform random distribution. In contrast, in the nucleus basalis, the cholinergic neurons had a spatial distribution of greater regularity than a uniform random distribution. Second, a quarter of the caudate-putamen is composed of axonal bundles, yet the spatial distribution of cholinergic neurons remained clustered when axonal bundles were accounted for. However, comparison with an inhomogeneous Poisson distribution showed that the nucleus basalis and caudate-putamen findings could be explained by density gradients in those structures. Third, the number of cholinergic neurons varies as a function of the volume of a specific brain region but cell body volume is constant across regions. The results of the present investigation provide topographic descriptions of cholinergic somata distribution and axonal conduits, and demonstrate spatial differences in cognitive control networks. The study provides a comprehensive digital database of the total population of ChAT-positive neurons in the reported structures, with the x,y,z coordinates of each neuron at micrometer resolution. This information is important for future digital cellular atlases and computational models of the forebrain cholinergic system enabling models based on actual spatial geometry
Activity-dependent refinement of the developing visual system. A comparative study across retinal ganglion cell populations and target nuclei
The formation of the mammalian visual system is a complex process that takes place in several phases and includes neurogenesis, axon guidance, axonal refinement and circuit assembly. The last stage of this process occurs after birth but before eye opening. During this period, axon terminals from retinal ganglion cells (RGCs) first extensively arborize in the different visual nuclei and then refine and establish appropriate connections. It is known that the spontaneous activity generated in the immature retina during perinatal ages plays an important role in this axonal refinement process but it is not clear to what extent such retinal activity differentially influences the refinement of the distinct populations of RGCs when they project to specific visual nuclei. To address this issue we have generated conditional mouse lines to alter spontaneous activity in different populations of RGCs and we have analyzed the projection patterns of RGCs in different visual nuclei in each of these mouse lines. Our results show that the alteration of spontaneous activity in RGCs affects axon refinement in the image-forming nuclei such as the lateral geniculate nucleus and the superior colliculus, supporting previous publications. Interestingly, we also observed that, although to a lesser extent than in the image-forming nuclei, retinal spontaneous activity correlation is important for the refinement of RGC axons in the non-image-forming nuclei such as the pretectal olive nucleus or the suprachiasmatic nucleus
The spatial dynamics of insulin-regulated GLUT4 dispersal
Insulin regulates glucose homeostasis by stimulation of glucose transport into adipose and muscle tissues through the regulated trafficking of glucose transporter 4 (GLUT4). In response to insulin GLUT4 rapidly translocates from intracellular storage sites to the plasma membrane where it facilitates glucose uptake. Significant impairments in glucose transport and GLUT4 trafficking are a major hallmark of diabetes mellitus type II. Recent advances in light microscopy techniques enabled the study of GLUT4 dynamics in the plasma membrane and it was reported that the transporter was clustered in the basal state and insulin stimulation resulted in GLUT4 dispersal.
The main aim of this study was to develop a microscopy-based assay to study and quantify insulin-stimulated GLUT4 dispersal dynamics in the plasma membrane. Insulin-stimulated GLUT4 dispersal has only been observed in adipocytes and therefore we have chosen this model as a starting point to investigate the molecular mechanisms behind GLUT4 clustering and dispersal. We explored a range of cluster analysis methods to find the most suitable way to quantify GLUT4 clustering dynamics. Furthermore, this project aimed to optimise super resolution imaging in a variety of cell culture models to determine whether insulin-stimulated GLUT4 dispersal operates in skeletal and cardiac muscle and whether this process is affected by disease.
Using a range of approaches we showed that insulin results in GLUT4 translocation and dispersal within the plasma membrane of 3T3-L1 adipocytes. We found that AMPK activation attenuated insulin-stimulated glucose uptake in 3T3-L1 adipocytes and also GLUT4 dispersal. It was observed that cholesterol depletion resulted in increased glucose uptake rates and GLUT4 clustering. Knock down of the membrane-localised protein EFR3 that has previously been shown to be involved in glucose uptake resulted in disruption of GLUT4 dispersal in adipocytes. We also found that HeLa cells show similar insulin-stimulated GLUT4 dispersal as adipocytes and suggest that HeLa cells are a suitable experimental model for initial studies of GLUT4 trafficking and dispersal. Chronic insulin treatment was observed to induce a state of cellular insulin resistance in 3T3-L1 adipocytes and resulted in reduced GLUT4 translocation and a more clustered GLUT4 configuration for both basal and insulin-stimulated cells. This indicates that insulin resistance affects intracellular GLUT4 trafficking pathways as well as the organization of the transporter within the plasma membrane in adipocytes. Moreover, we found a negative correlation between adipocyte cell area and insulin-stimulated GLUT4 translocation.
