scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy

2020 ◽  
Author(s):  
Shayne Quinn ◽  
Lu Huang ◽  
Jason Kerkvliet ◽  
Joel Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Phosphatidylinositol 3 Kinase ◽  
Membrane Ruffling ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
Early Production ◽  
A Minor ◽  
Pharmacologic Inhibition

Abstract Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2020 ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
Early Production ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Minor Role ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

2012 ◽  
Vol 198 (3) ◽  
pp. 421-437 ◽  
Author(s):  
Nadine Schmidt ◽  
Sreya Basu ◽  
Stefan Sladecek ◽  
Sabrina Gatti ◽  
Jeffrey van Haren ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Three Dimensional ◽  
Postsynaptic Membrane ◽  
Dimensional Structure ◽  
Muscle Membrane ◽  
Synaptic Membrane ◽  
Dense Network ◽  
Phosphatidylinositol 3 Kinase ◽  
Neuronal Regulation

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3β, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

scholarly journals Crystal structure ofN-(quinolin-6-yl)hydroxylamine

2014 ◽  
Vol 70 (11) ◽  
pp. 322-324 ◽  
Author(s):  
Anuruddha Rajapakse ◽  
Roman Hillebrand ◽  
Sarah M. Lewis ◽  
Zachary D. Parsons ◽  
Charles L. Barnes ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Title Compound ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Asymmetric Unit ◽  
Π Interactions ◽  
Compound C ◽  
Independent Molecules ◽  
A Minor

The title compound, C9H8N2O, crystallized with four independent molecules in the asymmetric unit. The four molecules are linkedviaone O—H...N and two N—H...N hydrogen bonds, forming a tetramer-like unit. In the crystal, molecules are further linked by O—H...N and N—H...O hydrogen bonds forming layers parallel to (001). These layers are linkedviaC—H...O hydrogen bonds and a number of weak C—H...π interactions, forming a three-dimensional structure. The crystal was refined as a non-merohedral twin with a minor twin component of 0.319.

Mapping of monoclonal antibodies specific to P64k: A common antigen of several isolates of Neisseria meningitidis

2001 ◽  
Vol 47 (2) ◽  
pp. 158-164 ◽  
Author(s):  
C Nazábal ◽  
T Carmenate ◽  
S Cruz ◽  
S González ◽  
R Silva ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Three Dimensional ◽  
Structural Data ◽  
Dimensional Structure ◽  
Bacterial Surface ◽  
A Minor ◽  
The Relationship ◽  
Cell Elisa ◽  
Competition Assays

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.Key words: Neisseria meningitidis, P64k, monoclonal antibodies, epitope mapping.

scholarly journals The solid solution Ba5.78Pb1.22F12Cl2

IUCrData ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Matthias Weil
Keyword(s):  
Solid Solution ◽  
Metal Ions ◽  
Title Compound ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Site Symmetry ◽  
Fluoride Ions ◽  
Coordination Polyhedra ◽  
Space Group ◽  
A Minor

The title compound, hexabarium lead(II) dodecafluoride dichloride, is a solid solution in the system Pb7F12Cl2–Ba7F12Cl2and crystallizes isotypically with the ordered modification of the parent compounds in the space groupP-6. The coordination polyhedra of the three different metal sites are distorted tricapped trigonal prisms with F7Cl2coordination sets for two of these sites (Wyckoff positions 3kand 3j, each with site symmetrym..), and the remaining site being exclusively coordinated by fluoride ions (1a, -6..). By sharing faces, a three-dimensional structure is accomplished. The three metal sites have remarkably different occupancies by the two types of metal ions. Whereas the site on the 3kposition shows only a minor incorporation of Pb2+[occupancy ratio Ba:Pb = 0.93 (4):0.07 (4)], the 3jsite shows the highest amount of incorporated Pb2+[Ba:Pb = 0.71 (5):29 (5)]. The occupancy ratio with respect to the 1asite is Ba:Pb = 0.86 (5):0.14 (5).

Vasopressin-induced changes in the three-dimensional structure of toad bladder apical surface

1987 ◽  
Vol 253 (5) ◽  
pp. C707-C720 ◽  
Author(s):  
J. H. Hartwig ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Cell ◽  
Three Dimensional ◽  
Granular Cell ◽  
Cytochalasin D ◽  
Toad Bladder ◽  
Dimensional Structure ◽  
Apical Plasma Membrane ◽  
Apical Surface

The apical plasma membrane of toad bladder granular cells undergoes a rapid and dramatic increase in water permeability in response to vasopressin stimulation. Previous studies have shown that this permeability increase is accompanied by characteristic changes in the morphology of this membrane and that these changes may be involved in the hormonal response. In this report, we have used the technique of rapid freezing and freeze drying to obtain high resolution stereo images of the surface of the granular cell apical plasma membrane before and during vasopressin stimulation. Using this approach, we confirmed that vasopressin induces a ridge-to-villus transformation of the cell surface even in the absence of osmotic water flow, but now show that this transformation occurs at least in part via a retraction of segments of preexisting ridges, rather than by the growth of new microvilli from the apical cell surface. This is also demonstrated by the finding that vasopressin induces the ridge-to-villus transformation of the cell surface even in the presence of cytochalasin D. In addition, the rapid-freeze, freeze-dry technique reveals that the surface glycocalyx of the epithelial cells consists of a complex, three-dimensional network of filaments that is heterogeneous among different cells. Finally, vasopressin-induced tubular invaginations of the apical plasma membrane were visualized in stereomicrographs, and the number and size of such invaginations were altered in the presence of cytochalasin D. These may represent surface images of vasopressin-induced exo- and endocytotic events that are related to membrane permeability changes.

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