1,721,060 research outputs found
Endoplasmic reticulum architecture: structures in flux
No full textThe endoplasmic reticulum (ER) is a dynamic pleiomorphic organelle containing continuous but distinct subdomains. The diversity of ER structures parallels its many functions, including secretory protein biogenesis, lipid synthesis, drug metabolism and Ca2+ signaling. Recent studies are revealing how elaborate ER structures arise in response to subtle changes in protein levels, dynamics, and interactions as well as in response to alterations in cytosolic ion concentrations. Subdomain formation appears to be governed by principles of self-organization. Once formed, ER subdomains remain malleable and can be rapidly transformed into alternative structures in response to altered conditions. The mechanisms that modulate ER structure are likely to be important for the generation of the characteristic shapes of other organelles
Neuromuscular Junction Dismantling in Amyotrophic Lateral Sclerosis
Full text linkNeuromuscular junction assembly and plasticity during embryonic, postnatal, and adult life are tightly regulated by the continuous cross-talk among motor nerve endings, muscle fibers, and glial cells. Altered communications among these components is thought to be responsible for the physiological age-related changes at this synapse and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS). ALS is characterized by the degeneration and death of motor neurons leading to skeletal muscle denervation, atrophy and, most often, death of the patient within five years from diagnosis. ALS is a non-cell autonomous disease as, besides motor neuron degeneration, glial cells, and possibly muscle fibers, play a role in its onset and progression. Here, we will review the recent literature regarding the mechanisms leading to neuromuscular junction disassembly and muscle denervation focusing on the role of the three players of this peripheral tripartite synapse
Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions
No full textSeveral fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize
Volume CLEM and conventional EM: a dual approach to investigate the mechanism of neuronal dysfunction in Angelman Syndrome
No full textAngelman Syndrome (AS) is a rare neurodevelopmental disorder arising from the loss of the maternal copy of UBE3A, a paternally imprinted gene at neuronal level. Similarly to AS, other neurological defects due to an unproper UBE3A dosage are directly associated with alterations in brain development and synapse ultrastructure. The nanometric resolution of electron microscopy (EM) has proven essential for studying their outcome at the network level and establishing meaningful correlations between synapses geometrical features and their function. To explore whether UBE3A loss effect on the developing connectome was cell-autonomous, we in-utero electroporated CRISPR/Cas9 constructs to silence the endogenous UBE3A expression in a subset of layer 2/3 pyramidal neurons of the mouse somatosensory cortex. By creating z-confined NIRB marks we were able to relocate dendrites of interest and analyze spines complexity three-dimensionally from volumetric SBF-SEM datasets. Furthermore, we exploited conventional EM to examine excitatory synapses structure in the cortical circuit of a classic AS model pan-neuronally depleted from UBE3A expression. We identified pre-synaptic compartment defects that seem to diminish over time to make room for later changes in the post-synapse. Overall, the combined use of these two models should help clarify whether the defects originate within the neurons or their wider network
Oocyte development and egg envelope formation in Oreochromis niloticus, a mouth-brooding cichlid fish
No full textThe development of the oocyte and of its associated follicle cells in the Nile tilapia, Oreochromis niloticus, has been examined by optical and transmission electron microscopy. During oocyte development the female gamete of Orochromis niloticus increases in size because of the accumulation of yolk in its cytoplasm. As the accumulation of yolk proceeds, the organization of cortex of the oocyte becomes very complex; all of the cytoplasmic organelles and several populations of vesicles can be found. On the other hand follicle cells also undergo a series of modifications: they first become cuboidal then cylindrical and their cytoplasm become densely populated with organelles. The mature egg of Oreochromis niloticus is surrounded by a thin acellular envelope (chorion) assembled during oocyte development. Biochemical analysis of isolated and purified chorions from mature females was also performed. SDS-PAGE under reducing conditions showed a reproducible pattern of three major polypeptides (121, 66 and 50 kD), most of which being glycosylated. The pattern of synthesis and assembly of the egg envelope in Oreochromis niloticus, a mouth-brooding cichlid fish, is also discussed
Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
No full textThe lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy
CLEM and Volume EM to study synapse architecture in Angelman Syndrome
No full textAngelman Syndrome (AS) is a neurogenetic disorder characterized by severe intellectual disability, speech impairment, seizure disorder, abnormal sleep patterns, motor dysfunction and unusually happy demeanor.
AS is caused by the loss of the maternal copy of the UBE3A gene, which is biallelically expressed in all cell types, but only maternally expressed in neurons due to epigenetic silencing of the paternal allele.
Studies in AS models suggest that the UBE3A gene, encoding for the ubiquitin-protein ligase E3A, is crucial for synaptic formation and maintenance, neuronal plasticity, learning and memory formation and, because of having only the maternal copy of UBE3A gene, neurons are quite vulnerable to its loss.
During development, the synapse is subjected to fine modification ranging from activity-dependent dendritic spine enlargement to changes in size and shape of the postsynaptic density. Similar modifications are at the bases of synaptic plasticity.
To study the effect of UBE3A inactivation on the functional organization of synapses and neuronal connectivity in AS, we will combine sparse in utero electroporation of layer 2/3 pyramidal neurons of the mouse somatosensory cortex with CRISPR/Cas9-mediated genome editing to inactivate endogenous Ube3a. The fluorescent protein tdTomato will be used as a filler to identify and select electroporated neurons. We will use Serial Block Face Scanning Electron Microscopy and CLEM to generate volumetric dataset to reconstruct electroporated neurons and to quantitatively study synapses in their environment, finally connecting the geometry of synapses with their functional state
vCLEM: bridging the gap between high-resolution structural imaging and functional analysis of synapses
No full textUnderstanding the sophisticated details of synaptic structures is crucial when studying
the mechanisms of neuronal communication and brain organization in health and
disease. Traditional imaging techniques often fail to meet the necessity of providing
both the detailed ultrastructure and the specific protein localization we need for
comprehensive synaptic studies.
Correlative Light and Electron Microscopy (CLEM) addresses this challenge by imaging
the same exact biological structure with two, or more, imaging modalities, thus
combining the strengths of light with the resolution of electron microscopy. Volume
CLEM (vCLEM) adds a higher level of complexity to this already intricated method,
introducing the invaluable benefits of the third dimension.
We use vCLEM to study Angelman Syndrome (AS), a neurogenic disorder resulting from
the loss of the maternal copy of the UBE3A gene. Taking advantage of CRISPR/Cas9-
mediated genome editing endogenous Ube3a is inactivated via sparse in utero
electroporation (IUE) of layer 2/3 pyramidal neurons of the mouse somatosensory
cortex.
The correlative workflow is complex and involves several steps: first, a confocal
microscope is used to identify electroporated fluorescent neurons in the brain section
and acquire images; next, Near Infra-Red Banding (NIRB) is conducted to generate
spatial coordinates for locating the electroporated neurons along the x, y, and z axes;
the sample is processed for the Serial Block-Face Scanning Electron Microscope (SBF-
SEM), which is then exploited to create volumetric datasets.
This approach represents a significant advancement in the study of synapses and
allows us to gain a comprehensive understanding of the effect of the loss of Ube3a gene
on synapse ultrastructure and brain connectivity during development
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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