Modeling large-scale dynamic processes in the cell: polarization, waves, and division

2014 ◽  
Vol 47 (3) ◽  
pp. 221-248 ◽  
Author(s):  
David Sept ◽  
Anders E. Carlsson
Keyword(s):  
Large Scale ◽  
Super Resolution ◽  
Cell Polarization ◽  
Fluorescent Labeling ◽  
Detailed Knowledge ◽  
Biophysical Modeling ◽  
Quantitative Understanding ◽  
Actin Waves ◽  
Super Resolution Microscopy ◽  
Compare And Contrast

AbstractThe past decade has witnessed significant developments in molecular biology techniques, fluorescent labeling, and super-resolution microscopy, and together these advances have vastly increased our quantitative understanding of the cell. This detailed knowledge has concomitantly opened the door for biophysical modeling on a cellular scale. There have been comprehensive models produced describing many processes such as motility, transport, gene regulation, and chemotaxis. However, in this review we focus on a specific set of phenomena, namely cell polarization, F-actin waves, and cytokinesis. In each case, we compare and contrast various published models, highlight the relevant aspects of the biology, and provide a sense of the direction in which the field is moving.

scholarly journals Density Distribution Maps: A Novel Tool for Subcellular Distribution Analysis and Quantitative Biomedical Imaging

Sensors ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 1009
Author(s):  
Ilaria De Santis ◽  
Michele Zanoni ◽  
Chiara Arienti ◽  
Alessandro Bevilacqua ◽  
Anna Tesei
Keyword(s):  
Density Distribution ◽  
Large Scale ◽  
Super Resolution ◽  
Spatial Location ◽  
Relative Displacement ◽  
Distribution Analysis ◽  
Laboratory Equipment ◽  
Imaging Device ◽  
Distribution Maps ◽  
Super Resolution Microscopy

Subcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects’ absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions; (b) empower subcellular molecules distributions analysis; (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.

scholarly journals Influenza A virus surface proteins are organized to help penetrate host mucus

2019 ◽  
Author(s):  
Michael D. Vahey ◽  
Daniel A. Fletcher
Keyword(s):  
Sialic Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Spatial Organization ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Viral Envelope ◽  
Surface Proteins ◽  
Virus Surface ◽  
Super Resolution Microscopy

AbstractInfluenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating an organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus’s conflicting needs to both penetrate mucus and stably attach to the underlying cells, and could contribute to the prevalence of the filamentous phenotype in clinical isolates of IAV.

scholarly journals Influenza A virus surface proteins are organized to help penetrate host mucus

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael D Vahey ◽  
Daniel A Fletcher
Keyword(s):  
Sialic Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Spatial Organization ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Viral Envelope ◽  
Surface Proteins ◽  
Virus Surface ◽  
Super Resolution Microscopy

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus’s conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

scholarly journals Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

2021 ◽  
Vol 22 (13) ◽  
pp. 6730
Author(s):  
Paweł Matryba ◽  
Kacper Łukasiewicz ◽  
Monika Pawłowska ◽  
Jacek Tomczuk ◽  
Jakub Gołąb
Keyword(s):  
Large Scale ◽  
Rapid Development ◽  
Super Resolution ◽  
Molecular Probes ◽  
Dimensional Structure ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Optical Clearing ◽  
Super Resolution Microscopy ◽  
Tissue Optical Clearing

The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.

scholarly journals Application of Super-Resolution and Advanced Quantitative Microscopy to the Spatio-Temporal Analysis of Influenza Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 233 ◽  
Author(s):  
Emma Touizer ◽  
Christian Sieben ◽  
Ricardo Henriques ◽  
Mark Marsh ◽  
Romain F. Laine
Keyword(s):  
Influenza Virus ◽  
Host Cell ◽  
Virus Replication ◽  
Large Scale ◽  
Super Resolution ◽  
Living Cells ◽  
Influenza Viruses ◽  
Animal Populations ◽  
Influenza Virus Replication ◽  
Super Resolution Microscopy

With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies.

