scholarly journals Architecture of the human PI4KIIIα lipid kinase complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13720-13725 ◽  
Author(s):  
Joshua A. Lees ◽  
Yixiao Zhang ◽  
Michael S. Oh ◽  
Curtis M. Schauder ◽  
Xiaoling Yu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Active Sites ◽  
Structural Complexity ◽  
Signaling Molecules ◽  
Cell Physiology ◽  
Lipid Kinase ◽  
Membrane Interaction ◽  
Regulatory Subunits ◽  
Cryo Electron Microscopy

Plasma membrane (PM) phosphoinositides play essential roles in cell physiology, serving as both markers of membrane identity and signaling molecules central to the cell’s interaction with its environment. The first step in PM phosphoinositide synthesis is the conversion of phosphatidylinositol (PI) to PI4P, the precursor of PI(4,5)P2 and PI(3,4,5)P3. This conversion is catalyzed by the PI4KIIIα complex, comprising a lipid kinase, PI4KIIIα, and two regulatory subunits, TTC7 and FAM126. We here report the structure of this complex at 3.6-Å resolution, determined by cryo-electron microscopy. The proteins form an obligate ∼700-kDa superassembly with a broad surface suitable for membrane interaction, toward which the kinase active sites are oriented. The structural complexity of the assembly highlights PI4P synthesis as a major regulatory junction in PM phosphoinositide homeostasis. Our studies provide a framework for further exploring the mechanisms underlying PM phosphoinositide regulation.

scholarly journals Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures of Enterococcus gallinarum and Streptococcus gordonii Septa Revealed by Cryo-Electron Microscopy of Vitreous Sections

2006 ◽  
Vol 188 (18) ◽  
pp. 6652-6660 ◽  
Author(s):  
Benoît Zuber ◽  
Marisa Haenni ◽  
Tânia Ribeiro ◽  
Kathrin Minnig ◽  
Fátima Lopes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Granular Layer ◽  
Streptococcus Gordonii ◽  
Periplasmic Space ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cryo Electron Microscopy ◽  
Enterococcus Gallinarum ◽  
Peptidoglycan Layer

ABSTRACT High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

scholarly journals Tyrosine kinase signaling in and on the endoplasmic reticulum

2020 ◽  
Vol 48 (1) ◽  
pp. 199-205 ◽  
Author(s):  
Hesso Farhan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Signaling Molecules ◽  
Cell Physiology ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Cellular Processes

Tyrosine kinases are signaling molecules that are common to all metazoans and are involved in the regulation of many cellular processes such as proliferation and survival. While most attention has been devoted to tyrosine kinases signaling at the plasma membrane and the cytosol, very little attention has been dedicated to signaling at endomembranes. In this review, I will discuss recent evidence that we obtained on signaling of tyrosine kinases at the surface of the endoplasmic reticulum (ER), as well as in the lumen of this organelle. I will discuss how tyrosine kinase signaling might regulate ER proteostasis and the implication thereof to general cell physiology.

Topography of signaling molecules as detected by electron microscopy on plasma membrane sheets isolated from non-adherent mast cells

2007 ◽  
Vol 328 (1-2) ◽  
pp. 139-151 ◽  
Author(s):  
Pavel Lebduška ◽  
Jan Korb ◽  
Magda Tůmová ◽  
Petr Heneberg ◽  
Petr Dráber
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Mast Cells ◽  
Signaling Molecules

scholarly journals Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

2020 ◽  
Author(s):  
Oleksiy Kovtun ◽  
Veronica Kane Dickson ◽  
Bernard T. Kelly ◽  
David. J. Owen ◽  
John A. G. Briggs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Protein Composition ◽  
Binding Mode ◽  
Coated Vesicles ◽  
Conformational Rearrangement ◽  
Multiple Interfaces ◽  
Cryo Electron Microscopy ◽  
Subtomogram Averaging ◽  
Key Steps

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

scholarly journals The structure of Macrophage Expressed Gene-1, a phagolysosome immune effector that is activated upon acidification

