scholarly journals Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Nathan A McDonald ◽  
Abigail L Lind ◽  
Sarah E Smith ◽  
Rong Li ◽  
Kathleen L Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Spatial Organization ◽  
Myosin Ii ◽  
Super Resolution ◽  
Protein Composition ◽  
Molecular Architecture ◽  
Contractile Ring ◽  
Super Resolution Microscopy ◽  
Multiple Signaling ◽  
Signaling Components

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

scholarly journals Phosphorylated paxillin and FAK constitute subregions within focal adhesions

2020 ◽  
Author(s):  
Michael Bachmann ◽  
Artiom Skripka ◽  
Bernhard Wehrle-Haller ◽  
Martin Bastmeyer
Keyword(s):  
Focal Adhesion ◽  
Structural Organization ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Multiple Signaling

AbstractIntegrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. We used structured illumination microscopy (SIM) to analyze spatial organization of paxillin phosphorylation (pPax) within adhesions. We found that pPax and focal adhesion kinase (FAK) form spot-like, spatially defined clusters within adhesions in several cell lines. In contrast, other adhesion proteins showed no consistent organization in such clusters. Live-cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify distance to each other dynamically. Moreover, we show that the distance between separate clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural organization within focal adhesions and demonstrate its regulation and dynamics.

scholarly journals Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

2021 ◽  
Author(s):  
Zachary A. McDargh ◽  
Shuyuan Wang ◽  
Harvey F. Chin ◽  
Sathish Thiyagarajan ◽  
Erdem Karatekin ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Striated Muscle ◽  
Protein Complexes ◽  
Myosin Ii ◽  
Force Production ◽  
Super Resolution ◽  
Building Blocks ◽  
Contractile Ring ◽  
Ring Model ◽  
Self Assembling

During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

scholarly journals Building the cytokinetic contractile ring in an early embryo: initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

2021 ◽  
Author(s):  
Chelsea Garno ◽  
Zoe H. Irons ◽  
Courtney M. Gamache ◽  
Xufeng Wu ◽  
Charles B. Shuster ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Animal Cell ◽  
Myosin Ii ◽  
Super Resolution ◽  
Order Structure ◽  
Contractile Ring ◽  
Sea Urchin Embryos ◽  
Structural Progression ◽  
Filament Network ◽  
Septin Filament

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide, for the first time, the visualization of septin filament higher order structure in an animal cell CR.

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 The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ana Rita Costa ◽  
Sara C Sousa ◽  
Rita Pinto-Costa ◽  
José C Mateus ◽  
Cátia DF Lopes ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Myosin Ii ◽  
Super Resolution ◽  
Loss Of Function ◽  
Axon Diameter ◽  
Heavy Chains ◽  
Axonal Diameter ◽  
Muscle Myosin ◽  
Super Resolution Microscopy ◽  
Actin Rings

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

scholarly journals The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy

2015 ◽  
Vol 128 (20) ◽  
pp. 3714-3719 ◽  
Author(s):  
L. Nahidiazar ◽  
M. Kreft ◽  
B. van den Broek ◽  
P. Secades ◽  
E. M. M. Manders ◽  
...  
Keyword(s):  
Super Resolution ◽  
Molecular Architecture ◽  
Super Resolution Microscopy

scholarly journals The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Hieng Chiong Tie ◽  
Alexander Ludwig ◽  
Sara Sandin ◽  
Lei Lu
Keyword(s):  
Optical Imaging ◽  
Golgi Complex ◽  
Spatial Organization ◽  
Super Resolution ◽  
Spatial Separation ◽  
Dense Packing ◽  
Golgi Stack ◽  
Central Disk ◽  
En Face ◽  
Super Resolution Microscopy

It is unclear how the two principal functions of the Golgi complex, processing and transport, are spatially organized. Studying such spatial organization by optical imaging is challenging, partially due to the dense packing of stochastically oriented Golgi stacks. Using super-resolution microscopy and markers such as Giantin, we developed a method to identify en face and side views of individual nocodazole-induced Golgi mini-stacks. Our imaging uncovered that Golgi enzymes preferentially localize to the cisternal interior, appearing as a central disk or inner-ring, whereas components of the trafficking machinery reside at the periphery of the stack, including the cisternal rim. Interestingly, conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi. In contrast, bulky cargos were found only at the rim. Our study therefore directly demonstrates the spatial separation of processing and transport functions within the Golgi complex.

scholarly journals Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (10) ◽  
pp. 1813-1825 ◽  
Author(s):  
F. Motegi ◽  
K. Nakano ◽  
I. Mabuchi
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Myosin Ii ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Actin Ring ◽  
Medial Region ◽  
Division Site ◽  
Medial Cortex ◽  
Actin Cables ◽  
Mutant Cells

Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring). Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring. Here, we investigated the mechanism of myosin-II assembly at the division site in S. pombe cells. First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3. Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells. It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis. Next, we investigated the dynamics of myosin-II during mitosis in S. pombe cells. In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots. The Myo2 dots and fibers then fused with each other to form a medial cortical network. Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region. Finally these structures were packed into a thin contractile ring. In mutant cells that cannot form the F-actin ring such as cdc3(ts), cdc8(ts) and cdc12(ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells. Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation. A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells. The Myo2dIQs could assemble into a normal-shaped ring in these cells. Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.

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