145 research outputs found
The Brain Invaders: Widespread Neuronal Dispersion in Developing and Adult Mammalian Brain
Cell migration is the principal determinant of the final brain cytoarchitecture. However, we have only limited knowledge about the migratory pathways and mechanisms utilized during brain development. In m y thesis I present new and unexpected findings about neuronal migration in the developing forebrain. I analyzed and characterized a novel type of neuronal migration, so called tangential chain migration that was first identified in the subventricular zone (SVZ) in the adult mouse brain. I developed an in vitro migration assay using Matrigel as a substrate, which allowed me to analyze dynamics and mechanism of S V Z cell translocation. I demonstrated that neurons migrating in chains move rapidly along each other in the absence of glia, implying that chain migration is different from previously described radial migration. Using the in vitro assay I discovered two regions in the embryonic mouse ventral forebrain containing large numbers of tangentially migrating neurons. I studied migratory potential of these embryonic cells after transplantation into the adult brain. I showed that cells from the medial ganglionic eminence (MGE) demonstrate a novel migratory behavior. M G E cells disperse through the adult brain tissue and differentiate into GABAergic neurons. I showed that M G E cells could disperse even in experimentally lesioned adult brain. Thus, the unique migratory behavior of M G E cells might open new clinical approaches to the repair of damaged brain. Finally, I show that widespread migration of M G E cells reflects their normal behavior in the developing brain. I analyzed migratory potential of M G E cells in vitro and by ultrasound guided transplantation in vivo. These experiments revealed large-scale directional migration of M G E cells into the developing neocortex in embryonic mouse brain. I propose that a ventro-dorsal permissive gradient detected in the developing forebrain might be the principal mechanism guiding M G E cell migration dorsally. In summary, I revealed a population of embryonic neuronal precursors with unprecedented capacity to migrate long distances both in the developing and adult mammalian brain
Making microscopy count: quantitative light microscopy of dynamic processes in living plants
First published: April 2016This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Cell theory has officially reached 350 years of age as the first use of the word âcellâ in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication âMicrographia: or some physiological definitions of minute bodiesâ. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the sub-cellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more inter-disciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking
Single molecule fluorescence measurements of complex systems
2017 Summer.Includes bibliographical references.Single molecule methods are powerful tools for investigating the properties of complex systems that are generally concealed by ensemble measurements. Here we use single molecule fluorescent measurements to study two different complex systems: 1/Æ noise in quantum dots and diffusion of the membrane proteins in live cells. The power spectrum of quantum dot (QD) fluorescence exhibits 1/Æ noise, related to the intermittency of these nanosystems. As in other systems exhibiting 1/Æ noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of 1/ÆÎČ noise for QDs decays with the observation time. We show that the traditional description of the power spectrum with a single exponent is incomplete and three additional critical exponents characterize the dependence on experimental time. A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal
Calcium Signalling during Primary Angiogenic Sprouting in Zebrafish
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a critical step for the formation of a functional vascular system during embryonic development. Furthermore, dysregulation of the vascular patterning is associated with more than 70 different diseases, including cancer, myocardial infarction, stroke and ocular disorders, such as macular degeneration. Studies of endothelial cells (ECs) in vitro and in vivo, have revealed the importance of vascular endothelial growth factor (VEGF) signalling together with the activation of the Dll4/Notch pathway to regulate ECâs differentiation into tip vs stalk cells as well as EC migration and proliferation during angiogenesis.
Similarities between the endothelial tip cell and the axonal growth cone are well established. The two cell types share not only a similar anatomical structure, but also common molecular pathways and respond to the same molecular cues. Ca2+ signalling especially through the L-type Ca2+ channel (LTCC) is crucial to regulate the axonal turning in order to promote attraction or repulsion in response to a molecular cue. In ECs, increase in cytosolic Ca2+ concentration (Ca2+i) is a key regulator of migration, proliferation, contraction, gene expression and other biological aspects. Despite the similarities between neuronal and vascular systems, the role of Ca2+ signalling through the LTCC during vascular formation and angiogenesis is still poorly understood.
