scholarly journals TDP-43 proteinopathy impairs neuronal mRNP granule mediated postsynaptic local translation and mRNA metabolism

2019 ◽  
Author(s):  
Chia-En Wong ◽  
Kuen-Jer Tsai
Keyword(s):  
Imaging Techniques ◽  
Super Resolution ◽  
Protein Translation ◽  
Local Protein Synthesis ◽  
Mrna Metabolism ◽  
Molecular Approaches ◽  
Super Resolution Microscopy ◽  
First Time ◽  
Activity Dependent ◽  
Local Protein

AbstractLocal protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartments is essential for synaptic remodelling and plasticity in the neuronal cells. Misregulation in these processes caused by TDP-43 proteinopathy lead to neurodegenerative diseases such frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Using biochemical analysis and imaging techniques including super-resolution microscopy, we provide evidences for the first time of the postsynaptic localization of TDP-43 in the mammalian synapses; and we show TDP-43 as a component of neuronal mRNP granules. With activity stimulation and different molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, and activation of local protein translation as long as impairments in models of TDP-43 proteinopathy. This study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Interphase Human Chromosome Exhibits Out of Equilibrium Glassy Dynamics

2017 ◽  
Author(s):  
Guang Shi ◽  
Lei Liu ◽  
Changbong Hyeon ◽  
D. Thirumalai
Keyword(s):  
Ostwald Ripening ◽  
Imaging Techniques ◽  
Mean Square Displacement ◽  
Super Resolution ◽  
Spatial Distance ◽  
Base Pairs ◽  
Glassy Dynamics ◽  
Distance Information ◽  
Contact Maps ◽  
Super Resolution Microscopy

AbstractThe structural organization of the condensed chromosomes is being revealed using chromosome conformation capture experiments and super-resolution imaging techniques. Fingerprints of their three-dimensional organization on length scale from about hundred kilo base pairs to millions of base pairs have emerged using advances in Hi-C and super-resolution microscopy. To determine the poorly understood dynamics of human interphase chromosomes, we created the Chromosome Copolymer Model (CCM) by representing the chromosomes as a self-avoiding polymer with two loci types corresponding to euchromatin and heterochromatin. Using advanced clustering algorithms we establish quantitatively that the simulated contact maps for chromosomes 5 and 10 and those inferred from Hi-C experiments are in agreement. Ward Linkage Matrix (WLM), constructed from spatial distance information, shows that the Topologically Associated Domains (TADs) and compartments predicted from simulations are in agreement with inferred WLM computed using data from super-resolution microscopy experiments. Glassy dynamics is manifested in the stretched exponential relaxation of the structure factor and caging in the mean square displacement of individual loci, ∆i(t) ∼ tα with 0 < α < 1. Remarkably, the distribution of α, is extremely broad suggestive of highly heterogeneous dynamics, which is also reflected in the large cell-to-cell variations in the contact maps. Chromosome organization is hierarchical involving the formation of chromosome droplets (CDs) on short genomic scale followed by coalescence of the CDs, reminiscent of Ostwald ripening. We propose that glassy landscapes for the condensed active chromosomes might provide a balance between genomic conformational stability and biological functions.

scholarly journals Microglia perform local protein synthesis at perisynaptic and phagocytic structures

2021 ◽  
Author(s):  
Michael J. Vasek ◽  
Jelani D. Deajon-Jackson ◽  
Yating Liu ◽  
Haley W. Crosby ◽  
Jiwon Yi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
De Novo ◽  
Protein Translation ◽  
Local Translation ◽  
Slice Preparation ◽  
Local Protein Synthesis ◽  
Phagocytic Cups ◽  
Regulate Protein ◽  
Lysosomal Proteins ◽  
Local Protein

AbstractRecent studies have illuminated the importance of several key signaling pathways in regulating the dynamic surveillance and phagocytic activity of microglia. Yet little is known about how these signals result in the assembly of phagolysosomal machinery near targets of phagocytosis, especially in processes distal from the microglial soma. Neurons, astrocytes, and oligodendrocytes locally regulate protein translation within distal processes. Therefore, we tested whether there is regulated local translation within peripheral microglia processes (PeMPs). We show that PeMPs contain ribosomes which engage in de novo protein synthesis, and these associate with a subpool of transcripts involved in pathogen defense, motility, and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis. Collectively, these data argue for a regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial function.

