scholarly journals Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1

2006 ◽  
Vol 172 (5) ◽  
pp. 705-717 ◽  
Author(s):  
Ishara Azmi ◽  
Brian Davies ◽  
Christian Dimaano ◽  
Johanna Payne ◽  
Debra Eckert ◽  
...  
Keyword(s):  
Protein Composition ◽  
Surface Protein ◽  
Multivesicular Body ◽  
Proper Function ◽  
Cellular Functions ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Evolutionarily Conserved

In eukaryotes, the multivesicular body (MVB) sorting pathway plays an essential role in regulating cell surface protein composition, thereby impacting numerous cellular functions. Vps4, an ATPase associated with a variety of cellular activities, is required late in the MVB sorting reaction to dissociate the endosomal sorting complex required for transport (ESCRT), a requisite for proper function of this pathway. However, regulation of Vps4 function is not understood. We characterize Vta1 as a positive regulator of Vps4 both in vivo and in vitro. Vta1 promotes proper assembly of Vps4 and stimulates its ATPase activity through the conserved Vta1/SBP1/LIP5 region present in Vta1 homologues across evolution, including human SBP1 and Arabidopsis thaliana LIP5. These results suggest an evolutionarily conserved mechanism through which the disassembly of the ESCRT proteins, and thereby MVB sorting, is regulated by the Vta1/SBP1/LIP5 proteins.

scholarly journals Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Chun-Che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ubiquitin Binding ◽  
Stimulation Of ◽  
Cargo Sorting

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

scholarly journals Unraveling the Molecular Tumor-Promoting Regulation of Cofilin-1 in Pancreatic Cancer

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 725
Author(s):  
Silke D. Werle ◽  
Julian D. Schwab ◽  
Marina Tatura ◽  
Sandra Kirchhoff ◽  
Robin Szekely ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Surface Protein ◽  
Aurora Kinase ◽  
Mechanistic Modeling ◽  
Cellular Functions ◽  
Actin Remodeling ◽  
Protein Levels ◽  
Regulation Model

Cofilin-1 (CFL1) overexpression in pancreatic cancer correlates with high invasiveness and shorter survival. Besides a well-documented role in actin remodeling, additional cellular functions of CFL1 remain poorly understood. Here, we unraveled molecular tumor-promoting functions of CFL1 in pancreatic cancer. For this purpose, we first show that a knockdown of CFL1 results in reduced growth and proliferation rates in vitro and in vivo, while apoptosis is not induced. By mechanistic modeling we were able to predict the underlying regulation. Model simulations indicate that an imbalance in actin remodeling induces overexpression and activation of CFL1 by acting on transcription factor 7-like 2 (TCF7L2) and aurora kinase A (AURKA). Moreover, we could predict that CFL1 impacts proliferation and apoptosis via the signal transducer and activator of transcription 3 (STAT3). These initial model-based regulations could be substantiated by studying protein levels in pancreatic cancer cell lines and human datasets. Finally, we identified the surface protein CD44 as a promising therapeutic target for pancreatic cancer patients with high CFL1 expression.

scholarly journals Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

2020 ◽  
Author(s):  
Chun-che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Evolutionarily Conserved ◽  
Carboxyl Terminal ◽  
Yeast Homolog ◽  
Direct Regulation

AbstractEndosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) form intralumenal vesicles (ILVs) during the conversion of endosomes to multivesicular bodies (MVBs). The assembly and disassembly of an ESCRT-III polymer facilitates membrane remodeling and scission during this process. The ESCRT-III-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) promotes ESCRT-III assembly and inhibits disassembly of ESCRT-III filaments by impeding Vps4, a AAA-ATPase that dismantles ESCRT-III polymers. Here we show that the evolutionarily conserved “V domain” of Bro1-family proteins directly stimulate Vps4 ATPase activity and this activity is enhanced by interaction with ubiquitin. Surprisingly, a carboxyl-terminal fragment of Bro1 containing the V domain supports ILV formation but not sorting of cargo into ILVs, revealing that these two processes can be uncoupled. These studies implicate Bro1 as a factor coordinating cargo sorting with direct regulation of Vps4 to modulate ESCRT-III driven ILV formation during MVB biogenesis.

scholarly journals Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

2021 ◽  
Author(s):  
Naresh Damuka ◽  
Miranda Orr ◽  
Paul W. Czoty ◽  
Jeffrey L. Weiner ◽  
Thomas J. Martin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Ex Vivo ◽  
Neuroblastoma Cells ◽  
Proper Function ◽  
Brain Functions ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

scholarly journals Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 692
Author(s):  
Giulia Franzoni ◽  
Antonio Anfossi ◽  
Chiara Grazia De Ciucis ◽  
Samanta Mecocci ◽  
Tania Carta ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Macrophage Polarization ◽  
Surface Protein ◽  
Antimicrobial Activities ◽  
Toll Like Receptor ◽  
Activation Markers ◽  
Class I Mhc ◽  
Toll Like Receptor 2

