scholarly journals Resolving kinesin stepping: one head at a time

2019 ◽  
Author(s):  
Willi L. Stepp ◽  
Zeynep Ökten
Keyword(s):  
Super Resolution ◽  
Transport Processes ◽  
Random Coil ◽  
Tail Domain ◽  
The Past ◽  
C Terminus ◽  
Range Transport ◽  
Processive Stepping ◽  
Super Resolution Microscopy ◽  
First Time

SummaryKinesins are well-known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin’ activity have thus been focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the ‘heads’. The distal random coil at the C-terminus, termed ‘tail domain’, is proposed to mediate this autoinhibition, however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild type conformation selectively impacts one head in the heterodimer but not the other. Our results provide key insights into the regulated kinesin stepping that had escaped experimental scrutiny.

scholarly journals Resolving kinesin stepping: one head at a time

2019 ◽  
Vol 2 (5) ◽  
pp. e201900456 ◽  
Author(s):  
Willi L Stepp ◽  
Zeynep Ökten
Keyword(s):  
Super Resolution ◽  
Transport Processes ◽  
Random Coil ◽  
Tail Domain ◽  
The Past ◽  
C Terminus ◽  
Range Transport ◽  
Processive Stepping ◽  
Super Resolution Microscopy ◽  
First Time

Kinesins are well known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin’ activity have, thus, been the focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the “heads.” The distal random coil at the C terminus, termed “tail domain,” is proposed to mediate this autoinhibition; however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild-type conformation selectively impacts one head in the heterodimer but not the other. Our results provide insights into the regulated kinesin stepping that had escaped experimental scrutiny so far.

scholarly journals Super-resolution microscopy: successful applications in centrosome study and beyond

2019 ◽  
Vol 5 (5-6) ◽  
pp. 235-243 ◽  
Author(s):  
Jingyan Fu ◽  
Chuanmao Zhang
Keyword(s):  
Molecular Basis ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
The Past ◽  
Eukaryotic Species ◽  
Organizing Center ◽  
Cilia And Flagella ◽  
Super Resolution Microscopy

AbstractCentrosome is the main microtubule-organizing center in most animal cells. Its core structure, centriole, also assembles cilia and flagella that have important sensing and motility functions. Centrosome has long been recognized as a highly conserved organelle in eukaryotic species. Through electron microscopy, its ultrastructure was revealed to contain a beautiful nine-symmetrical core 60 years ago, yet its molecular basis has only been unraveled in the past two decades. The emergence of super-resolution microscopy allows us to explore the insides of a centrosome, which is smaller than the diffraction limit of light. Super-resolution microscopy also enables the compartmentation of centrosome proteins into different zones and the identification of their molecular interactions and functions. This paper compiles the centrosome architecture knowledge that has been revealed in recent years and highlights the power of several super-resolution techniques.

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 Single-photon avalanche diode imagers in biophotonics: review and outlook

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Claudio Bruschini ◽  
Harald Homulle ◽  
Ivan Michel Antolovic ◽  
Samuel Burri ◽  
Edoardo Charbon
Keyword(s):  
Single Photon ◽  
Photon Counting ◽  
Super Resolution ◽  
Cmos Technology ◽  
Time Resolved ◽  
Avalanche Diode ◽  
The Past ◽  
Fast Pace ◽  
On Chip ◽  
Super Resolution Microscopy

Abstract Single-photon avalanche diode (SPAD) arrays are solid-state detectors that offer imaging capabilities at the level of individual photons, with unparalleled photon counting and time-resolved performance. This fascinating technology has progressed at a very fast pace in the past 15 years, since its inception in standard CMOS technology in 2003. A host of architectures have been investigated, ranging from simpler implementations, based solely on off-chip data processing, to progressively “smarter” sensors including on-chip, or even pixel level, time-stamping and processing capabilities. As the technology has matured, a range of biophotonics applications have been explored, including (endoscopic) FLIM, (multibeam multiphoton) FLIM-FRET, SPIM-FCS, super-resolution microscopy, time-resolved Raman spectroscopy, NIROT and PET. We will review some representative sensors and their corresponding applications, including the most relevant challenges faced by chip designers and end-users. Finally, we will provide an outlook on the future of this fascinating technology.

scholarly journals Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Yingying Jing ◽  
Chenshuang Zhang ◽  
Bin Yu ◽  
Danying Lin ◽  
Junle Qu
Keyword(s):  
Biological Activities ◽  
Super Resolution ◽  
Spatiotemporal Resolution ◽  
The Past ◽  
Relevant Field ◽  
Living Species ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Conventional Light Microscopy

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.

scholarly journals Shedding Structured Light on Molecular Immunity: The Past, Present and Future of Immune Cell Super Resolution Microscopy

2021 ◽  
Vol 12 ◽  
Author(s):  
Timothy M. Johanson ◽  
Christine R. Keenan ◽  
Rhys S. Allan
Keyword(s):  
Immune System ◽  
Immune Cell ◽  
Structured Light ◽  
Super Resolution ◽  
The Past ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
The Way

In the two decades since the invention of laser-based super resolution microscopy this family of technologies has revolutionised the way life is viewed and understood. Its unparalleled resolution, speed, and accessibility makes super resolution imaging particularly useful in examining the highly complex and dynamic immune system. Here we introduce the super resolution technologies and studies that have already fundamentally changed our understanding of a number of central immunological processes and highlight other immunological puzzles only addressable in super resolution.

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 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.

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
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