Dendritic Spine Shape Analysis: A Clustering Perspective

Functional properties of neurons are strongly coupled with their morphology. Changes in neuronal activity alter morphological characteristics of dendritic spines. First step towards understanding the structure-function relationship is to group spines into main spine classes reported in the literature. Shape analysis of dendritic spines can help neuroscientists understand the underlying relationships. Due to unavailability of reliable automated tools, this analysis is currently performed manually which is a time-intensive and subjective task. Several studies on spine shape classification have been reported in the literature, however, there is an on-going debate on whether distinct spine shape classes exist or whether spines should be modeled through a continuum of shape variations. Another challenge is the subjectivity and bias that is introduced due to the supervised nature of classification approaches. In this paper, we aim to address these issues by presenting a clustering perspective. In this context, clustering may serve both confirmation of known patterns and discovery of new ones. We perform cluster analysis on two-photon microscopic images of spines using morphological, shape, and appearance based features and gain insights into the spine shape analysis problem. We use histogram of oriented gradients (HOG), disjunctive normal shape models (DNSM), morphological features, and intensity profile based features for cluster analysis. We use x-means to perform cluster analysis that selects the number of clusters automatically using the Bayesian information criterion (BIC). For all features, this analysis produces 4 clusters and we observe the formation of at least one cluster consisting of spines which are difficult to be assigned to a known class. This observation supports the argument of intermediate shape types.Comment: Accepted for BioImageComputing workshop at ECCV 201

3D-surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semi-supervised deep learning

Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nm range and strong contrast for membranous structures without requirement for labeling or chemical fixation. The short acquisition time and the relatively large volumes acquired allow for fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D-segmentation pipeline based on semi-supervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells

Automated 4D analysis of dendritic spine morphology: applications to stimulus-induced spine remodeling and pharmacological rescue in a disease model

Uncovering the mechanisms that regulate dendritic spine morphology has been limited, in part, by the lack of efficient and unbiased methods for analyzing spines. Here, we describe an automated 3D spine morphometry method and its application to spine remodeling in live neurons and spine abnormalities in a disease model. We anticipate that this approach will advance studies of synapse structure and function in brain development, plasticity, and disease