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

Current trends in mathematical modeling of tumor–microenvironment interactions: a survey of tools and applications

2010 ◽  
Vol 235 (4) ◽  
pp. 411-423 ◽  
Author(s):  
Katarzyna A Rejniak ◽  
Lisa J McCawley
Keyword(s):  
Mathematical Modeling ◽  
Protein Interactions ◽  
Computational Models ◽  
Chemical Species ◽  
Tumor Stroma ◽  
Cellular Functions ◽  
Complex Interactions ◽  
Expanding Population

In its simplest description, a tumor is comprised of an expanding population of transformed cells supported by a surrounding microenvironment termed the tumor stroma. The tumor microcroenvironment has a very complex composition, including multiple types of stromal cells, a dense network of various extracellular matrix (ECM) fibers interpenetrated by the interstitial fluid and gradients of several chemical species that either are dissolved in the fluid or are bound to the ECM structure. In order to study experimentally such complex interactions between multiple players, cancer is dissected and considered at different scales of complexity, such as protein interactions, biochemical pathways, cellular functions or whole organism studies. However, the integration of information acquired from these studies into a common description is as difficult as the disease itself. Computational models of cancer can provide cancer researchers with invaluable tools that are capable of integrating the complexity into organizing principles as well as suggesting testable hypotheses. We will focus in this Minireview on mathematical models in which the whole cell is a main modeling unit. We will present a current stage of such cell-focused mathematical modeling incorporating different stromal components and their interactions with growing tumors, and discuss what modeling approaches can be undertaken to complement the in vivo and in vitro experimentation.

Generation of Slow Network Oscillations in the Developing Rat Hippocampus After Blockade of Glutamate Uptake

2007 ◽  
Vol 98 (4) ◽  
pp. 2324-2336 ◽  
Author(s):  
Adriano Augusto Cattani ◽  
Valérie Delphine Bonfardin ◽  
Alfonso Represa ◽  
Yehezkel Ben-Ari ◽  
Laurent Aniksztejn
Keyword(s):  
Nmda Receptors ◽  
Glutamate Uptake ◽  
Pyramidal Cells ◽  
Cell Types ◽  
Proper Function ◽  
Network Oscillations ◽  
Bath Application ◽  
Hippocampal Network

Cell-surface glutamate transporters are essential for the proper function of early cortical networks because their dysfunction induces seizures in the newborn rat in vivo. We have now analyzed the consequences of their inhibition by dl-TBOA on the activity of the developing CA1 rat hippocampal network in vitro. dl-TBOA generated a pattern of recurrent depolarization with an onset and decay of several seconds' duration in interneurons and pyramidal cells. These slow network oscillations (SNOs) were mostly mediated by γ-aminobutyric acid (GABA) in pyramidal cells and by GABA and N-methyl-d-aspartate (NMDA) receptors in interneurons. However, in both cell types SNOs were blocked by NMDA receptor antagonists, suggesting that their generation requires a glutamatergic drive. Moreover, in interneurons, SNOs were still generated after the blockade of NMDA-mediated synaptic currents with MK-801, suggesting that SNOs are expressed by the activation of extrasynaptic NMDA receptors. Long-lasting bath application of glutamate or NMDA failed to induce SNOs, indicating that they are generated by periodic but not sustained activation of NMDA receptors. In addition, SNOs were observed in interneurons recorded in slices with or without the strata pyramidale and oriens, suggesting that the glutamatergic drive may originate from the radiatum and pyramidale strata. We propose that in the absence of an efficient transport of glutamate, the transmitter diffuses in the extracellular space to activate extrasynaptic NMDA receptors preferentially present on interneurons that in turn activate other interneurons and pyramidal cells. This periodic neuronal coactivation may contribute to the generation of seizures when glutamate transport dysfunction is present.

Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

Neuromuscular target recognition by a homophilic interaction of connectin cell adhesion molecules in Drosophila

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1433-1441 ◽  
Author(s):  
A. Nose ◽  
T. Umeda ◽  
M. Takeichi
Keyword(s):  
Cell Adhesion ◽  
Synapse Formation ◽  
Target Recognition ◽  
Surface Protein ◽  
Transgenic Lines ◽  
A Cell ◽  
Neuromuscular Connectivity ◽  
Recognition Molecule

Drosophila Connectin (CON) is a cell surface protein of the leucine-rich repeat family. During the formation of neuromuscular connectivity, CON is expressed on the surface of a subset of embryonic muscles and on the growth cones and axons of the motoneurons that innervate these muscles, including primarily SNa motoneurons and their synaptic targets (lateral muscles). In vitro, CON can mediate homophilic cell adhesion. In this study, we generated transgenic lines that ectopically expressed CON on all muscles. In the transformant embryos and larvae, SNa motoneurons often inappropriately innervated a neighboring non-target muscle (muscle 12) that ectopically expressed CON. Furthermore, the ectopic synapse formation was dependent on the endogenous CON expression on the SNa motoneurons. These results show that CON can function as an attractive and homophilic target recognition molecule in vivo.

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

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