scholarly journals The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

2021 ◽  
Author(s):  
John H. Henson ◽  
Bakary Samasa ◽  
Charles B. Shuster ◽  
Athula H. Wikramanayake
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Super Resolution ◽  
Cell Types ◽  
Early Embryo ◽  
Ultrastructural Organization ◽  
C Terminus ◽  
Pathway Activation ◽  
Vegetal Cortex ◽  
Canonical Wnt

AbstractWnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

scholarly journals Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs.

1991 ◽  
Vol 114 (5) ◽  
pp. 1017-1028 ◽  
Author(s):  
E Houliston ◽  
R P Elinson
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Rana Pipiens ◽  
Cortical Microtubules ◽  
Cell Biol ◽  
Vegetal Cortex ◽  
Almost All ◽  
Cytoplasmic Side ◽  
Cytokeratin Filaments

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.

scholarly journals The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

2018 ◽  
Author(s):  
António T. Pedrosa ◽  
Korinn N. Murphy ◽  
Ana T. Nogueira ◽  
Amanda J. Brinkworth ◽  
Tristan R. Thwaites ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Vertical Displacement ◽  
Cell Types ◽  
Host Protein ◽  
Specific Domain ◽  
Type Iii Effector ◽  
Type Iii ◽  
C Terminus

AbstractThe human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions required interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and dynamics.

scholarly journals An agent-based model of molecular aggregation at the cell membrane

2019 ◽  
Author(s):  
Juliette Griffié ◽  
Ruby Peters ◽  
Dylan M. Owen
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Super Resolution ◽  
Cell Types ◽  
Signalling Pathways ◽  
Molecular Aggregation ◽  
Agent Based Model ◽  
Agent Based ◽  
Molecular Clustering ◽  
In Cells

AbstractMolecular clustering at the plasma membrane has long been identified as a key process and is associated with regulating signalling pathways across cell types. Recent advances in microscopy, in particular the rise of super-resolution, have allowed the experimental observation of nanoscale molecular clusters in the plasma membrane. However, modelling approaches capable of recapitulating these observations are in their infancy, partly because of the extremely complex array of biophysical factors which influence molecular distributions and dynamics in the plasma membrane. We propose here a highly abstracted approach: an agent-based model dedicated to the study of molecular aggregation at the plasma membrane. We show that when molecules are modelled as though they can act (diffuse) in a manner which is influenced by their molecular neighbourhood, many of the distributions observed in cells can be recapitulated, even though such sensing and response is not possible for real membrane molecules. As such, agent-based offers a unique platform which may lead to a new understanding of how molecular clustering in extremely complex molecular environments can be abstracted, simulated and interpreted using simple rules.Author summaryMolecular aggregation in cell membranes is a key component of cellular machinery, involved across cell types in inter-cellular communication and signalling pathway initiation. As such, understanding the underlying mechanisms and molecule cluster characteristics at a more theoretical level is a pre-requisite. Complete descriptive molecular models have proven impossible to realise due to the overall complexity of the processes involved, highlighting the need for novel approaches. While conceptual models have been shown to be powerful tools and are routinely used in other fields with high level of complexity such as social sciences or economics, they are overall lacking from the literature when it comes to cell studies. We suggest in this work that the same principle applies to cell biology and in particular, the study of molecular clustering. We propose here a general model, independent of cell types or signalling pathways: an agent-based model dedicated to molecular clustering in the plasma membrane. We show we are able to recapitulate molecular aggregation similar to observations in cells while new properties are highlighted by our model, for instance, clustering is a digitised process.

scholarly journals Wild-type and mutant ferroportins do not form oligomers in transfected cells

2006 ◽  
Vol 396 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Ana Sofia Gonçalves ◽  
Françoise Muzeau ◽  
Rand Blaybel ◽  
Gilles Hetet ◽  
Fathi Driss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Kidney ◽  
Cell Types ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Form ◽  
Hek 293 ◽  
C Terminus ◽  
293 Cells ◽  
Iron Export

Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.

scholarly journals A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation

2021 ◽  
Author(s):  
Charles E Mackay ◽  
Miranda Floen ◽  
M Dennis Leo ◽  
Raquibul Hasan ◽  
Carlos Fernandez-Pena ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Coiled Coil ◽  
Cell Types ◽  
Systemic Blood Pressure ◽  
Sk Channels ◽  
Double Knockout ◽  
Signaling Complex ◽  
C Terminus

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and primarily via the activation of nitric oxide synthase (NOS), with a smaller contribution from small-conductance Ca2+-activated K+ (SK) channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates non-selective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility and demonstrate that this occurs through the formation of an interdependent signaling complex with PC-2 in endothelial cells. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to Ca2+ influx, NOS and SK channel activation, vasodilation and a reduction in blood pressure.

scholarly journals Megadalton-node assembly by binding of Skb1 to the membrane anchor Slf1

2014 ◽  
Vol 25 (17) ◽  
pp. 2660-2668 ◽  
Author(s):  
Lin Deng ◽  
Ruth Kabeche ◽  
Ning Wang ◽  
Jian-Qiu Wu ◽  
James B. Moseley
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Cell Cycle Progression ◽  
Cell Types ◽  
Lipid Binding ◽  
In Vitro Assays ◽  
Limiting Factor ◽  
C Terminus ◽  
Node Formation

The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types.

scholarly journals MICAL-L1 is required for cargo protein delivery to the cell surface

Biology Open ◽  
2021 ◽  
Vol 10 (6) ◽  
Author(s):  
R. Sikora ◽  
P. Bun ◽  
L. Danglot ◽  
M. Alqabandi ◽  
P. Bassereau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Trafficking ◽  
Protein Delivery ◽  
Close Association ◽  
Super Resolution ◽  
Small Gtpase ◽  
C Terminus ◽  
Membrane Tubulation

ABSTRACT Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0252845
Author(s):  
Chelsea Garno ◽  
Zoe H. Irons ◽  
Courtney M. Gamache ◽  
Quenelle McKim ◽  
Gabriela Reyes ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Animal Cell ◽  
Myosin Ii ◽  
Super Resolution ◽  
Early Embryo ◽  
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-like 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 the visualization of an apparent septin filament network with the CR structure of an animal cell.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

The extracellular matrix of echinoderm and amphibian eggs: Visualization in quick-frozen, deep-etched, and rotary-shadowed specimens

1990 ◽  
Vol 48 (3) ◽  
pp. 60-61
Author(s):  
Barry Bonnell ◽  
Carolyn Larabell ◽  
Douglas Chandler
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Crystalline Structure ◽  
Cortical Granule ◽  
Two Dimensional ◽  
Small Peptides ◽  
Deep Etching ◽  
Quick Freezing ◽  
Acrosomal Reaction

Eggs of many species including those of echinoderms, amphibians and mammals exhibit an extensive extracellular matrix (ECM) that is important both in the reception of sperm and in providing a block to polyspermy after fertilization.In sea urchin eggs there are two distinctive coats, the vitelline layer which contains glycoprotein sperm receptors and the jelly layer that contains fucose sulfate glycoconjugates which trigger the acrosomal reaction and small peptides which act as chemoattractants for sperm. The vitelline layer (VL), as visualized by quick-freezing, deep-etching, and rotary-shadowing (QFDE-RS), is a fishnet-like structure, anchored to the plasma membrane by short posts. Orbiting above the VL are horizontal filaments which are thought to anchor the thicker jelly layer to the egg. Upon fertilization, the VL elevates and is transformed by cortical granule secretions into the fertilization envelope (FE). The rounded casts of microvilli in the VL are transformed into angular peaks and the envelope becomes coated inside and out with sheets of paracrystalline protein having a quasi-two dimensional crystalline structure.

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