살아있는 뉴런과 동물에서 mRNA 관찰에 대한 연구

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 물리·천문학부(물리학전공), 2021.8. 이병훈.mRNA는 유전자 발현의 첫번째 산물이면서, 리보솜과 함께 단백질을 합성한다. 특히 뉴런에서, 몇몇 RNA들은 자극에 의해 만들어지고, 뉴런의 특정 부분으로 수송되어 국소적으로 단백질 양을 조절할 수 있게 한다. 최근 mRNA 표지 기술의 발전으로 살아있는 세포에서 단일 mRNA를 관찰하는 것이 가능해졌다. 이 연구에서, 우리는 RNA 이미징 기술을 이용해, 기억 형성과 상기할 때 활성화된 뉴런의 집합을 찾는 것 뿐 아니라, 뉴런의 축삭돌기에서 mRNA가 어떻게 수송되는지를 관찰했다. 이 논문의 첫 부분에서 우리는 신경 자극에 반응해서 만들어지는 것으로 알려진, Arc 유전자의 전사를 관찰하였다. 기억은 engram 혹은 기억 흔적 (memory trace)라고 불리는 뉴런들의 집합에 저장되어 있다고 생각된다. 그러나, 시간에 따라서 이런 기억 흔적세포들의 집합이 어떻게 변하고, 변화하면서도 어떻게 정보를 유지할 수 있는지 잘 알려져 있지 않다. 또한, 살아있는 동물에서, 기억 흔적세포를 긴 시간 동안 여러 번 찾아내는 것은 어려운 일이었다. 이 연구에서는 genetically-encoded RNA indicator (GERI) 기술을 사용해, 기억 흔적세포의 표식으로 널리 사용되는 Arc mRNA의 전사과정을 살아있는 쥐에서 관찰하였다. GERI를 이용함으로써, 기존 방법들의 한계점이었던 시간 제약 없이, 실시간으로 Arc를 발현하는 뉴런들을 찾아낼 수 있었다. 쥐에게 공간 공포 기억을 주고 나서 여러 번 기억을 상기시키는 행동실험 후에 Arc를 발현하는 세포를 식별했을 때, CA1에서는 Arc를 발현하는 세포가 이틀 후에는 더 이상 활성화되지 않았으나, RSC의 경우 4퍼센트의 뉴런들이 계속해서 활성화하는 것을 관찰했다. 신경활동과 유전자 발현을 같이 조사하기 위해, 쥐가 가상 환경을 탐험하고 있을 때 GERI와 칼슘 이미징을 동시에 진행하였다. 그 결과, 기억을 형성할 때와 상기시킬 때 Arc를 발현했던 뉴런들이 기억을 표상하는 것을 알아낼 수 있었다. 이처럼 GERI 기술을 이용해 살아있는 동물에서 유전자 발현된 세포를 찾아내는 방식은 다양한 학습 및 기억 과정에서 기억 흔적세포의 dynamics에 대해 알아낼 수 있을 것으로 기대된다. 이 논문의 두번째 부분에서, 우리는 세포 골격의 기본 구성 단위가 되는 β-actin의 mRNA를 축삭돌기에서 관찰하였다. mRNA의 국소화 (localization)를 통한 국소 단백질 합성은 축삭돌기 (axon)의 성장과 재생에 중요한 역할이 있다고 알려져 있다. 하지만, 아직 mRNA의 국소화가 축삭돌기에서 어떻게 조절되고 있는지 잘 알려져 있지 않다. 이 문제를 해결하기 위해서, 우리는 모든 β-actin mRNA가 형광으로 표지된 유전자 변형 쥐를 이용해, 살아있는 축삭돌기에서 β-actin mRNA를 관찰하였다. 이 쥐의 뉴런을 축삭을 구분해 줄 수 있는 미세유체 장치 (microfluidic device)에 배양한 뒤에, β-actin mRNA를 관찰하고 추적을 진행했다. 축삭은 세포 몸통으로부터 길게 자라기 때문에 mRNA가 먼 거리를 수송되어야 함에도 불구하고, 대부분의 mRNA가 수상돌기에 비해 덜 움직이고 작은 영역에서 움직이는 것을 보았다. 우리는 β-actin mRNA가 주로 축삭돌기의 가지가 될 수 있는 filopodia 근처와, 시냅스가 만들어지는 bouton 근처에 국소화되는 것을 관찰했다. Filopodia와 bouton이 actin이 풍부한 부분으로 알려져 있기 때문에, 우리는 액틴 필라멘트와 β-actin mRNA의 움직임간에 연관성을 조사했다. 흥미롭게도, 우리는 β-actin mRNA가 액틴 필라멘트와 같이 국소화 되고, β-actin mRNA가 액틴 필라멘트 안에서 sub-diffusive한 움직임을 보였으며, 먼 거리를 움직이던 mRNA도 액틴 필라멘트에 고정되는 모습도 확인할 수 있었다. 축삭에서 β-actin mRNA 움직임을 본 이번 관찰은 mRNA 수송 및 국소화에 대한 생물물리학 적 메커니즘의 기반이 될 수 있을 것이다.mRNA is the first product of the gene expression and facilitates the protein synthesis. Especially in neurons, some RNAs are transcribed in response to stimuli and transported to the specific region, altering local proteome for neurons to function normally. Recent advances of mRNA labeling techniques allowed us to observe the single mRNAs in live cells. In this thesis, we applied RNA imaging technique not only to identify the neuronal ensemble that activated during memory formation and retrieval, but also to traffic mRNAs transported to the axon. In the first part of the thesis, we observed the transcription site of Arc gene, one of the immediate-early gene, which is rapidly transcribed upon the neural stimuli. Because of the characteristic of expressing in response to stimuli, Arc is widely used as a marker for memory trace cells thought to store memories. However, little is known about the ensemble dynamics of these cells because it has been challenging to observe them repeatedly over long periods of time in vivo. To overcome this limitation, we present a genetically-encoded RNA indicator (GERI) technique for intravital chronic imaging of endogenous Arc mRNA. We used our GERI to identify Arc-positive neurons in real time without the time lag associated with reporter protein expression in conventional approaches. We found that Arc-positive neuronal populations rapidly turned over within two days in CA1, whereas ~4% of neurons in the retrosplenial cortex consistently expressed Arc upon contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the overlapping population of neurons expressing Arc during encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA imaging approach has potential to unravel the dynamics of engram cells underlying various learning and memory processes. In the second part of this thesis, we imaged β-actin mRNAs, which can generate a cytoskeletal protein, β-actin, through translation. Local protein synthesis has a critical role in axonal guidance and regeneration. Yet it is not clearly understood how the mRNA localization is regulated in axons. To address these questions, we investigated mRNA motion in live axons using a transgenic mouse that expresses fluorescently labeled endogenous β-actin mRNA. By culturing hippocampal neurons in a microfluidic device that allows separation of axons from dendrites, we performed single particle tracking of β-actin mRNA selectively in axons. Although axonal mRNAs need to travel a long distance, we observed that most axonal mRNAs show much less directed motion than dendritic mRNAs. We found that β-actin mRNAs frequently localize at the neck of filopodia which can grow as axon collateral branches and at varicosities where synapses typically occur. Since both filopodia and varicosities are known as actin-rich areas, we investigated the dynamics of actin filaments and β-actin mRNAs simultaneously by using high-speed dual-color imaging. We found that axonal mRNAs colocalize with actin filaments and show sub-diffusive motion within the actin-rich regions. The novel findings on the dynamics of β-actin mRNA will shed important light on the biophysical mechanisms of mRNA transport and localization in axons.1. INTRODUCTION, 1 1.1. Neuronal ensemble, 1 1.2. Immediate-early Gene (IEG), 3 1.3. Methods for IEG-positive neurons, 3 1.4. Two-photon microscope, 5 1.5. References, 7 2. IMAGING ARC mRNA TRANSCRIPTION SITES IN LIVE MICE, 9 2.1. Introduction, 9 2.2. Materials and Methods, 10 2.3. Results and Discussion, 18 2.4. References, 26 3. NEURONS EXPRESSING ARC mRNA DURING REPEATED MEMORY RETRIEVALS, 28 3.1. Introduction, 28 3.2. Results and Discussion, 28 3.3. References, 35 4. NEURAL ACTIVITIES OF ARC+ NEURONS, 36 4.1. Introduction, 36 4.2. Materials and Methods, 37 4.3. Results and Discussion, 38 4.4. References, 52 5. AXONAL mRNA DYNAMICS IN LIVE NEURONS, 54 5.1. Introduction, 54 5.2. Materials and Methods, 55 5.3. Results and Discussion, 59 5.4. References, 70 6. CONCLUSION AND OUTLOOK, 72 ABSTRACT IN KOREAN (국문초록), 76박