We also report that insulin did not stimulate the reorganisation of the transferrin receptor in the plasma membrane of HeLa cells suggesting that insulin-stimulated GLUT4 dispersal did not originate from endosomal compartments in HeLa cells and that this observed effect may be specific for GLUT4. Finally, we observed that insulin did not affect GLUT4 distribution in the membrane of a commercially available model of skeletal muscle from healthy and diabetic donors. Sortilin is a sorting receptor involved in the formation of GLUT4 containing vesicles and levels of this protein were found to be reduced in skeletal muscle myotubes derived from a diabetic donor.
Finally, we discovered that insulin stimulated GLUT4 dispersal also operates in stem cell-derived cardiomyocytes and have investigated GLUT4 dispersal in a variety of in vitro models of cardiac muscle tissue.
Taken together, this thesis has detailed several novel findings regarding the regulation of GLUT4 clustering in adipose and muscle tissues. A robust assay to measure GLUT4 dispersal has been established and molecular mechanisms behind the observed GLUT4 clustering dynamics have been described in adipocytes. Furthermore GLUT4 clustering was characterised in several cell culture models of skeletal and cardiac muscle for the first time
In Situ Transcriptome Accessibility Sequencing (INSTA-seq)
Subcellular RNA localization regulates spatially polarized cellular processes, but unbiased investigation of its control in vivo remains challenging. Current hybridization-based methods cannot differentiate small regulatory variants, while in situ sequencing is limited by short reads. We solved these problems using a bidirectional sequencing chemistry to efficiently image transcript-specific barcode in situ , which are then extracted and assembled into longer reads using NGS. In the Drosophila retina, genes regulating eye development and cytoskeletal organization were enriched compared to methods using extracted RNA. We therefore named our method In Situ Transcriptome Accessibility sequencing (INSTA-seq). Sequencing reads terminated near 3’ UTR cis -motifs (e.g. Zip48C, stau ), revealing RNA-protein interactions. Additionally, Act5C polyadenylation isoforms retaining zipcode motifs were selectively localized to the optical stalk, consistent with their biology. Our platform provides a powerful way to visualize any RNA variants or protein interactions in situ to study their regulation in animal development
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Optical imaging methods for the study of disease models from the nano to the mesoscale
The visualisation of disease phenotypes allows scientists to study fundamental mechanisms of disease. Optical imaging methods are useful not only to observe anatomical features of biological samples, but also to infer interactions between molecular species using fluorescence labelling. This thesis presents the development of imaging and analysis tools to study biological questions in three models of disease, with samples ranging from the sub-cellular to the organ scale.
First, the role of the alpha-synuclein (a-syn) protein, whose dysfunction is a hallmark of Parkinson’s Disease, was studied with respect to vesicle trafficking at the synapse. Synaptic vesicles are ∼40 nm in diameter; imaging vesicles therefore requires methods with resolution below the diffraction limit. Single-molecule localisation microscopy (SMLM), which circumvents the diffraction limit by separating fluorophore emission in time to localise individual molecules in space with ∼20 nm precision, was thus implemented to study a-syn in purified synaptic boutons. A software package was developed to analyse the colocalisation of a-syn with internalised vesicles, and the clustering of a-syn under differing synaptic calcium levels. The colocalisation of a-syn and internalised vesicles was found to be temperature independent, suggesting that a-syn is involved in non-canonical trafficking mechanisms. Ground truth simulations from a synaptosome model were used to benchmark two cluster analysis methods. Both methods applied on the experimental data showed that a-syn becomes less clustered at low synaptic calcium levels.
