Super-resolution confocal live imaging microscopy (SCLIM) — Cutting-edge technology in cell biology

2013 ◽  
Author(s):  
Akihiko Nakano
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Live Imaging ◽  
Cutting Edge

scholarly journals Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258111
Author(s):  
Sofia Rodriguez-Gallardo ◽  
Kazuo Kurokawa ◽  
Susana Sabido-Bozo ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Chain Length ◽  
Cell Biology ◽  
Super Resolution ◽  
Live Imaging ◽  
Live Cell ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Er Export ◽  
Cargo Sorting

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by Super-resolution Confocal Live Imaging Microscopy (SCLIM) v1

2021 ◽  
Author(s):  
Sofia Rodriguez-Gallardo † ◽  
Kazuo Kurokawa †* ◽  
Susana Sabido-Bozo ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Chain Length ◽  
Cell Biology ◽  
Super Resolution ◽  
Live Imaging ◽  
Live Cell ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Er Export ◽  
Cargo Sorting

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37ºC). ER export is restored by shifting down to permissive temperature (24ºC) and progressive incorporation of the two different types of cargos into the fluorescently labeled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

scholarly journals Carl Singer (1945-2013) – Life on a Plate: Yeast genetics meetings and micromanipulators

2021 ◽  
Author(s):  
Harry Singer ◽  
Terrance G Cooper
Keyword(s):  
Molecular Biology ◽  
Cell Biology ◽  
Cutting Edge ◽  
Genetic Maps ◽  
Yeast Genetics

Abstract Micromanipulators, more than any other instrument, opened the early doors to developing the powerful genetics of yeast that underlies much of the molecular work today. The ability to separate the spores of a tetrad and analyze their phenotypes generated the genetic maps and biology upon which subsequent cloning, sequencing, cutting edge molecular and cell biology depended. This work describes the development of those micromanipulators from garage to barn to factory and the developer of the sophisticated instruments we use today. For more than 30 years Carl Singer and his family were staunch and generous supporters of the International Conferences on Yeast Genetics and Molecular Biology meetings both in Europe and America. Carl Singer's displays at meetings became a traditional fixture and engaged the appetites of many students and advanced researchers to employ a technique that many perceived as too complicated or difficult, but which he made simple and easy to learn. His experiences also document a sketch of the international yeast meetings, their venues and how they developed through the years.

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

Abstract 524: Identification of Roles for the Rho-Specific Guanine Nucleotide Dissociation Inhibitor (RhoGDI) Ly-Gdi in Platelet Function

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Anh T Ngo ◽  
Marisa L Thierheimer ◽  
Özgün Babur ◽  
Anne D Rocheleau ◽  
Xiaolin Nan ◽  
...  
Keyword(s):  
Platelet Function ◽  
Rho Gtpases ◽  
Cell Biology ◽  
Rho Gtpase ◽  
Thrombus Formation ◽  
Super Resolution ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Morphological Alterations ◽  
Guanine Nucleotide Dissociation Inhibitor

Introduction: Upon activation, platelets undergo specific morphological alterations critical to hemostatic plug and thrombus formation via actin cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in regulating platelet function. Methods and Hypothesis: Through an approach combining pharmacology, cell biology and systems biology methods we assessed the hypothesis that RhoGDI proteins regulate Rho GTPase-driven platelet functions downstream of platelet integrin and glycoprotein receptors. Results: We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular staining and super resolution microscopy assays find that Ly-GDI displays an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI in platelets, as Ly-GDI is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Notably, inhibition of PKC diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Conclusion: In conclusion, our results support roles for Ly-GDI as a localized regulator of Rho GTPases in platelets and link PKC and Rho GTPase signaling systems to platelet function.

scholarly journals Digital Spindle: A New Way to Explore Mitotic Functions by Whole Cell Data Collection and a Computational Approach

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1255
Author(s):  
Norio Yamashita ◽  
Masahiko Morita ◽  
Hideo Yokota ◽  
Yuko Mimori-Kiyosue
Keyword(s):  
Cell Biology ◽  
Information Science ◽  
Three Dimensional ◽  
Light Sheet ◽  
Live Imaging ◽  
Three Dimensions ◽  
Complex Data ◽  
Spatiotemporal Resolution ◽  
Whole Cell ◽  
Light Sheet Microscopy

From cells to organisms, every living system is three-dimensional (3D), but the performance of fluorescence microscopy has been largely limited when attempting to obtain an overview of systems’ dynamic processes in three dimensions. Recently, advanced light-sheet illumination technologies, allowing drastic improvement in spatial discrimination, volumetric imaging times, and phototoxicity/photobleaching, have been making live imaging to collect precise and reliable 3D information increasingly feasible. In particular, lattice light-sheet microscopy (LLSM), using an ultrathin light-sheet, enables whole-cell 3D live imaging of cellular processes, including mitosis, at unprecedented spatiotemporal resolution for extended periods of time. This technology produces immense and complex data, including a significant amount of information, raising new challenges for big image data analysis and new possibilities for data utilization. Once the data are digitally archived in a computer, the data can be reused for various purposes by anyone at any time. Such an information science approach has the potential to revolutionize the use of bioimage data, and provides an alternative method for cell biology research in a data-driven manner. In this article, we introduce examples of analyzing digital mitotic spindles and discuss future perspectives in cell biology.

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

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
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.

FRET in Cell Biology: Still Shining in the Age of Super‐Resolution?

ChemPhysChem ◽  
2010 ◽  
Vol 12 (3) ◽  
pp. 484-490 ◽  
Author(s):  
Hernán E. Grecco ◽  
Peter J. Verveer
Keyword(s):  
Cell Biology ◽  
Super Resolution
Sign in / Sign up

Export Citation Format

Share Document