Frozen-hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure

Cryo-electron tomography and small-angle X-ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen-hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of similar to 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are similar to 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X-ray scattering of whole chromosomes shows a main scattering peak at similar to 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis

MaLeFiSenta: Machine Learning for FilamentS Identification and Orientation in the ISM

Filament identification became a pivotal step in tackling fundamental problems in various fields of Astronomy. Nevertheless, existing filament identification algorithms are critically user-dependent and require individual parametrization. This study aimed to adapt the neural networks approach to elaborate on the best model for filament identification that would not require fine-tuning for a given astronomical map. First, we created training samples based on the most commonly used maps of the interstellar medium obtained by Planck and Herschel space telescopes and the atomic hydrogen all-sky survey HI4PI. We used the Rolling Hough Transform, a widely used algorithm for filament identification, to produce training outputs. In the next step, we trained different neural network models. We discovered that a combination of the Mask R-CNN and U-Net architecture is most appropriate for filament identification and determination of their orientation angles. We showed that neural network training might be performed efficiently on a relatively small training sample of only around 100 maps. Our approach eliminates the parametrization bias and facilitates filament identification and angle determination on large data sets..

The Nanoarchitecture of the Outer Hair Cell Lateral Wall: Structural Correlates of Electromotility

Proper mammalian hearing depends on an outer hair cell-based mechanism that amplifies the sound-induced travelling waves in the cochlea. Outer hair cells (OHCs) contribute to this cochlear amplification through their electromotile property—voltage-dependent somatic length changes that can operate at acoustic frequencies. This unique form of motility is driven by prestin, a member of the solute carrier 26 family of anion transporters that is highly expressed along the OHC lateral plasma membrane. The lateral plasma membrane is supported by a cortical actin-spectrin lattice and a smooth ER system known as lateral cisternae to form a regular layered structure along the entire OHC lateral wall. The detailed structural organization of each layer and how they interact to transduce prestin conformational changes into whole-cell motility are not well understood. In this dissertation, I combine cryogenic sample preparation methods and electron tomography to elucidate the functional architecture of the OHC lateral wall complex. In chapter 1, I review the biology of the mammalian auditory system. In chapter 2, I detail how the combined methodological approach used can preserve and reveal the three-dimensional nano-architectures in cells at near-native state. In chapter 3, I describe the successful use of this methodology to elucidate the structure-function relationships in a comparable model structure, the glycocalyx on the surface of enterocytes. In Chapter 4, I provide the details on the organization of each layer of the OHC lateral wall complex and how they are structurally integrated. I show that the lateral plasma membrane contains closely tiled microdomains of orthogonally packed putative prestin protein complexes. The cortical lattice connects the plasma membrane to the adjacent lateral cisternae through two independent cross-bridging components. The lateral cisternae are in turn integrated through inter and intra-cisternal cross-bridging systems. Finally, mitochondria are attached to the lateral cisternae through another set of linker elements. By quantifying the dimensions of each of these components and mapping their distribution I provide a detailed blueprint of the nano-architecture of the OHC electromotile apparatus and discuss how its cohesive structure allows effective transmission of forces generated by prestin to the rest of the cell to drive cochlear amplification

NOGO-A/RTN4A and NOGO-B/RTN4B are simultaneously expressed in epithelial, fibroblast and neuronal cells and maintain ER morphology

Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.Peer reviewe

Electron Cryotomography

Electron cryotomography (ECT) is an emerging technology that allows thin samples such as macromolecular complexes and small bacterial cells to be imaged in 3-D in a nearly native state to “molecular” (~4 nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal filaments, cell wall elements, motility machines, chemoreceptor arrays, internal compartments, and other ultrastructures. This article describes the technique and summarizes its contributions to bacterial cell biology. For comparable recent reviews, see (Subramaniam 2005; Jensen and Briegel 2007; Murphy and Jensen 2007; Li and Jensen 2009). For reviews on the history, technical details, and broader application of electron tomography in general, see for example (Subramaniam and Milne 2004; Lucić et al. 2005; Leis et al. 2008; Midgley and Dunin-Borkowski 2009)