Second, the spatiotemporal association of ESCRT-II, a protein complex whose role in the budding of the human immunodeficiency virus (HIV) was previously considered dispensable, and the HIV polyprotein Gag was studied during viral egress using novel image analysis tools. A nearest-neighbour analysis showed the ESCRT-II protein EAP45 colocalises with Gag similarly to ALIX, a protein well known to be involved in HIV budding. However, upon deletion of EAP45’s N-terminus, its colocalisation with Gag was significantly impaired, highlighting the importance of this EAP45 domain in linking to Gag. Single particle tracking was used to trace the trajectories of EAP45 and Gag in live cells, and an algorithm was developed to visualise the simultaneous motion of two particles; these analyses revealed three types of potential dynamic interaction between EAP45 and Gag.
Finally, an open-source instrument to visualise phenotypes from large organs in 3D was developed for the study of chronic obstructive pulmonary disease (COPD) models. The instrument implements Optical Projection Tomography, a technique which can reconstruct cross-sectional slices of a transparent object from its orthographic projections, using off-the- shelf components and novel ImageJ plugins for artefact correction and volume reconstructions. Excised and cleared mouse lungs were imaged in which high order airways can be discerned with 50 μm resolution. The raw lung data, instructions for building the instrument, the free ImageJ plugins, and a detailed software manual are available in an online repository to encourage the widespread use of OPT for imaging large samples.Gates Cambridg
Characterizing Crown Struture of Three Interior Northwest Conifer Species Using Terrestrial Laser Scanning
Emerging interests in wildland fire behavior and risk, bioenergy utilization, carbon sequestration, and wildlife conservation increasingly rely on accurate assessments of the amount and location of biomass within the dominant plants on the landscape, often at finer scales than traditional methods have provided. At the tree scale, current studies often distribute biomass uniformly through simple volumes (e.g., cones and cylinders). However, biomass is heterogeneous at a variety of scales from needle clusters to groups of trees. This thesis presents techniques for using terrestrial laser scanning data to define crown profiles and describe within-crown heterogeneity in Pseudotusga menziesii, Pinus ponderosa, and Abies lasiocarpa of the Interior Northwest. Crown profiles were modeled using parametric curves applied to crown-length normalized laser point clouds, dimensioned by height above ground and distance from bole-centroids. A crown-base metric was derived from the laser data and compared to conventional field measurements. For all species, a modified Weibull curve fit crown points with significantly smaller error than a beta curve, cone, or cylinder; crown profile Weibull curves were species-specific and not interchangeable without producing signifcantly greater error. Within-crown patterning was described using a 3-D form of the Ripley’s K function. Ripley’s K analysis detected maximum clustering occuring at scales of 1.25 – 2.50 percent of crown length (e.g., 25-50 cm radius clusters in a 20 meter crown). P. ponderosa demonstrated clustering over the largest range of scales and to the greatest degree, while A. lasiocarpa exhibited clustering over the smallest range of scales. The scale of clustering did not change when points roughly corresponding to branchwood were excluded from the analysis. This study provides groundwork for predicting the spatial distribution of biomass with tree crowns. Limitations of the work include uncertainty regarding the impacts of occlusion of inner crowns and the relationships between laser points and foliage-branch elements, and the lack of spatial explicitness inherent to Ripley’s K. Future work should examine these issues with an eye toward refinement of predictive models linking traditional biomass allometry with spatial arrangement of canopy material
Spatiotemporal coordination of signaling at single molecule resolution
Advances in live-cell single-molecule imaging and modeling over the past decade have invited the closer study of biological structure and dynamics at the nanoscale. The higher resolution of these single-molecule experiments results in finely-grained datasets that can feed detailed quantitative models. Likewise, single-molecule models can account for microscopic details such as noise and heterogeneity inherent to diffusional and chemical processes, which are often neglected in models based on bulk concentrations. Examining microscale biological structures at single molecule resolution in living cells has led to new findings, such as the dynamic regulation of nanoscale structure. I cover three topics from the perspective of single molecules. Chapters 1-3 are on modeling the spatiotemporal coordination of both spontaneous and pheromone-guided yeast polarity establishment. Chapter 4 is on computational modeling and analysis for a technique called Binder/Tag, which we applied to study the conformational dynamics of the protein Src kinase in living cells. Chapter 5 is on modeling clustering-mediated activation of immunoreceptors, using the phagocytic receptor FcγRIIA as a prototypical example.Doctor of Philosoph
Spatial, temporal and functional molecular architecture of the munc18-syntaxin interaction
Regulation of soluble N-ethylmaleimide-sensitive fusion protein attachment protein
receptors (SNARE) mediated exocytosis is dependent upon four key proteins; the
vesicular SNARE synaptobrevin, target SNAREs SNAP-25 and syntaxin and the
Sec1/Munc18 (SM) protein munc18-1. Despite the munc18-1-syntaxin interaction being
central to regulated vesicle exocytosis the spatial and temporal pattern of their molecular
distribution and interaction in neuroendocrine and neuronal cells remains undefined.