scholarly journals A toolbox of anti-mouse and rabbit IgG secondary nanobodies

2017 ◽  
Author(s):  
Tino Pleiner ◽  
Mark Bates ◽  
Dirk Görlich
Keyword(s):  
Large Scale ◽  
Ethical Problem ◽  
Super Resolution ◽  
Superior Performance ◽  
Affinity Tags ◽  
Site Specific ◽  
E Coli ◽  
Rabbit Igg ◽  
Secondary Antibodies ◽  
Super Resolution Microscopy

AbstractPolyclonal anti-IgG secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in E. coli and could thus make secondary antibody-production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore-coupling. We demonstrate their superior performance in Western Blotting, both in peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and super-resolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols and even allow multi-target localization with primary IgGs from the same species and of the same class.

scholarly journals The WAVE complex associates with sites of saddle membrane curvature

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Anne Pipathsouk ◽  
Rachel M. Brunetti ◽  
Jason P. Town ◽  
Brian R. Graziano ◽  
Artù Breuer ◽  
...  
Keyword(s):  
Cell Shape ◽  
Large Scale ◽  
Super Resolution ◽  
Membrane Curvature ◽  
Cell Morphogenesis ◽  
Cell Behaviors ◽  
Complex Forms ◽  
Super Resolution Microscopy ◽  
Wave Complex ◽  
Organizing Principles

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

scholarly journals Covalent protein labeling by SpyTag-SpyCatcher in fixed cells for super-resolution microscopy

2017 ◽  
Author(s):  
Veronica Pessino ◽  
Rose Citron ◽  
Siyu Feng ◽  
Bo Huang
Keyword(s):  
Fusion Proteins ◽  
Gene Editing ◽  
Protein Structures ◽  
Super Resolution ◽  
High Specificity ◽  
Fluorescent Labeling ◽  
Protein Labeling ◽  
Intracellular Proteins ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy

AbstractLabeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. While the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We have also applied this method to endogenous proteins via gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.

scholarly journals Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication

2019 ◽  
Author(s):  
Nidhi Vishnoi ◽  
Karthigeyan Dhanasekeran ◽  
Madeleine Chalfant ◽  
Ivan Surovstev ◽  
Mustafa K. Khokha ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Congenital Heart ◽  
Super Resolution ◽  
State Level ◽  
Fluorescent Labeling ◽  
Nuclear Pore ◽  
Centriole Duplication ◽  
Pericentriolar Material ◽  
Super Resolution Microscopy

AbstractNUP188 encodes a scaffold component of the nuclear pore complex (NPC) and has been implicated as a congenital heart disease gene through an ill-defined function at centrioles. Here, we explore the mechanisms that physically and functionally segregate Nup188 between the pericentriolar material (PCM) and NPCs throughout the cell cycle. Pulse-chase fluorescent labeling approaches indicate that Nup188 populates centrosomes with newly synthesized protein that does not exchange with NPCs even after mitotic NPC breakdown. In addition, the steady-state level of Nup188 at centrosomes is controlled by the sensitivity of the PCM pool, but not the NPC pool, to proteasomal degradation. Proximity-labeling and super-resolution microscopy supports that Nup188 interacts with components of PCM including Cep192 and the centriolar satellite component, PCM1. Consistent with this, Nup188 plays a role in centriole duplication at or upstream of Sas6 loading. Together, our data establish Nup188 as a functional component of PCM and potentially provides insight into the pathogenesis of congenital heart disease.

scholarly journals Graphene-enabled, spatially controlled electroporation of adherent cells for live-cell super-resolution microscopy

2019 ◽  
Author(s):  
Seonah Moon ◽  
Wan Li ◽  
Ke Xu
Keyword(s):  
Mammalian Cells ◽  
Single Layer ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Biological Research ◽  
Super Resolution Microscopy ◽  
Affinity Probes ◽  
Intracellular Targets

AbstractThe incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, reliable fluorescent labeling of intracellular targets remains a challenge. Here, utilizing the unique mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent live cells and in situ super-resolution microscopy on the same device. ∼90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate excellent spatiotemporal controls, which, in combination with the ready patternablity of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate. We thus open up a new pathway to the microscopic manipulation and visualization of live cells.

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