Multispectral fingerprinting for improved in vivo cell dynamics analysis

Background: Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks. Results: We found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets. Conclusions: Our results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cell's position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and pattern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously film and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo

Filopodyan: An open-source pipeline for the analysis of filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/ VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.J.L. Gallop and V. Urbančič are supported by the Wellcome Trust (WT095829AIA). J. Mason and B. Richier are supported by the European Research Council (281971). C.E. Holt is supported by the Wellcome Trust (program grant 085314) and the European Research Council (advanced grant 322817). The Gurdon Institute is funded by the Wellcome Trust (203144) and Cancer Research UK (C6946/A24843)

Wnt/PCP controls spreading of Wnt/β-catenin signals by cytonemes in vertebrates

This is the author accepted manuscript.The final version is available from eLife Sciences Publications via the DOI in this record.Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/PCP autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 gets activated. Ror2/PCP signaling leads to induction of cytonemes, which mediate transport of Wnt8a to neighboring cells. In the Wnt receiving cells, Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, the murine intestinal crypt, and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.This project was funded by the Living Systems Institute, the University of Exeter and the Boehringer Ingelheim Foundation to SS. Studies in the DMV lab are supported by the National Research Foundation of Singapore and National Medical Research Council under its STAR Award Program. JR and AS were supported by the Impuls- und Vernetzungsfond of the Helmholtz Association. GUN was funded by the Deutsche Forschungsgemeinschaft (SFB 1324, projects A6 and Z2, GRK2039) and Helmholtz Association Program STN

Imaging of Dynamic Changes of the Actin Cytoskeleton in Microextensions of Live NIH3T3 Cells with a GFP Fusion of the F-Actin Binding Domain of Moesin

BACKGROUND: The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin. RESULTS: C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent. CONCLUSIONS: Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex