scholarly journals Cytoskeleton mechanics determine resting size and activation dynamics of platelets

2018 ◽  
Author(s):  
Aastha Mathur ◽  
Sandra Raquel Correia ◽  
Serge Dmitrieff ◽  
Romain Gibeaux ◽  
Iana Kalinina ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Morphological Changes ◽  
Super Resolution ◽  
Elastic Response ◽  
List Type ◽  
Platelet Size ◽  
Activation Dynamics ◽  
Marginal Band ◽  
Super Resolution Microscopy ◽  
Cell Fragments

SummaryPlatelets are cell fragments of various size that help maintain hemostasis. The way platelets respond during a clotting process is known to depend on their size, with important physiological consequences. We characterized the cytoskeleton of platelets as a function of their size. In resting Human and Mice platelets, we find a quadradic law between the size of a platelet and the amount of microtubule polymer it contains. We further estimate the length and number of microtubules in the marginal band using Electron and Super-resolution microscopy. In platelets activated with ADP, the marginal band coils as a consequence of cortical contraction driven by actin. We observe that this elastic coiling response is accompanied by a reversible shortening of the marginal band. Moreover, larger platelets have a higher propensity to coil. These results establish the dynamic equilibrium that is responsible for platelet size and differential response on a more quantitative level.HighlightsPlatelet size scales consistently with amount of polymerized tubulin in both mouse and human.Polymerized actin is required for ADP-induced marginal band coiling.Upon activation, the marginal band exhibits a reversible visco-elastic response involving shortening.Larger marginal bands have a higher propensity to coil than shorter ones.In briefThe cytoskeleton is adapted to platelet size and its mechanical properties determine propensity of a platelet to undergo morphological changes in response to agonists.

scholarly journals Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

2021 ◽  
Author(s):  
Matthew D. Lycas ◽  
Aske L. Ejdrup ◽  
Andreas T. Sørensen ◽  
Nicolai O. Haahr ◽  
Søren H. Jørgensen ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Striatal Slices ◽  
D2 Autoreceptor ◽  
Molecular Components ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Excitatory Drive

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

scholarly journals Continuous rearrangement of the postsynaptic gephyrin scaffolding domain: a super-resolution quantified and energetic approach

2017 ◽  
Author(s):  
Pamela C. Rodriguez ◽  
Leandro G. Almeida ◽  
Antoine Triller
Keyword(s):  
Neuronal Activity ◽  
Single Molecule ◽  
Morphological Changes ◽  
Super Resolution ◽  
Scaffold Protein ◽  
Synaptic Function ◽  
Inhibitory Synapses ◽  
Spatial Reorganization ◽  
Nanoscopic Structure ◽  
Super Resolution Microscopy

AbstractSynaptic function and plasticity requires a delicate balance between overall structural stability and the continuous rearrangement of the components that make up the presynaptic active zone and the postsynaptic density (PSD). Photoactivated localization microscopy (PALM) has provided a detailed view of the nanoscopic structure and organization of some of these synaptic elements. Still lacking, are tools to address the morphing and stability of such complexes at super-resolution. We describe an approach to quantify morphological changes and energetic states of multimolecular assemblies over time. With this method, we studied the scaffold protein gephyrin, which forms postsynaptic clusters that play a key role in the stabilization of receptors at inhibitory synapses. Postsynaptic gephyrin clusters exhibit an internal microstructure composed of nanodomains. We found, that within the PSD, gephyrin molecules continuously undergo spatial reorganization. This dynamic behavior depends on neuronal activity and cytoskeleton integrity. The proposed approach also allowed access to the effective energy responsible for the tenacity of the PSD despite molecular instability.Significant statementSuper-resolution microscopy has become an important tool for the study of biological systems, allowing detailed, nano-scale structural reconstruction, single molecule tracking, particle counting, and interaction studies. However, quantification tools that take full advantage of the information provided by this technology are still lacking. We describe a novel quantification method to obtain information related to the size, directionality, dynamics, and stability of clustered structures from super-resolution microscopy. With this method, we studied the stability of gephyrin clusters, the main inhibitory scaffold protein. We found that gephyrin molecules continuously undergo reorganization based on neuronal activity and changes in the cytoskeleton.

