scholarly journals Single Cell Center of Mass for the Analysis of BMP Receptor Heterodimers Distributions

2021 ◽  
Author(s):  
Hendrik Boog ◽  
Rebecca Medda ◽  
Elisabetta Ada Cavalcanti-Adam
Keyword(s):  
Signaling Pathways ◽  
Single Cells ◽  
Center Of Mass ◽  
Control Cell ◽  
Culture Conditions ◽  
Cell Polarization ◽  
Receptor Complexes ◽  
Cell Shapes ◽  
Distribution Studies ◽  
Receptor Distribution

At the plasma membrane, transmembrane receptors are at the interface between cells and their environment. They allow sensing and transduction of chemical and mechanical extracellular signals. The spatial distribution of receptors and the specific recruitment of receptor subunits to the cell membrane is crucial for the regulation of signaling and cell behavior. However, it is challenging to define what regulates such spatial patterns for receptor localization, as cell shapes are extremely diverse when cells are maintained in standard culture conditions. Bone morphogenic protein receptors (BMPRs) are serine-threonine kinases, which build heteromeric complexes of BMPRI and II. These are especially interesting targets for receptor distribution studies, since the signaling pathways triggered by BMPR-complexes depends on their dimerization mode. They might exist as pre-formed complexes, or assemble upon binding of BMP, triggering cell signaling which leads to differentiation or migration. In this work we analyzed BMPR receptor distributions in single cells grown on micropatterns, which allows not only to control cell shape, but also the distribution of intracellular organelles and protein assemblies. We developed a script called ComRed (Center Of Mass Receptor Distribution), which uses center of mass calculations to analyze the shift and spread of receptor distributions according to the different cell shapes. ComRed was tested by simulating changes in experimental data, showing that shift and spread of distributions can be reliably detected. Our ComRed-based analysis of BMPR-complexes indicates that receptor distribution depends on cell polarization. The absence of a coordinated internalization after addition of BMP suggests that a rapid and continual recycling of BMPRs occurs. Receptor complexes formation and localization in cells induced by BMP might yield insights into the local regulation of different signaling pathways.

scholarly journals Single Cell Center of Mass for the Analysis of BMP Receptor Heterodimers Distributions

2021 ◽  
Vol 7 (11) ◽  
pp. 219
Author(s):  
Hendrik Boog ◽  
Rebecca Medda ◽  
Elisabetta Ada Cavalcanti-Adam
Keyword(s):  
Signaling Pathways ◽  
Single Cells ◽  
Center Of Mass ◽  
Control Cell ◽  
Culture Conditions ◽  
Cell Polarization ◽  
Receptor Complexes ◽  
Cell Shapes ◽  
Distribution Studies ◽  
Receptor Distribution

At the plasma membrane, transmembrane receptors are at the interface between cells and their environment. They allow sensing and transduction of chemical and mechanical extracellular signals. The spatial distribution of receptors and the specific recruitment of receptor subunits to the cell membrane is crucial for the regulation of signaling and cell behavior. However, it is challenging to define what regulates such spatial patterns for receptor localization, as cell shapes are extremely diverse when cells are maintained in standard culture conditions. Bone morphogenetic protein receptors (BMPRs) are serine-threonine kinases, which build heteromeric complexes of BMPRI and II. These are especially interesting targets for receptor distribution studies, since the signaling pathways triggered by BMPR-complexes depends on their dimerization mode. They might exist as preformed complexes, or assemble upon binding of BMP, triggering cell signaling which leads to differentiation or migration. In this work we analyzed BMPR receptor distributions in single cells grown on micropatterns, which allow not only to control cell shape, but also the distribution of intracellular organelles and protein assemblies. We developed a script called ComRed (Center Of Mass Receptor Distribution), which uses center of mass calculations to analyze the shift and spread of receptor distributions according to the different cell shapes. ComRed was tested by simulating changes in experimental data showing that shift and spread of distributions can be reliably detected. Our ComRed-based analysis of BMPR-complexes indicates that receptor distribution depends on cell polarization. The absence of a coordinated internalization after addition of BMP suggests that a rapid and continual recycling of BMPRs might occur. Receptor complexes formation and localization in cells induced by BMP might yield insights into the local regulation of different signaling pathways.