This study provides evidence that the LTCC regulates EC migration during the primary angiogenic sprouting of the intersegmental vessels (ISV) in zebrafish embryos. The stimulation of the LTCC strongly increased EC migration from the dorsal aorta (DA), resulting in an overbranching phenotype, while the downregulation of the channel reduced the EC migration and proliferation compromising the ISV formation. Additionally, I observed that LTCC synergistically interacts with the canonical transient receptor potential-1 (TRPC1) Ca2+ channel to promote ISV development, suggesting the importance of Ca2+ fluxes through the plasma membrane during angiogenesis. Furthermore, mRNA-expression analysis of VEGF signalling and Dll4/Notch-pathway components revealed the importance of the LTCC during angiogenesis: perturbation of LTCC conductance, but not TRPC1, increased the mRNA-expression level of the VEGF and Dll4/Notch pathway components, compromising the angiogenic behaviour of ECs. Taken together, this study demonstrates that Ca2+ fluxes through plasma membrane of endothelial cells represent an integral part of angiogenic process. Moreover, like axon growth cone, the endothelial migration requires a tight regulation of Ca2+ signaling.Die Angiogenese, die Entstehung neuer BlutgefĂ€Ăe aus vorbestehenden BlutgefĂ€Ăen, stellt einen wichtigen Schritt bei der Formation eines funktionalen vaskulĂ€ren Systems wĂ€hrend der embryonalen Entwicklung dar. Fehlregulierungen bei der Entstehung des vaskulĂ€ren Geflechts sind auĂerdem mit einer Vielzahl von Krankheiten, wie Krebs, Herzinfarkt und SchlaganfĂ€llen und Augenerkrankungen wie Makuladegeneration und mehr als 70 anderen Krankheiten assoziiert. Sowohl in vitro als auch in vivo Studien an Endothelzellen (EZ) zeigten einen wichtigen Einfluss des Vascular endothelial growth factor - (VEGF) Signalweges sowie der Aktivierung des Dll4/Notch-Signalweges auf die Regulierung der Differenzierung von EZ zu Spitzen- oder Stielzellen, auf die Migration von EZ sowie deren Proliferation wĂ€hrend der Angiogenese.
Gemeinsamkeiten zwischen der endothelialen Spitzenzelle und dem axonalen Wachstumskegel sind seit langem bekannt. Die beiden Zelltypen weisen nicht nur eine Àhnliche anatomische Struktur, sondern auch gemeinsame molekulare Signalwege auf und reagieren auf dieselben Botenstoffe.
Die Signaltransduktion durch Calciumionen, vorallem ĂŒber den L-Typ Calciumkanal (LTCC), spielt eine wichtige Rolle bei der axonalen Wegfindung und beeinflusst entweder die Anziehung oder die AbstoĂung der axonalen Zelle als Antwort auf Botenstoffe. In EZ ist ein Anstieg der zytosolischen Calcium-Konzentration (Ca2+i) unter anderem ein SchlĂŒsselreiz fĂŒr die Migration, Proliferation und Kontraktion der EZ sowie die Expression bestimmter Gene. Trotz der Gemeinsamkeiten zwischen dem neuronalen und vaskulĂ€ren System, ist vor allem ĂŒber die Rolle der Ca2+-Signaltransduktion ĂŒber den LTCC bei der Formation des vaskulĂ€ren Systems und der Angiogenese wenig bekannt.
Die hier vorliegende Studie zeigt, dass der LTCC die Migration von EZ wĂ€hrend der ersten angiogenetischen Aussprossung intersegmentaler BlutgefĂ€Ăe (ISV) in Zebrafisch-Embryonen reguliert. Eine Stimulation des LTCC fĂŒhrte zu einem starken Anstieg der Migration von EZ aus der dorsalen Aorta (DA) und resultierte in einer erhöhten Verzweigung entstehender BlutgefĂ€Ăe. Die Herunterregulation des Kanals reduzierte die Migration und Proliferation von EZ und fĂŒhrte zu einer BeeintrĂ€chtigung der Ausbildung von ISV. Des Weiteren konnte nachgewiesen werden, dass der LTCC bei der Entstehung von ISV synergistisch mit dem kanonischen transient receptor potential-1 (TRPC1) Ca2+-Kanal wechselwirkt, was die entscheidende Rolle des Ca2+-Fluxes durch die Plasmamembran wĂ€hrend der Angiogenese beweist.
Insgesamt zeigt die hier vorliegende Arbeit, dass Ca2+ - Ströme durch die Plasmamembran von Endothelzellen einen wichtigen Bestandteil des angiogenetischen Prozesses darstellen. Desweiteren ist fĂŒr die Migration von Endothelzellen, wie beim axonalen Wachstumskegel, eine engmaschige Regulation von Calcium - Signalwegen vonnöten
IST Austria Thesis
Cytoplasm is a gel-like crowded environment composed of tens of thousands of macromolecules, organelles, cytoskeletal networks and cytosol. The structure of the cytoplasm is thought to be highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules is very restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the jammed nature of the cytoplasm at the microscopic scale, large-scale reorganization of cytoplasm is essential for important cellular functions, such as nuclear positioning and cell division. How such mesoscale reorganization of the cytoplasm is achieved, especially for very large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, has only begun to be understood.