The cartography of cell motion

Cell motility plays an important role throughout biology, the polymerisation of actin being fundamental in producing protrusive force. However, it is increasingly apparent that intracellular pressure, arising from myosin-II contraction, is a co-driver of motility. In its extreme form, pressure manifests itself as hemispherical protrusions, referred to as blebs, where membrane is torn from the underlying cortex. Although many components and signalling pathways have been identified, we lack a complete model of motility, particularly of the regulation and mechanics of blebbing. Advances in microscopy are continually improving the quality of time series image data, but the absence of highthroughput tools for extracting quantitative numbers remains an analysis bottle-neck. We develop the next generation of the successful QuimP software designed for automated analysis of motile cells, producing quantitative spatio-temporal maps of protein distributions and changes in cell morphology. Key to QuimP's new functionality, we present the Electrostatic Contour Migration Method (ECMM) that provides high resolution tracking of local deformation with better uniformity and efficiency than rival methods. Photobleaching experiments are used to give insight into the accuracy and limitations of in silico membrane tracking algorithms. We employ ECMM to build an automated protrusion tracking method (ECMM-APT) sensitive not only to pseudopodia, but also the complex characteristics of high speed blebs. QuimP is applied to characterising the protrusive behaviour of Dictyostelium, induced to bleb by imaging under agar. We show blebs are characterised by distinct speed-displacement distributions, can reach speeds of 4.9μm/sec, and preferentially form at the anks during chemotaxis. Significantly, blebs emerge from at to concave membrane regions suggesting curvature is a major determinant of bleb location, size, and speed. We hypothesise that actin driven pseudopodia at the leading edge induce changes in curvature and therefore membrane tension, positive curvature inhibiting blebbing at the very front, and negative curvature enhancing blebbing at the sides. This possibly provides the necessary space for rear advancement. Furthermore, bleb kymographs reveal a retrograde shift of the cortex at the point of bleb expansion, suggesting inward contractive forces acting on the cortex even at concave regions. Strains defficient in phospholipid signalling show impaired chemotaxis and blebbing. Finally, we present further applications of QuimP, for example, we conclusively show that dishevelled is not polarised during Xenopus gastrulation, contrary to hypotheses in the literature