Targeting Mcl-1 Enhances the Activity of Tyrosine Kinase Inhibitor Gilteritinib in FLT3 Mutated AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Vivian Ruvolo ◽  
Wenjing Tao ◽  
Paul Hughes ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Progenitor Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Mapk Signaling ◽  
Culture Conditions ◽  
Beta Catenin ◽  
Adaptive Resistance

Anti-apoptotic Bcl-2 proteins play critical roles in AML cell and AML stem/progenitor cell survival and drug resistance, hence are relevant therapeutic targets. Indeed, the combination of the selective Bcl-2 inhibitor venetoclax (VEN) with a hypomethylating agent elicits CR/CRi rates of > 65%, is well tolerated by elderly AML patients, and obtained FDA approval. However, despite of the major improvement in response rates, survival extension was limited and most patients ultimately relapsed largely due to the development of resistant disease. Molecular analysis of treated patients revealed that primary and adaptive resistance to VEN-based combinations was frequently characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS (DiNardo CD et al., Blood 2020). FLT3 is one of the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways such as RAS/MAPK, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients treated with TKIs ultimately relapse and adapt to TKI therapy by reactivating the MAPK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 expression can be induced by VEN treatment and is a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML, targeting AML cells and stem/progenitor cells. To determine if targeting Mcl-1 enhances the activity of TKIs in FLT3 mutated AML, we treated MV4-11 and Molm13 cells with Mcl-1 inhibitor AMG176 and TKI gilteritinib (GIL) and observed synergism, as defined by combination index < 1 in both cells. Mechanistic studies demonstrated that GIL markedly decreased Mcl-1 and antagonized AMG176-induced Mcl-1 induction. GIL and its combination with AMG176 also decreased Bcl-xL. Although Bcl-2 protein levels were largely not changed in MV4-11 cells, we found both single treatment and the combination greatly decreased Bcl-2 associated athanogene (BAG) proteins BAG1, BAG3, and BAG4 at the RNA level, which needs to be confirmed at the protein level. The BAG proteins are a family of chaperone regulators and BAG1 was reported to bind and enhance the activity of multiple proteins known to support cells survival, including Bcl-2 (Takayama S et al., Cell 1995). Interestingly, GIL treatment greatly diminished the levels of beta-catenin and its target protein c-Myc, consistent with our previous report that FLT3 regulates beta-catenin signaling (Xiang et al., CCR, 2018). We have generated Mcl-1 overexpressing (OE) and VEN-resistance (VEN-R) MV4-11 and Molm13 cells. The Mcl-1 OE cells are highly resistant to VEN and the VEN-R cells expressed high levels of Mcl-1. Combined inhibition of AMG176 and GIL synergistically induced cell death in Mcl-1 OE and VEN-R resistant cells. Although the expression is low in AML cells we tested, BCL2A1 is also known as a resistant factor to VEN. We generated BCL2A1 OE MV4-11 and Molm13 cells and demonstrated that combined inhibition of FLT3 and Mcl-1 was highly effective in these cells as well. Western blot analysis revealed that GIL effectively decreased Mcl-1 in Mcl-1 OE and VEN-R and BCL2A1 in BCL2A1 OE MV4-11 cells. Next, we treated FLT3 mutated AML patient samples harboring both, ITD and D835 mutations, from 2 patients who had both failed VEN-based therapy and from 1 patient with ITD mutation, with AMG176 and GIL under MSC co-culture conditions. Synergy was observed in all samples in AML blasts and AML stem/progenitor cells. Collectively, our data demonstrate that targeting Mcl-1 enhances the activity of GIL in FLT3 mutated AML, including those resistant to/relapsed from VEN-based therapy, findings that may warrant clinical evaluation. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals COL2A1 Is a Novel Biomarker of Melanoma Tumor Repopulating Cells

Biomedicines ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 360
Author(s):  
Bhavana Talluri ◽  
Kshitij Amar ◽  
Michael Saul ◽  
Tasnim Shireen ◽  
Vjollca Konjufca ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Line ◽  
Aerobic Glycolysis ◽  
Parental Control ◽  
Control Cell ◽  
Differentially Expressed Gene ◽  
Gene Clusters ◽  
Rna Seq ◽  
Novel Biomarkers ◽  
Gene Regulatory

