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

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.

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Radial F-actin Organization During Early Neuronal Development

10.1101/372813 ◽

2018 ◽

Author(s):

Durga Praveen Meka ◽

Robin Scharrenberg ◽

Bing Zhao ◽

Theresa König ◽

Irina Schaefer ◽

...

Keyword(s):

Neuronal Development ◽

Super Resolution ◽

Actin Dynamics ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Actin Organization ◽

Organizing Center ◽

Super Resolution Microscopy ◽

Radial Organization

AbstractThe centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center (Farina et al., 2016), raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here we report, using super-resolution microscopy and live-cell imaging, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoconversion/photoactivation experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin towards the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers; hence sustaining initial neuronal development.

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Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia

10.1101/2020.08.20.260414 ◽

2020 ◽

Author(s):

Jérémy Magescas ◽

Sani Eskinazi ◽

Michael V. Tran ◽

Jessica L. Feldman

Keyword(s):

Sensory Neurons ◽

Super Resolution ◽

Spatial Separation ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Tissue Specific ◽

C Elegans ◽

Organizing Center ◽

Pericentriolar Material ◽

Specific Degradation

SummaryDuring mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis while SPD-2/CEP192 and PCMD-1, which are essential in the zygote, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans [1-3], we find evidence for “centriole-less PCM” at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules towards the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

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Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169286 ◽

1994 ◽

Vol 52 ◽

pp. 310-311

Author(s):

M.B. Braunfeld ◽

M. Moritz ◽

B.M. Alberts ◽

J.W. Sedat ◽

D.A. Agard

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Vital Role ◽

Complex Nature ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Nucleation Sites ◽

Dimensional Reconstruction ◽

Organizing Center ◽

Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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MINSTED fluorescence localization and nanoscopy

Nature Photonics ◽

10.1038/s41566-021-00774-2 ◽

2021 ◽

Author(s):

Michael Weber ◽

Marcel Leutenegger ◽

Stefan Stoldt ◽

Stefan Jakobs ◽

Tiberiu S. Mihaila ◽

...

Keyword(s):

Standard Deviation ◽

Super Resolution ◽

Diffraction Limit ◽

Stimulated Emission ◽

Far Field ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Molecular Scale ◽

Localization Precision ◽

Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

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The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control

Journal of Cell Science ◽

10.1242/jcs.256537 ◽

2021 ◽

Author(s):

Xiaolei Gao ◽

Saturnino Herrero ◽

Valentin Wernet ◽

Sylvia Erhardt ◽

Oliver Valerius ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Cell Types ◽

Spindle Pole ◽

Receptor Protein ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Spindle Pole Bodies ◽

Organizing Center ◽

Ring Complex ◽

Core Components

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) were described in many cell types. Functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans additional MTOCs were discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates SPB outer plaque with sMTOC activities. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest polo-like kinase as a regulator of MTOC activities and as a scaffolding unit through interaction with γ-tubulin ring complex receptors.

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Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

Australian Journal of Chemistry ◽

10.1071/ch13499 ◽

2014 ◽

Vol 67 (2) ◽

pp. 179 ◽

Cited By ~ 16

Author(s):

Donna R. Whelan ◽

Thorge Holm ◽

Markus Sauer ◽

Toby D. M. Bell

Keyword(s):

Cell Biology ◽

Super Resolution ◽

Diffraction Limit ◽

Stochastic Optical Reconstruction Microscopy ◽

Resolution Imaging ◽

Optical Reconstruction ◽

Set Up ◽

Microscopy Techniques ◽

Super Resolution Microscopy ◽

Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

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Super-resolution microscopy and its applications in neuroscience

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545817300014 ◽

2017 ◽

Vol 10 (05) ◽

pp. 1730001 ◽

Cited By ~ 3

Author(s):

Xuecen Wang ◽

Jiahao Wang ◽

Xinpei Zhu ◽

Yao Zheng ◽

Ke Si ◽

...

Keyword(s):

Optical Microscopy ◽

Super Resolution ◽

Diffraction Limit ◽

Biological Science ◽

Basic Principles ◽

Suitable Tool ◽

Subcellular Processes ◽

Conventional Microscopy ◽

Super Resolution Microscopy

Optical microscopy promises researchers to see most tiny substances directly. However, the resolution of conventional microscopy is restricted by the diffraction limit. This makes it a challenge to observe subcellular processes happened in nanoscale. The development of super-resolution microscopy provides a solution to this challenge. Here, we briefly review several commonly used super-resolution techniques, explicating their basic principles and applications in biological science, especially in neuroscience. In addition, characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.

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

Light Science & Applications ◽

10.1038/s41377-019-0191-5 ◽

2019 ◽

Vol 8 (1) ◽

Cited By ~ 31

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.

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The Nanoscopic Organization of Synapse Structures: A Common Basis for Cell Communication

Membranes ◽

10.3390/membranes11040248 ◽

2021 ◽

Vol 11 (4) ◽

pp. 248

Author(s):

Xiaojuan Yang ◽

Wim Annaert

Keyword(s):

Plasma Membranes ◽

Current Knowledge ◽

Super Resolution ◽

Diffraction Limit ◽

Stimulated Emission ◽

Synaptic Proteins ◽

Resolving Power ◽

Structured Illumination Microscopy ◽

Membrane Contact Sites ◽

Super Resolution Microscopy

Synapse structures, including neuronal and immunological synapses, can be seen as the plasma membrane contact sites between two individual cells where information is transmitted from one cell to the other. The distance between the two plasma membranes is only a few tens of nanometers, but these areas are densely populated with functionally different proteins, including adhesion proteins, receptors, and transporters. The narrow space between the two plasma membranes has been a barrier for resolving the synaptic architecture due to the diffraction limit in conventional microscopy (~250 nm). Various advanced super-resolution microscopy techniques, such as stimulated emission depletion (STED), structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM), bypass the diffraction limit and provide a sub-diffraction-limit resolving power, ranging from 10 to 100 nm. The studies using super-resolution microscopy have revealed unprecedented details of the nanoscopic organization and dynamics of synaptic molecules. In general, most synaptic proteins appear to be heterogeneously distributed and form nanodomains at the membranes. These nanodomains are dynamic functional units, playing important roles in mediating signal transmission through synapses. Herein, we discuss our current knowledge on the super-resolution nanoscopic architecture of synapses and their functional implications, with a particular focus on the neuronal synapses and immune synapses.

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

Frontiers in Chemistry ◽

10.3389/fchem.2021.746900 ◽

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.

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