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The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.
Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.
The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.
Copyright © 2019 Elsevier Inc. All rights reserved.
Na-K-2Cl cotransporter-1 (NKCC1) mediates the electroneutral transport of Na, K, and Cl and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.
Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
Annexin proteins function as Ca-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.
© 2019 McCulloch et al.
Integrins are α/β heterodimers that interconvert between inactive and active states. In the active state the α/β cytoplasmic domains recruit integrin-activating proteins and separate the transmembrane and cytoplasmic (TMcyto) domains (unclasped TMcyto). Conversely, in the inactive state the α/β TMcyto domains bind integrin-inactivating proteins, resulting in the association of the TMcyto domains (clasped TMcyto). Here, we report the isolation of integrin cytoplasmic tail interactors using either lipid bicelle-incorporated integrin TMcyto domains (α5, αM, αIIb, β1, β2 and β3 integrin TMcyto) or a clasped, lipid bicelle-incorporated αMβ2 TMcyto. Among the proteins found to preferentially bind clasped rather than the isolated αM and β2 subunits was L-plastin (LCP1, also known as plastin-2), which binds to and maintains the inactive state of αMβ2 integrin and thereby regulates leukocyte adhesion to integrin ligands under flow. Our findings offer a global view on cytoplasmic proteins interacting with different integrins and provide evidence for the existence of conformation-specific integrin interactors.
© 2018. Published by The Company of Biologists Ltd.
Porphyromonas gingivalis is a keystone bacterium in the oral microbial communities that elicits a dysbiosis between the microbiota and the host. Therefore, inhibition of this organism in dental plaques has been one of the strategies for preventing and treating chronic periodontitis. We previously identified a Streptococcal ArcA derived Anti-P gingivalils Peptide (SAPP) that in vitro, is capable of repressing the expression of several virulence genes in the organism. This leads to a significant reduction in P gingivalis virulence potential, including its ability to colonize on the surface of Streptococcus gordonii, to invade human oral epithelial cells, and to produce gingipains. In this study, we showed that SAPP had minimal cytotoxicity to human oral keratinocytes and gingival fibroblasts. We observed that SAPP directly bound to the cell surface of P gingivalis, and that alterations in the sequence at the N-terminus of SAPP diminished its abilities to interact with P gingivalis cells and repressed the expression of virulence genes. Most strikingly, we demonstrated using an ex-vivo assay that besides its inhibitory activity against P gingivalis colonization, SAPP could also reduce the levels of several other oral Gram-negative bacteria strongly associated with periodontitis in multispecies biofilms. Our results provide a platform for the development of SAPP-targeted therapeutics against chronic periodontitis.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Quantum dots are nanometer-sized semiconductors that have size-tunable, narrow emission bands, high quantum yields, and are resistant to photobleaching. Ligand-conjugated quantum dots enable the real time visualization of membrane proteins and have revealed that membrane diffusion dynamics are intrinsic to protein regulation, are susceptible to the level of membrane cholesterol, and are altered in genetic variants linked to disease, suggesting a mise en place approach to neuropsychopharmacology.
Ca/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu is present in CaMKII complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu directly interacts with purified Thr286-autophosphorylated (activated) CaMKII However, the binding of CaMKII to this CTD fragment is reduced by the addition of excess Ca/calmodulin or by additional CaMKII autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKII is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKII with full-length mGlu expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKII with mGlu in heterologous cells. First, cell-surface biotinylation studies showed that CaMKII increases the surface expression of mGlu Second, using Ca fluorimetry and single-cell Ca imaging, we found that CaMKII reduces the initial peak of mGlu-mediated Ca mobilization by about 25% while doubling the relative duration of the Ca signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.