Reticulon Homology Domain Containing Protein Families of the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the largest membrane bound organelle in a cell and has multiple responsibilities. Execution of the various duties performed by the ER requires it to be shaped in a rather complex and intricate manner. ER’s two major structural motives, namely sheets and tubules, play very complex yet not fully understood role in giving ER its overall structure and function. The ratio of sheet and tubule conformations differ significantly within cell types and during cell cycle. Such a balance is possible only with a well-functioning set of factors that constantly communicate with each other throughout a cell cycle. These factors are specifically responsible for either shaping the ER sheets or tubules in addition to factors that keep the dynamic nature of the ER sound. During mitosis, ER undergoes a major transformation in its structure, where the sheet-tubule ratio shifts more towards tubules. Specific factors keep this process sound by acting actively during the stage of mitosis for proper cell division to occur. Although research on such factors are still on-going, many in-depth details on such factors (e.g. their precise localization) and their mechanism of action plus novel factors for ER shaping still needs to be resolved using techniques involving high end light and electron microscopy. In addition, a constant battle in data analysis for answering key questions also persists. Development of tools to study and analyse data on the lines of image analysis and processing is an unmet need that needs simultaneous attention. The research in this thesis focuses on three family of proteins that we uncover as responsible candidates in shaping the ER. To aid the study, this thesis also discusses the development of a software platform for analysis of microscopic data generated during this study. In this research, Reticulon family of proteins (RTN) were characterised using high-end microscopic techniques. We showed RTN4A and RTN4B to localize to ER tubules and sheet edges using pre-embedding immuno electron microscopy (immuno-EM) and electron tomography. Using qPCR, RTN4A and RTN4B were observed to be the most expressed isoforms in neurons and epithelial cells respectively. FAM134C, a poorly characterised protein was identified as one of the RTN4B interacting proteins. FAM134C localised to the ER where it specifically resided at high curvature ER (sheet edges and tubules) similar to RTN4B. FAM134C, similar to the RTN4B also had the capability to promote ER tubules upon overexpression. In addition, another family of proteins belonging to receptor expression enhancing protein (REEP), namely REEP3 and REEP4 were studied for shaping ER during mitotic stage of cell cycle. REEP3 and REEP4 collectively were observed both in tubulating peripheral ER during mitosis and clearing tubular ER from the chromatin for a normal mitosis to take place. Collectively, this work elaborates on proteins belonging to three classes that shape and position the ER specifically either in interphase or during stages of cell division. Our findings also throws light on the role of different domains in each of these proteins such as the reticulon homology domain (RHD) that was observed to be present in all these proteins under study. The RHD previously known for inserting partially and unsymmetrically in the outer leaflet of the ER gives a strong indication for proteins like RTN4B and FAM134C to localize to ER thus tubulating ER upon overexpression conditions. We uncovered the RHD’s crucial role in ER shaping and positioning in REEP3/4 during mitosis.Endoplasma- eli solulimakalvosto (engl. endoplasmic reticulum, ER) on solun suurin kalvon rajoittama organelli, ja sillä on useita tehtäviä. Pystyäkseen suoriutumaan eri tehtävistään ER:n rakenne on monimuotoinen ja alati muuntautuva. ER:n päärakenteet, laatat ja tubulukset, muodostavat monimutkaisen verkoston, eikä niiden kaikkia toimintoja täysin vielä tunneta. Laattojen ja tubulusten määrällinen suhde on erilainen eri solutyypeissä ja solusyklin vaiheissa. ER:n toimivan tasapainon saavuttamiseksi tarvitaan useita tekijöitä, jotka ovat vuorovaikutuksessa keskenään koko solusyklin ajan. Osa näistä tekijöistä osallistuu ER:n rakenteen muokkaamiseen ja osa on vastuussa ER:n dynaamisesta luonteesta. Solunjakautumisen aikana ER:n rakenne muuttuu, ja tubulaarisia rakenteita muodostuu suhteellisesti enemmän. Eri tekijät toimivat aktiivisesti solunjakautumisen eri vaiheissa mahdollistaen näin solujen jakautumisen. Nämä tekijät ovat edelleen tutkimuksen kohteena, ja yksityiskohtien esim. tarkan paikantumisen ja toimintamekanismin selvittämiseksi sekä vielä tuntemattomien, ER:n rakenteeseen vaikuttavien tekijöiden löytämiseksi, on käytettävä kehittyneitä tekniikoita kuten valo- ja elektronimikroskopiaa. Myös tietoaineiston analysoinnin täytyy edelleen kehittyä pystyäksemme vastaamaan tärkeisiin kysymyksiin, ja sen vuoksi sekä kuvankäsittelyyn että kuvien analysointiin tarvittavien ohjelmien kehittämiseen on kiinnitettävä erityistä huomiota. Tässä väitöskirjatutkimuksessa tutkittiin kolmea proteiiniperhettä, joiden osoitettiin vaikuttavan ER:n rakenteeseen. Tutkimuksen aikana otettujen mikroskooppikuvien analysointi oli tämän tutkimuksen kannalta oleellista ja tästä johtuen työssä käsitellään myös kuvankäsittelyohjelmiston kehittämistä. Tässä väitöskirjatutkimuksessa karakterisoitiin Reticulon-proteiineja (RTN) uusilla, kehittyneillä mikroskooppisilla tekniikoilla. Immuunielektronimikroskopialla ja elektronitomografialla osoitettiin RTN4A:n ja RTN4B:n paikallistuvan ER:n tubuluksiin ja laattojen reunoille. Kvantitatiivisella polymeraasiketjureaktiolla pystyttiin osoittamaan, että RTN4A on eniten ilmennetty muunnos hermosoluissa ja RTN4B vastaavasti pintakudossoluissa. Vähän tutkittu proteiini, FAM134C, tunnistettiin yhdeksi RTN4B:n kanssa vuorovaikutuksessa olevista proteiineista ja se paikallistettiin samankaltaisiin ER:n rakenteisiin kuin RTN4B (laattojen reunat ja tubulukset). FAM134C:n ja RTN4B:n ylituotto aikaansai tubulaaristen rakenteiden muodostamista. Lisäksi, ER:n rakenneproteiiniryhmän REEP (engl. receptor expression enhancing protein) proteiinien REEP3 ja REEP4 vaikutusta ER:n rakenteeseen tutkittiin solunjakautumisvaiheen aikana. Solunjakautumisvaiheessa REEP3 ja REEP4 paikallistettiin tubulaariseen, perifeeraaliseen ER:ään. Nämä proteiinit tarvittiin myös tubulaarisen ER:n irrottamiseksi kromatiinista normaalin solunjakautumisen aikaansaamiseksi. Tämä tutkimus syventää tietoja kolmen ER:ä muokkaavan proteiiniperheen proteiineista ja niiden paikallistumisesta ja vaikutuksista ER:n rakenteeseen niin kasvuvaiheessa kuin solunjakautumisen eri vaiheissa. Tulokset antavat myös lisätietoa eri domeenien rooleista näissä proteiineissa, esim. retikulonihomologia-domeenista (RHD), jonka löydettiin kaikista näistä proteiineista. RHD:n tiedetään olevan osittain kaksoiskalvorakenteen sisällä ja siten aiheuttavan kalvojen kaarevoitumista: tämä havaittiin myös ER:n tubuloitumisena ylituotettaessa RTN4B:tä tai FAM134C:tä. Tutkimuksen mukaan RHD:lla oli ratkaiseva rooli ER:n REEP 3/4 rakenneproteiinien toiminnassa solunjakautumisen aikana