Using in vitro and molecular approaches this thesis shows that disruption of the munc18-
1-syntaxin-N-terminal interaction results in significant changes in syntaxin localisation,
membrane-proximal vesicle dynamics and fusion efficiency within neuroendocrine cells.
Using the super-resolution techniques Ground State Depletion-Individual molecule return
(GSDIM) Microscopy and Photoactivation Localisation Microscopy (PALM) this thesis
has demonstrated that the spatial distribution of single munc18-1 molecules is non-random
and that few munc18-1 molecules are required for exocytosis to proceed in
neuroendocrine cells. Furthermore, targeted disruption of the N-terminal interaction
resulted only in a reorganisation of interaction with syntaxin with no change in the
molecular spatial pattern of secretory vesicles, syntaxin or munc18-1. Single molecule
imaging PALM (sptPALM) enabled the investigation of the complex spatio-temporal
behaviours of single munc18-1 molecules in living neuroendocrine cells. Spatially
resolved maps of single munc18-1 molecules demonstrated that munc18-1 exhibits a
caged motion within areas of the plasma membrane and were found to move between
molecular storage depots distinct from vesicle docking sites. To explore the precise
spatial and temporal sequence of interactions between syntaxin and munc18-1 in living
neurons, super-resolution imaging techniques PALM and sptPALM were employed. Two
kinetically and spatially distinct populations of munc18-1 molecules co-exist within a
living neuron and munc18-1 requires syntaxin to traffic efficiently in axons but not for its
retention in nerve terminals. Moreover, Fluorescence Correlation Spectroscopy (FCS)
revealed that the majority of munc18-1 molecules do not interact with syntaxin in nerve
terminals and the diffusion rate of syntaxin is significantly slowed down upon neuronal
depolarisation
Exploring coherence and disorder: an analysis of spatial patterning within the neuromesodermal progenitor niche
How regulatory frameworks control cellular identity and organisation via cell-cell
communication is a poorly understood yet fundamental process in development.
Different signalling pathway regulatory mechanisms can create a variety of spatial
patterns of transcription factor (TF) expression and differentiation, however
quantitatively assessing multicellular organisation in 3D has only recently been made
possible due to advances in imaging and image analysis tools. Downstream analysis
methods are still in their infancy and require further development to utilise the newly
available information.
Neuromesodermal progenitors (NMPs) are a bipotent population of cells in the post
gastrulation epiblast that self-renew while allocating cells to neural and mesodermal
tissues of the trunk. Gradients of Retinoic acid, Wnt, and FGF signalling direct the
neural vs mesoderm cell fate decision and regionalise the axial progenitor niches,
but the spatial patterning of TF expression has not been quantified. Further, previous
work shows that the Notch signalling pathway also regulates the cell fate decision in
NMPs, but this is not well characterised and it’s unknown if Notch contributes to any
TF patterning.
I aimed to use systems biology inspired analysis methods to investigate the role of
Notch signalling in NMP fate and patterning. First, I investigated the pro-neural effect
of Notch inhibition in NMPs and identified which Notch components are expressed.
Then, I developed quantitative analysis methods that show differential spatial
patterning of key TF fate markers in NMP niches in vitro and in vivo. Finally, I
explored how Notch influences this patterning, overall providing a framework for
future work to analyse spatial gene expression data