scholarly journals Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

2020 ◽  
Author(s):  
Tobias C. Kunz ◽  
Ralph Götz ◽  
Shiqiang Gao ◽  
Markus Sauer ◽  
Vera Kozjak-Pavlovic
Keyword(s):  
Morphological Changes ◽  
Membrane Surface ◽  
Cell Signalling ◽  
Super Resolution ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Intermembrane Space ◽  
Membrane Bound ◽  
Super Resolution Microscopy ◽  
First Time

AbstractMitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signalling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to ageing and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED and SIM have been applied for cristae visualisation.Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labelled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labelling and ExM as a tool for studying internal structure of mitochondria.

scholarly journals Sparse deconvolution of high-density super-resolution images

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Siewert Hugelier ◽  
Johan J. de Rooi ◽  
Romain Bernex ◽  
Sam Duwé ◽  
Olivier Devos ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Super Resolution ◽  
Penalized Regression ◽  
High Density ◽  
Wide Field ◽  
Cellular Processes ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Simulated Images ◽  
Norm Penalty

Abstract In wide-field super-resolution microscopy, investigating the nanoscale structure of cellular processes, and resolving fast dynamics and morphological changes in cells requires algorithms capable of working with a high-density of emissive fluorophores. Current deconvolution algorithms estimate fluorophore density by using representations of the signal that promote sparsity of the super-resolution images via an L1-norm penalty. This penalty imposes a restriction on the sum of absolute values of the estimates of emitter brightness. By implementing an L0-norm penalty – on the number of fluorophores rather than on their overall brightness – we present a penalized regression approach that can work at high-density and allows fast super-resolution imaging. We validated our approach on simulated images with densities up to 15 emitters per μm-2 and investigated total internal reflection fluorescence (TIRF) data of mitochondria in a HEK293-T cell labeled with DAKAP-Dronpa. We demonstrated super-resolution imaging of the dynamics with a resolution down to 55 nm and a 0.5 s time sampling.

scholarly journals Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain

2020 ◽  
Vol 40 (1_suppl) ◽  
pp. S67-S80 ◽  
Author(s):  
Dániel P Varga ◽  
Ákos Menyhárt ◽  
Balázs Pósfai ◽  
Eszter Császár ◽  
Nikolett Lénárt ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Super Resolution ◽  
Potassium Uptake ◽  
Extracellular Potassium ◽  
Spreading Depolarization ◽  
Selective Elimination ◽  
Intact Brain ◽  
Super Resolution Microscopy ◽  
The Brain

Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signaling and to reduce the incidence of spreading depolarization (SD) during cerebral ischemia. However, the mechanisms through which microglia interfere with SD remained unexplored. Here, we identify microglia as essential modulators of the induction and evolution of SD in the physiologically intact brain in vivo. Confocal- and super-resolution microscopy revealed that a series of SDs induced rapid morphological changes in microglia, facilitated microglial process recruitment to neurons and increased the density of P2Y12 receptors (P2Y12R) on recruited microglial processes. In line with this, depolarization and hyperpolarization during SD were microglia- and P2Y12R-dependent. An absence of microglia was associated with altered potassium uptake after SD and increased the number of c-fos-positive neurons, independently of P2Y12R. Thus, the presence of microglia is likely to be essential to maintain the electrical elicitation threshold and to support the full evolution of SD, conceivably by interfering with the extracellular potassium homeostasis of the brain through sustaining [K+]e re-uptake mechanisms.

scholarly journals Novel insight into the potential pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication mutations in patients with Pelizaeus–Merzbacher disease

2021 ◽  
Author(s):  
Ruoyu Duan ◽  
Liuju Li ◽  
Huifang Yan ◽  
Miao He ◽  
Kai Gao ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Morphological Changes ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Potential Pathogenicity ◽  
Pelizaeus Merzbacher Disease ◽  
Super Resolution Microscopy ◽  
First Time ◽  
Insight Into

Abstract Among the hypomyelinating leukodystrophies, Pelizaeus–Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ~ 70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients’ and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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