In this thesis, I focus on the recent advances in elucidating the molecular, cellular and biophysical principles underlying cytoplasmic organization across different scales, structures and species. First, I outline which of these principles have been identified by reductionist approaches, such as in vitro reconstitution assays, where boundary conditions and components can be modulated at ease. I then describe how the theoretical and experimental framework established in these reduced systems have been applied to their more complex in vivo counterparts, in particular oocytes and embryonic syncytial structures, and discuss how such complex biological systems can initiate symmetry breaking and establish patterning.
Specifically, I examine an example of large-scale reorganizations taking place in zebrafish embryos, where extensive cytoplasmic streaming leads to the segregation of cytoplasm from yolk granules along the animal-vegetal axis of the embryo. Using biophysical experimentation and theory, I investigate the forces underlying this process, to show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the embryo. This wave functions in segregation by both pulling cytoplasm animally and pushing yolk granules vegetally. Cytoplasm pulling is mediated by bulk actin network flows exerting friction forces on the cytoplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. This study defines a novel role of bulk actin polymerization waves in embryo polarization via cytoplasmic segregation. Lastly, I describe the cytoplasmic reorganizations taking place during zebrafish oocyte maturation, where the initial segregation of the cytoplasm and yolk granules occurs. Here, I demonstrate a previously uncharacterized wave of microtubule aster formation, traveling the oocyte along the animal-vegetal axis. Further research is required to determine the role of such microtubule structures in cytoplasmic reorganizations therein.
Collectively, these studies provide further evidence for the coupling between cell cytoskeleton and cell cycle machinery, which can underlie a core self-organizing mechanism for orchestrating large-scale reorganizations in a cell-cycle-tunable manner, where the modulations of the force-generating machinery and cytoplasmic mechanics can be harbored to fulfill cellular functions
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Investigating Metabolic Activities and Phenotypes in Biological Systems with Vibrational Probes and Raman Techniques
In this dissertation, the emerging stimulated Raman scattering (SRS) microscopy in combination with various vibrational tags was extensively used to explore various aspects of biological systems. New techniques as well as new Raman active materials were also developed to facilitate the applications of SRS in biology.
Chapter one introduces and comprehensively reviews vibrational tags that have been developed to date in combination with imaging techniques and their applications in biological sciences to investigate metabolism in living organisms.
Chapter two studies lipotoxicity, a phenomenon that is well known but poorly understood. The study found phase separation can form on ER membrane in cells treated with long chain fatty acids due to the high transition temptation of their metabolites. It was also found that the phase separation severely disturbs normal distribution of ER membrane proteins because of hydrophobic mismatching. As the result, ER normal structure is disrupted, luminal space is collapsed, and interconnectivity of ER that ensures normal ER functions is lost. Additionally, ER stress sensor IRE1α was found to be activated directly by the formation of phase separation, which triggers apoptosis and ultimately leads to cell death.
Chapter three describes the development of a new method termed as metabolic activity phenotyping (MAP) that acquires quantitative measurements of metabolic activities of individual cells, which is essential to understanding questions in diverse fields in biology. To achieve the goal, an automatic system was designed and built that improves the acquisition speed by more than 100 times compared to commercially available instruments. A set of vibrational probes with deuterium labeling was also carefully selected to enable accurate measurement of metabolic flux. Combining the merits of high throughput measurements and vibrational tags, MAP was applied to investigate the metabolic activity differences among various cancer cells, to study the heterogeneity of drug efficacy, and to facilitate breast cancer subtyping.
Chapter four describes the development and application of a new class of Raman active nanoparticles, or Rdots. These Rdots were generated by non-covalently incorporating small molecule Raman probe into polymeric nanoparticles. The resulted Rdots are of compact size (~20 nm) and preserve all Raman spectral features of the small molecule probes used. Rdots were compared to other existing Raman active materials including SERS nanoparticles, and Rdots surpass all the other materials in terms of brightness. In addition, Rdots also possess narrow spectral linewidth (< 3 nm), making them ideal for multiplexed imaging. In the study, Rdots were used as immunostaining reporters to visualize cytoskeleton networks and surface markers in cell and tissue samples
Mitochondrial dynamics during mouse oocyte maturation
Mitochondria provide the primary source of ATP for the oocyte and pre-implantation embryo and undergo a number of redistributions during oocyte maturation which may be related to developmental competence. The experiments presented in this thesis aim to examine the changes in distribution and function of mitochondria during the transition from the germinal vesicle stage to the mature metaphase II arrested egg.