Imaging and Computational Methods for Exploring Sub-cellular Anatomy

The ability to create large-scale high-resolution models of biological tissue provides an excellent opportunity for expanding our understanding of tissue structure and function. This is particularly important for brain tissue, where the majority of function occurs at the cellular and sub-cellular level. However, reconstructing tissue at sub-cellular resolution is a complex problem that requires new methods for imaging and data analysis. In this dissertation, I describe a prototype microscopy technique that can image large volumes of tissue at sub-cellular resolution. This method, known as Knife-Edge Scanning Microscopy (KESM), has an extremely high data rate and can capture large tissue samples in a reasonable time frame. We can therefore image complete systems of cells, such as whole small animal organs, in a matter of days. I then describe algorithms that I have developed to cope with large and complex data sets. These include methods for improving image quality, tracing filament networks, and constructing high-resolution anatomical models. These methods are highly parallel and designed to allow users to segment and visualize structures that are unique to high-throughput microscopy data. The resulting models of large-scale tissue structure provide much more detail than those created using standard imaging and segmentation techniques

Nessys:A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures

Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However, current methods for segmenting nuclei in 3D tissues are not designed for situations in which nuclei are densely packed, nonspherical, or heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture. Here, we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree-structured ridge-tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer's memory. We consistently obtain accurate segmentation rates of >90%, even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation. We have also generated a live reporter of the NE that can be used to track live cells in 3 dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation. We provide these tools in an open-access user-friendly format

Acquisition and Mining of the Whole Mouse Brain Microstructure

Charting out the complete brain microstructure of a mammalian species is a grand challenge. Recent advances in serial sectioning microscopy such as the Knife- Edge Scanning Microscopy (KESM), a high-throughput and high-resolution physical sectioning technique, have the potential to finally address this challenge. Nevertheless, there still are several obstacles remaining to be overcome. First, many of these serial sectioning microscopy methods are still experimental and are not fully automated. Second, even when the full raw data have been obtained, morphological reconstruction, visualization/editing, statistics gathering, connectivity inference, and network analysis remain tough problems due to the unprecedented amounts of data. I designed a general data acquisition and analysis framework to overcome these challenges with a focus on data from the C57BL/6 mouse brain. Since there has been no such complete microstructure data from any mammalian species, the sheer amount of data can overwhelm researchers. To address the problems, I constructed a general software framework for automated data acquisition and computational analysis of the KESM data, and conducted two scientific case studies to discuss how the mouse brain microstructure from the KESM can be utilized. I expect the data, tools, and studies resulting from this dissertation research to greatly contribute to computational neuroanatomy and computational neuroscience