scholarly journals CD82 and Gangliosides Tune CD81 Membrane Behavior

2021 ◽  
Vol 22 (16) ◽  
pp. 8459
Author(s):  
Laurent Fernandez ◽  
Morgane Malrieu ◽  
Christine Bénistant ◽  
Patrice Dosset ◽  
Eric Rubinstein ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Protein Interactions ◽  
Mammary Epithelial Cells ◽  
Protein Composition ◽  
Mammary Epithelial ◽  
Protein Protein Interactions ◽  
Membrane Behavior ◽  
New Information ◽  
Lateral Segregation

Tetraspanins are a family of transmembrane proteins that form a network of protein–protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.

scholarly journals Exocyst Dynamics During Vesicle Tethering and Fusion

2018 ◽  
Author(s):  
Syed Mukhtar Ahmed ◽  
Hisayo Nishida-Fukuda ◽  
Yuchong Li ◽  
W. Hayes McDonald ◽  
Claudiu Gradinaru ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Mammary Epithelial Cells ◽  
Dynamic Equilibrium ◽  
Correlation Spectroscopy ◽  
Mammary Epithelial ◽  
High Speed Imaging ◽  
Arrival Times

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of Sec3 occurs prior to fusion, whereas other subunits depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.

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.

scholarly journals The NHR1 Domain of Neuralized Binds Delta and Mediates Delta Trafficking and Notch Signaling

2007 ◽  
Vol 18 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Cosimo Commisso ◽  
Gabrielle L. Boulianne
Keyword(s):  
Plasma Membrane ◽  
Notch Signaling ◽  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Neural Development ◽  
Ring Domain ◽  
Protein Protein Interactions ◽  
Necessary And Sufficient ◽  
Conserved Residue ◽  
Notch Ligands

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized1, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein–protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.

Proteomics Study in Urolithiasis

2020 ◽  
Vol 17 (2) ◽  
pp. 88-94
Author(s):  
Manavi Jain ◽  
Paramveer Yadav ◽  
Priyadarshini
Keyword(s):  
Protein Interactions ◽  
Kidney Diseases ◽  
Stone Formation ◽  
Protein Composition ◽  
Protein Crystal ◽  
World Population ◽  
Protein Protein Interactions ◽  
Bioinformatic Tools ◽  
Proteomic Study ◽  
Stone Matrix

Urolithiasis, which is the presence of stones in the urinary tract, has long been linked with a higher risk of causing chronic kidney diseases and associated illnesses, such as diabetes-affecting 12% of the world population. This clinical condition arises due to the supersaturation of urine and alterations in the expression of cellular and urinary proteins. The renal stone mineral composition has been well understood and incorporated as a routine part of stone removal, however, the protein composition, an essential fraction of the stone matrix has been inadequately understood and not adeptly established. Stone proteomics consists of a number of techniques including crystal analysis using X-ray diffractometry and IR spectroscopy, sample purification, identification and characterization of proteins using high throughput mass spectrometric methods. However, not many studies have utilized the data obtained from these experiments to assign functional significance to associated identified proteins. Protein network analysis using bioinformatic tools such as STRING to study protein-protein interactions will enable researchers to get better insight into stone formation mechanics. Hence, a comprehensive proteomic study of kidney stone matrix will help in deciphering protein-crystal pathways generating novel information useful for clinical application.

scholarly journals The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 986
Author(s):  
Monique D. Appelman ◽  
Marion J.D. Robin ◽  
Esther W.M. Vogels ◽  
Christie Wolzak ◽  
Winnie G. Vos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bile Salt ◽  
Protein Interactions ◽  
Basolateral Membrane ◽  
Sodium Taurocholate ◽  
Salt Transport ◽  
Protein Protein Interactions ◽  
Cell Surface Biotinylation ◽  
Detergent Resistant Membranes ◽  
Entry Receptor

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein–protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.

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