Expansion Microscopy: Scalable and Convenient Super-Resolution Microscopy

2019 ◽  
Vol 35 (1) ◽  
pp. 683-701 ◽  
Author(s):  
Paul W. Tillberg ◽  
Fei Chen
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Resolving Power ◽  
Relative Ease ◽  
Nanoscale Structures ◽  
Physical Form ◽  
Tissue Specimens ◽  
Super Resolution Microscopy

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick.

scholarly journals Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca2+ channel distances

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Janus RL Kobbersmed ◽  
Andreas T Grasskamp ◽  
Meida Jusyte ◽  
Mathias A Böhme ◽  
Susanne Ditlevsen ◽  
...  
Keyword(s):  
Super Resolution ◽  
Release Site ◽  
Stochastic Simulations ◽  
Short Term ◽  
Short Term Plasticity ◽  
Release Probability ◽  
Vesicular Release ◽  
Dependent Inhibition ◽  
Super Resolution Microscopy ◽  
Activity Dependent

Chemical synaptic transmission relies on the Ca2+-induced fusion of transmitter-laden vesicles whose coupling distance to Ca2+ channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Here, using electron- and super-resolution microscopy at the Drosophila neuromuscular junction we quantitatively map vesicle:Ca2+ channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. We show that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca2+-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. We propose activity-dependent inhibition of vesicle un-priming or release site activation as novel facilitation mechanisms.

scholarly journals Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Laser Illumination ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractNon-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

scholarly journals Alternative polyadenylation and differential expression of Shank mRNAs in the synaptic neuropil

2014 ◽  
Vol 369 (1633) ◽  
pp. 20130137 ◽  
Author(s):  
Irina Epstein ◽  
Georgi Tushev ◽  
Tristan J. Will ◽  
Irena Vlatkovic ◽  
Iván J. Cajigas ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Neurological Diseases ◽  
Postsynaptic Density ◽  
Alternative Polyadenylation ◽  
Messenger Rnas ◽  
Local Protein Synthesis ◽  
Neuronal Signalling ◽  
The Stability ◽  
Activity Dependent ◽  
Local Protein

The stability and dynamics of synapses rely on tight regulation of the synaptic proteome. Shank proteins, encoded by the three genes Shank1, Shank2 and Shank3 are scaffold molecules in the postsynaptic density of excitatory neurons that contribute to activity-dependent neuronal signalling. Mutations in the Shank genes are associated with neurological diseases. Using state-of-the-art technologies, we investigated the levels of expression of the Shank family messenger RNAs (mRNAs) within the synaptic neuropil of the rat hippocampus. We detected all three Shank transcripts in the neuropil of CA1 pyramidal neurons. We found Shank1 to be the most abundantly expressed among the three Shank mRNA homologues. We also examined the turnover of Shank mRNAs and predict the half-lives of Shank1, Shank2 and Shank3 mRNAs to be 18–28 h. Using 3′-end sequencing, we identified novel 3′ ends for the Shank1 and Shank2 3′ untranslated regions (3′ UTRs) that may contribute to the diversity of alternative polyadenylation (APA) for the Shank transcripts. Our findings consolidate the view that the Shank molecules play a central role at the postsynaptic density. This study may shed light on synaptopathologies associated with disruption of local protein synthesis, perhaps linked to mutations in mRNA 3′ UTRs or inappropriate 3′ end processing.

scholarly journals Recent Advances in Organelle-Targeted Fluorescent Probes

Molecules ◽  
2021 ◽  
Vol 26 (1) ◽  
pp. 217
Author(s):  
Na-Eun Choi ◽  
Ji-Yu Lee ◽  
Eun-Chae Park ◽  
Ju-Hee Lee ◽  
Jiyoun Lee
Keyword(s):  
Fluorescent Probes ◽  
Molecular Level ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Structural Features ◽  
Future Directions ◽  
Cellular Processes ◽  
Biochemical Pathways ◽  
Recent Advances ◽  
Super Resolution Microscopy

Recent advances in fluorescence imaging techniques and super-resolution microscopy have extended the applications of fluorescent probes in studying various cellular processes at the molecular level. Specifically, organelle-targeted probes have been commonly used to detect cellular metabolites and transient chemical messengers with high precision and have become invaluable tools to study biochemical pathways. Moreover, several recent studies reported various labeling strategies and novel chemical scaffolds to enhance target specificity and responsiveness. In this review, we will survey the most recent reports of organelle-targeted fluorescent probes and assess their general strategies and structural features on the basis of their target organelles. We will discuss the advantages of the currently used probes and the potential challenges in their application as well as future directions.

scholarly journals Cortical wiring by synapse-specific control of local protein synthesis

2021 ◽  
Author(s):  
Clémence Bernard ◽  
David Exposito-Alonso ◽  
Martijn Selten ◽  
Stella Sanalidou ◽  
Alicia Hanusz-Godoy ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Synapse Formation ◽  
Mrna Translation ◽  
Protein Translation ◽  
Synaptic Proteins ◽  
Local Synthesis ◽  
Local Protein Synthesis ◽  
Mouse Cerebral Cortex ◽  
A Cell ◽  
Local Protein

Neurons use local protein synthesis as a mechanism to support their morphological complexity, which requires independent control across multiple subcellular compartments including individual synapses. However, to what extent local translation is differentially regulated at the level of specific synaptic connections remains largely unknown. Here, we identify a signaling pathway that regulates the local synthesis of proteins required for the formation of excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves the regulation of the mTORC1 inhibitor Tsc2 by the receptor tyrosine kinase ErbB4, which enables the local control of mRNA translation in a cell type-specific and synapse-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins that regulates the formation of excitatory inputs on PV+ interneurons downstream of ErbB4 signaling. Our work demonstrates that local protein translation is regulated at the level of specific connections to control synapse formation in the nervous system.

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