scholarly journals Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Julien Gronnier ◽  
Jean-Marc Crowet ◽  
Birgit Habenstein ◽  
Mehmet Nail Nasir ◽  
Vincent Bayle ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Super Resolution ◽  
Lipid Binding ◽  
Single Particle Tracking ◽  
Structural Basis ◽  
Binding Motif ◽  
Supramolecular Organization ◽  
Hormonal Crosstalk ◽  
Definition Of

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.

scholarly journals Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

2020 ◽  
Author(s):  
Jia Hui Li ◽  
Paula Santos-Otte ◽  
Braedyn Au ◽  
Jakob Rentsch ◽  
Stephan Block ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Magnetic Nanoparticles ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Single Molecule Level ◽  
Membrane Components

AbstractThe plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available for nanoscale manipulation of membrane protein location at the single molecule level. Here, we report the use of fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to manipulate their movement. FMNPs allow single-particle tracking (SPT) at 10 nm spatial and 5 ms temporal resolution, and using a magnetic needle, we pull membrane components laterally through the membrane with femtonewton-range forces. In this way, we successfully dragged lipid-anchored and transmembrane proteins over the surface of living cells. Doing so, we detected submembrane barriers and in combination with super-resolution microscopy could localize these barriers to the actin cytoskeleton. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

scholarly journals Tracking of Quantum Dot-labeled CFTR Shows Near Immobilization by C-Terminal PDZ Interactions

2006 ◽  
Vol 17 (12) ◽  
pp. 4937-4945 ◽  
Author(s):  
Peter M. Haggie ◽  
Jung Kyung Kim ◽  
Gergely L. Lukacs ◽  
A. S. Verkman
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Quantum Dot ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Adaptor Protein ◽  
Single Particle Tracking ◽  
Binding Motif ◽  
Protein Fusion ◽  
Green Fluorescent

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100–200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.

scholarly journals Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

2016 ◽  
Vol 113 (9) ◽  
pp. E1152-E1161 ◽  
Author(s):  
Loes M. Stevers ◽  
Chan V. Lam ◽  
Seppe F. R. Leysen ◽  
Femke A. Meijer ◽  
Daphne S. van Scheppingen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Binding Site ◽  
Binding Motif ◽  
Protein Protein Interaction ◽  
Biochemical Assays ◽  
Binding Groove ◽  
Definition Of ◽  
R Domain

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.

scholarly journals Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

2021 ◽  
pp. 100607
Author(s):  
Ivana Malcova ◽  
Ladislav Bumba ◽  
Filip Uljanic ◽  
Darya Kuzmenko ◽  
Jana Nedomova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Binding ◽  
Membrane Localization ◽  
Effector Protein ◽  
Plasma Membrane Localization

scholarly journals pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

Polytypes and polytypism

1986 ◽  
Vol 176 (3-4) ◽  
Author(s):  
R. J. Angel
Keyword(s):  
Structural Variation ◽  
Structural Basis ◽  
Definition Of ◽  
Alloy Systems

AbstractThe definition of polytypism recommended by the IMA/IUCr joint committee on nomenclature is reviewed in the light of the structural variation observed in mineral and alloy systems. As a result of this review it is concluded that previous definitions of polytypism are too restrictive to be practical, and a definition which emphasises the structural basis of polytypism is proposed:“Polytypism arises from the different modes of stacking of one or more structurally compatible modules.”The various types of structural variation included under this definition are described using as examples minerals, ceramics, and alloys.

scholarly journals Membrane traffic between secretory compartments is differentially affected during mitosis.

1990 ◽  
Vol 1 (5) ◽  
pp. 415-424 ◽  
Author(s):  
T Kreiner ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Traffic ◽  
Human Growth ◽  
Secretory Proteins ◽  
Viral Membrane ◽  
Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

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