Mitochondrial distribution was monitored throughout oocyte maturation and accumulation of mitochondria around the first meiotic spindle was observed. This was dependent on the activities of microtubules and their associated motor proteins dynein and kinesin. Migration of the spindle to the oocyte cortex was accompanied by mitochondria but at polar body extrusion a dramatic reorganisation of mitochondria away from the cortical domain occurred, suggesting that a mechanism exists for retention of these important organelles in the oocyte during this asymmetric cell division. The role of the mitochondrial adapter proteins Trak and Miro in establishing redistribution of mitochondria was also addressed.
Finally, a novel recombinant FRET probe for measuring ATP was validated for use in oocytes. Use of this probe revealed alterations to both ATP levels and ATP consumption at different stages of oocyte maturation. Furthermore, whilst the first meiotic spindle was found to be dependent on mitochondrial activity to retain its structure and function, attempts to identify subcellular heterogeneity in ATP supply and demand related to the distribution of mitochondria around the spindle did not reveal any differences. However, the presence of cumulus cells surrounding the oocyte as part of a cumulus-oocyte complex was found to influence ATP levels in the oocyte; oocytes matured as part of a cumulus-oocyte complex had higher ATP levels than those observed in oocytes which had been denuded of cumulus cells. This was found to be dependent on the presence of gap junctional communication between the somatic and germ cell compartments, since inhibition of gap junctions abolished the higher ATP levels observed in cumulus enclosed oocytes
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Investigating Protein-lipid-membrane Interactions in Plant Cells using Bimolecular Fluorescence Complementation
The overall focus of this thesis is on the distribution of specific lipids and membrane proteins of the external and internal membranes of plant cells, in the context of the roles that those lipids and proteins may play in microbe-plant interactions. The work includes the development of several new tools, the refinement of some existing tools, and the highlighting of several poorly appreciated artifacts that are common to such studies.
This thesis is comprised of five chapters. In Chapter 1, I would like to step back to have a general overview, including initial questions we asked, the way we interpreted unexpected results. In Chapter 2 âManipulating Endoplasmic Reticulum-Plasma Membrane Tethering through BiFC interactions in plantsâ, I demonstrated that the heterogeneous network of patches produced in FLS2-StRem1.3 BiFC complexes corresponded to ER-PM tethering, which resulted from the non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3. This work confirmed that membrane targeting of integral membrane proteins (IMPs) such as FLS2 requires either co-translational or post-translational integration into the ER membrane before trafficking to their membrane destination. These observations suggest a re-visit of several previous studies which have reported heterogeneous patch-like distributions when using IMPs and PMPs in BiFC experiments.
In Chapter 3 âManipulating Tethering of Multivesicular Bodies and the Tonoplast to the Plasma Membrane Through BiFC Interactions in Plantsâ, I strengthened the evidence that the patch-like distributions observed when combining PtdIns(3)P biosensors with StREM1.3, resulted from tethering of MVBs and the tonoplast to the PM. I also observed that the membrane binding domains of the E3 ubiquitin-ligases SAUL1 (AtPUB44) and AtPUB43, could tether MVBs and the tonoplast to the PM, suggesting a possible functional role for these proteins in MVB-PM tethering, such as the secretion of exosomes.
Although my observations reported in Chapter 2 and Chapter 3 led to new insights into membrane organism in plant cells, they also highlighted the risk of using BiFC assays to study membrane protein interactions in plants, which without proper controls could lead to misinterpretation, or cause unrecognized alterations in cellular structure and membrane organization. In chapter 4 âFluorescent Protein mEos3.2 Shows Low Self-Association in Bimolecular Fluorescence Complementation Assays in Plantsâ, I show that the mEOS3.2 BiFC probe, split at residue 164E, also produced minimal non-specific detectable BiFC signals when transiently expressed in Nicotiana benthamiana leaf cortical cells, but produced excellent signals with interacting protein partners. I also demonstrated that the re-assembled mEos3.2 could still photo-convert from green to red, which aided in distinguishing specific BiFC signals from background, and could allow the visualization of BiFC complexes at nanometer spatial resolution using photo-activated localization microscopy (PALM) imaging.
In chapter 5 âIn vivo Super-Resolution Imaging of the Dynamics of PtdIns(4)P in the Plasma Membrane Of Plant Cellsâ, I successfully extended the application of mEos3.2 to study the spatiotemporal dynamics of a lipid species, PtdIns(4)P, in the plasma membrane of plant cells at single-molecule resolution using Single Particle Tracking PALM imaging (sptPALM). This work demonstrated the advantages of sptPALM compared to traditional imaging methods, such as Fluorescence Recovery After Photobleaching (FRAP), for studying the molecular dynamics of the plasma membrane. In addition, my work refined the specificity of the PtdIns(4)P biosensor FAPP1
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