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 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.

scholarly journals Mitochondrial Import of the ADP/ATP Carrier: the Essential TIM Complex of the Intermembrane Space Is Required for Precursor Release from the TOM Complex

2002 ◽  
Vol 22 (22) ◽  
pp. 7780-7789 ◽  
Author(s):  
Kaye N. Truscott ◽  
Nils Wiedemann ◽  
Peter Rehling ◽  
Hanne Müller ◽  
Chris Meisinger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Intermembrane Space ◽  
Hydrophobic Membrane ◽  
Tom Complex ◽  
Native Electrophoresis ◽  
Mitochondrial Intermembrane Space ◽  
First Time ◽  
Blue Native Electrophoresis ◽  
Step Mechanism ◽  
Blue Native

ABSTRACT The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.

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 The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Heike Rampelt ◽  
Iva Sucec ◽  
Beate Bersch ◽  
Patrick Horten ◽  
Inge Perschil ◽  
...  
Keyword(s):  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Transmembrane Segments ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
The Matrix ◽  
Mitochondrial Carrier Family ◽  
Mitochondrial Intermembrane Space

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

scholarly journals Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Patrina A Pellett ◽  
Felix Dietrich ◽  
Jörg Bewersdorf ◽  
James E Rothman ◽  
Grégory Lavieu
Keyword(s):  
Steady State ◽  
Transport Model ◽  
Vesicular Transport ◽  
Super Resolution ◽  
Glycosyl Transferases ◽  
Golgi Transport ◽  
Transport Vesicle ◽  
Membrane Bound ◽  
Soluble Aggregates ◽  
Super Resolution Microscopy

A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary.

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 MICOS and phospholipid transfer by Ups2–Mdm35 organize membrane lipid synthesis in mitochondria

2016 ◽  
Vol 213 (5) ◽  
pp. 525-534 ◽  
Author(s):  
Mari J. Aaltonen ◽  
Jonathan R. Friedman ◽  
Christof Osman ◽  
Bénédicte Salin ◽  
Jean-Paul di Rago ◽  
...  
Keyword(s):  
Lipid Synthesis ◽  
Membrane Lipid ◽  
Intermembrane Space ◽  
Lipid Transfer ◽  
Inner Mitochondrial Membrane ◽  
Phospholipid Transfer ◽  
Mitochondrial Intermembrane Space ◽  
In Trans ◽  
And Function ◽  
Specific Lipid

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2–Mdm35 complexes (SLMO2–TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2–Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2–Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

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 Neuronal Localization of SENP Proteins with Super Resolution Microscopy

2020 ◽  
Vol 10 (11) ◽  
pp. 778
Author(s):  
Luca Colnaghi ◽  
Andrea Conz ◽  
Luca Russo ◽  
Clara A. Musi ◽  
Luana Fioriti ◽  
...  
Keyword(s):  
Super Resolution ◽  
Structured Illumination ◽  
Neuronal Function ◽  
Structured Illumination Microscopy ◽  
Synaptic Markers ◽  
Specific Protease ◽  
Fine Regulation ◽  
And Function ◽  
Super Resolution Microscopy ◽  
First Time

SUMOylation of proteins plays a key role in modulating neuronal function. For this reason, the balance between protein SUMOylation and deSUMOylation requires fine regulation to guarantee the homeostasis of neural tissue. While extensive research has been carried out on the localization and function of small ubiquitin-related modifier (SUMO) variants in neurons, less attention has been paid to the SUMO-specific isopeptidases that constitute the human SUMO-specific isopeptidase (SENP)/Ubiquitin-Specific Protease (ULP) cysteine protease family (SENP1-3 and SENP5-7). Here, for the first time, we studied the localization of SENP1, SENP6, and SENP7 in cultured hippocampal primary neurons at a super resolution detail level, with structured illumination microscopy (SIM). We found that the deSUMOylases partially colocalize with pre- and post-synaptic markers such as synaptophysin and drebrin. Thus, further confirming the presence with synaptic markers of the negative regulators of the SUMOylation machinery.

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