2019 ◽  
Author(s):  
Siew Siew Pang ◽  
Charles Bayly-Jones ◽  
Mazdak Radjainia ◽  
Bradley A. Spicer ◽  
Ruby H.P. Law ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Mode Of Action ◽  
Membrane Attack Complex ◽  
Membrane Interaction ◽  
Immune Effector ◽  
Cryo Electron Microscopy

Macrophage Expressed Gene-1 (MPEG-1; also termed Perforin-2) is an endosomal / phagolysosomal perforin-like protein that is conserved across the metazoan kingdom and that functions within the phagolysosome to damage engulfed microbes. Like the Membrane Attack Complex and perforin, MPEG-1 has been postulated to form pores in target membranes, however, its mode of action remains to be established. We used single particle cryo-Electron Microscopy to determine the 2.4 Å structure of a hexadecameric assembly of MPEG-1 that displays the expected features of a soluble pre-pore complex. We further discovered that the MPEG-1 pre-pore-like assemblies can be induced to perforate membranes through mild acidification, such as would occur within maturing phagolysosomes. We next solved the 3.6 Å cryo-EM structure of MPEG-1 in complex with liposomes. Remarkably these data revealed that a C-terminal Multi-vesicular body of 12 kDa (MVB12)-associatedβ-prism (MABP) domain interacts with target membranes in a mode that positions the pore forming machinery of MPEG-1 to point away from the bound membrane. This unexpected mechanism of membrane interaction raises the intriguing possibility that MPEG-1 may be able to remain bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

scholarly journals The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Siew Siew Pang ◽  
Charles Bayly-Jones ◽  
Mazdak Radjainia ◽  
Bradley A. Spicer ◽  
Ruby H. P. Law ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mode Of Action ◽  
Membrane Interaction ◽  
Immune Effector ◽  
Cryo Electron Microscopy

Abstract Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

scholarly journals Molecular mechanism of the Orai channel activation

2018 ◽  
Author(s):  
Xiaofen Liu ◽  
Guangyan Wu ◽  
Yi Yu ◽  
Xiaozhe Chen ◽  
Renci Ji ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Plasma Membrane ◽  
Potential Gradient ◽  
State Structure ◽  
Channel Activation ◽  
Cryo Electron Microscopy ◽  
Cytosolic Side ◽  
Ion Conducting ◽  
Channel Assembly

The Orai channel is characterized by voltage independence, low conductance and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane. How the channel is activated and promotes Ca2+ permeation are not well understood. Here, we report the crystal structure and cryo-electron microscopy reconstruction of a Drosophila melanogaster Orai mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which six TM1 helices in the center form the ion-conducting pore, and six TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation and Ca2+ permeation.

Electron microscopy of endocardial cell populations

1978 ◽  
Vol 36 (2) ◽  
pp. 486-487
Author(s):  
T. G. Sarphie ◽  
C. R. Comer ◽  
D. J. Allen
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cellular Transformation ◽  
Cell Populations ◽  
Cell Surfaces ◽  
Kb Cells ◽  
Dna Viruses ◽  
Cell Cycles ◽  
Ultrastructural Studies ◽  
Endocardial Cell

Previous ultrastructural studies have characterized surface morphology during norma cell cycles in an attempt to associate specific changes with specific metabolic processes occurring within the cell. It is now known that during the synthetic ("S") stage of the cycle, when DNA and other nuclear components are synthesized, a cel undergoes a doubling in volume that is accompanied by an increase in surface area whereby its plasma membrane is elaborated into a variety of processes originally referred to as microvilli. In addition, changes in the normal distribution of glycoproteins and polysaccharides derived from cell surfaces have been reported as depreciating after cellular transformation by RNA or DNA viruses and have been associated with the state of growth, irregardless of the rate of proliferation. More specifically, examination of the surface carbohydrate content of synchronous KB cells were shown to be markedly reduced as the cell population approached division Comparison of hamster kidney fibroblasts inhibited by vinblastin sulfate while in metaphase with those not in metaphase demonstrated an appreciable decrease in surface carbohydrate in the former.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

Sign in / Sign up

Export Citation Format

Share Document