scholarly journals CombiFlow: Combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones

2021 ◽  
Author(s):  
Roos Houtsma ◽  
Nisha K. van der Meer ◽  
Kees Meijer ◽  
Linde Morsink ◽  
Shanna M. Hogeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Residual Disease ◽  
Expression Profiles ◽  
Clonal Evolution ◽  
Membrane Expression ◽  
Routine Diagnostics ◽  
Free Survival ◽  
Longitudinal Tracking ◽  
Plasma Membrane Expression

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.

Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation

2005 ◽  
Vol 288 (5) ◽  
pp. F930-F938 ◽  
Author(s):  
Lene N. Nejsum ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Jørgen Frøkiær ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Rat Kidney ◽  
Wild Type ◽  
Membrane Expression ◽  
Bidirectional Regulation ◽  
Plasma Membrane Expression ◽  
Intracellular Vesicles ◽  
Membrane Treatment ◽  
Microscopic Techniques

The present study examined the role of PKA and serine256 (S256) phosphorylation for AQP2 trafficking and recycling using cells transfected with wild-type AQP2 (AQP2-WT) or mutant AQP2 and high-resolution confocal microscopic techniques. In transiently transfected MDCK-C7 cells, stimulation with forskolin induced translocation of AQP2-WT to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H-89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H-89 treatment of AQP2-S256D (mimicking constitutively phosphorylated AQP2 and hence localized to the plasma membrane) resulted in redistribution of AQP2-S256D to intracellular vesicles, even in the presence of forskolin. Both PGE2 and dopamine stimulation induced endocytosis of AQP2-WT and AQP2-S256D, respectively, in forskolin-stimulated cells. Consistent with this, dopamine in the presence of vasopressin stimulated endocytosis of AQP2 in slices of rat kidney inner medulla without substantial dephosphorylation. In conclusion, these results strongly suggest that 1) S256 phosphorylation is necessary but not sufficient for AQP2 plasma membrane expression, 2) active PKA is required for AQP2 plasma membrane expression, 3) PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

2007 ◽  
Vol 292 (5) ◽  
pp. C1690-C1700 ◽  
Author(s):  
Neelakshi R. Jog ◽  
Madhavi J. Rane ◽  
George Lominadze ◽  
Gregory C. Luerman ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Immunoblot Analysis ◽  
Maximal Response ◽  
Initial Increase ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Latrunculin A ◽  
Specific Granules

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED50of 15, 31, and 28 nM, respectively, with maximal response at 10−7M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC50for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

scholarly journals Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

2019 ◽  
Vol 317 (2) ◽  
pp. F411-F418
Author(s):  
Casandra M. Monzon ◽  
Jeffrey L. Garvin
Keyword(s):  
Plasma Membrane ◽  
Control Period ◽  
Paracellular Pathway ◽  
Membrane Expression ◽  
No Donor ◽  
Charge Selectivity ◽  
Plasma Membrane Expression ◽  
Cgmp Signaling ◽  
Blocking Antibodies ◽  
Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

scholarly journals Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

2009 ◽  
Vol 296 (4) ◽  
pp. C857-C867 ◽  
Author(s):  
Silvia M. Uriarte ◽  
Neelakshi R. Jog ◽  
Gregory C. Luerman ◽  
Samrath Bhimani ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Human Neutrophils ◽  
Functional Responses ◽  
Membrane Expression ◽  
Granule Exocytosis ◽  
Plasma Membrane Expression ◽  
Microtubular Cytoskeleton ◽  
Latrunculin A ◽  
Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

2017 ◽  
Vol 19 (5) ◽  
pp. 1377-1386 ◽  
Author(s):  
Vineet Kumar ◽  
Tot Bui Nguyen ◽  
Beáta Tóth ◽  
Viktoria Juhasz ◽  
Jashvant D. Unadkat
Keyword(s):  
Plasma Membrane ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
In Cells

scholarly journals Low Plasma Membrane Expression of the Miltefosine Transport Complex Renders Leishmania braziliensis Refractory to the Drug

2009 ◽  
Vol 53 (4) ◽  
pp. 1305-1313 ◽  
Author(s):  
María P. Sánchez-Cañete ◽  
Luís Carvalho ◽  
F. Javier Pérez-Victoria ◽  
Francisco Gamarro ◽  
Santiago Castanys
Keyword(s):  
Plasma Membrane ◽  
Cutaneous Leishmaniasis ◽  
Oral Drug ◽  
Leishmania Braziliensis ◽  
Β Subunit ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Highly Sensitive ◽  
P Type ◽  
Transport Complex

ABSTRACT Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its β subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the β subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

scholarly journals Single-cell mutational profiling enhances the clinical evaluation of AML MRD

2020 ◽  
Vol 4 (5) ◽  
pp. 943-952 ◽  
Author(s):  
Asiri Ediriwickrema ◽  
Alexey Aleshin ◽  
Johannes G. Reiter ◽  
M. Ryan Corces ◽  
Thomas Köhnke ◽  
...  
Keyword(s):  
Single Cell ◽  
Residual Disease ◽  
Single Cells ◽  
Clonal Evolution ◽  
Clonal Diversity ◽  
Free Survival ◽  
Clonal Hematopoiesis ◽  
Mutational Profiling ◽  
Generation Sequencing ◽  
Relapse Rates

Abstract Although most patients with acute myeloid leukemia (AML) achieve clinical remission with induction chemotherapy, relapse rates remain high. Next-generation sequencing enables minimal/measurable residual disease (MRD) detection; however, clinical significance is limited due to difficulty differentiating between pre-leukemic clonal hematopoiesis and frankly malignant clones. Here, we investigated AML MRD using targeted single-cell sequencing (SCS) at diagnosis, remission, and relapse (n = 10 relapsed, n = 4 nonrelapsed), with a total of 310 737 single cells sequenced. Sequence variants were identified in 80% and 75% of remission samples for patients with and without relapse, respectively. Pre-leukemic clonal hematopoiesis clones were detected in both cohorts, and clones with multiple cooccurring mutations were observed in 50% and 0% of samples. Similar clonal richness was observed at diagnosis in both cohorts; however, decreasing clonal diversity at remission was significantly associated with longer relapse-free survival. These results show the power of SCS in investigating AML MRD and clonal evolution.

Sign in / Sign up

Export Citation Format

Share Document