Soft 3D-fibrin-gel selected tumor repopulating cells (TRCs) from the B16F1 melanoma cell line exhibit extraordinary self-renewal and tumor-regeneration capabilities. However, their biomarkers and gene regulatory features remain largely unknown. Here, we utilized the next-generation sequencing-based RNA sequencing (RNA-seq) technique to discover novel biomarkers and active gene regulatory features of TRCs. Systems biology analysis of RNA-seq data identified differentially expressed gene clusters, including the cell adhesion cluster, which subsequently identified highly specific and novel biomarkers, such as Col2a1, Ncam1, F11r, and Negr1. We validated the expression of these genes by real-time qPCR. The expression level of Col2a1 was found to be relatively low in TRCs but twenty-fold higher compared to the parental control cell line, thus making the biomarker very specific for TRCs. We validated the COL2A1 protein by immunofluorescence microscopy, showing a higher expression of COL2A1 in TRCs compared to parental control cells. KEGG pathway analysis showed the JAK/STAT, hypoxia, and Akt signaling pathways to be active in TRCs. Besides, the aerobic glycolysis pathway was found to be very active, indicating a typical Warburg Effect on highly tumorigenic cells. Together, our study revealed highly specific biomarkers and active cell signaling pathways of melanoma TRCs that can potentially target and neutralize TRCs.

scholarly journals Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

2018 ◽  
Vol 115 (19) ◽  
pp. E4377-E4385 ◽  
Author(s):  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Sitong Zhou ◽  
Michael Giannetto ◽  
David S. Gokhin ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Motor Activity ◽  
Control Cell ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
General Function ◽  
Actin Networks ◽  
Cell Shapes

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin–F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin–F-actin networks.

scholarly journals Surface functions during mitosis. III. Quantitative analysis of ligand-receptor movement into the cleavage furrow: diffusion vs. flow.

1982 ◽  
Vol 93 (3) ◽  
pp. 950-960 ◽  
Author(s):  
D E Koppel ◽  
J M Oliver ◽  
R D Berlin
Keyword(s):  
Cell Shape ◽  
Single Cells ◽  
Effective Diffusion ◽  
Membrane Receptors ◽  
Cleavage Furrow ◽  
Receptor Complex ◽  
Con A ◽  
Surface Distribution ◽  
Lectin Receptor ◽  
Receptor Complexes

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A-receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(-9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow.

scholarly journals Cdc42 and Par6–PKCζ regulate the spatially localized association of Dlg1 and APC to control cell polarization

2005 ◽  
Vol 170 (6) ◽  
pp. 895-901 ◽  
Author(s):  
Sandrine Etienne-Manneville ◽  
Jean-Baptiste Manneville ◽  
Sarah Nicholls ◽  
Michael A. Ferenczi ◽  
Alan Hall
Keyword(s):  
Total Internal Reflection ◽  
Biochemical Analysis ◽  
Control Cell ◽  
Leading Edge ◽  
Cell Polarization ◽  
Physical Interaction ◽  
Biological Processes ◽  
Polyposis Coli ◽  
Tumor Suppressor Proteins ◽  
Wide Range

Cell polarization is essential in a wide range of biological processes such as morphogenesis, asymmetric division, and directed migration. In this study, we show that two tumor suppressor proteins, adenomatous polyposis coli (APC) and Dlg1-SAP97, are required for the polarization of migrating astrocytes. Activation of the Par6–PKCζ complex by Cdc42 at the leading edge of migrating cells promotes both the localized association of APC with microtubule plus ends and the assembly of Dlg-containing puncta in the plasma membrane. Biochemical analysis and total internal reflection fluorescence microscopy reveal that the subsequent physical interaction between APC and Dlg1 is required for polarization of the microtubule cytoskeleton.

Partitioning of symbiotic Chlorella at host cell telophase in the green hydra symbiosis

1990 ◽  
Vol 329 (1252) ◽  
pp. 47-53 ◽  
Keyword(s):  
Cell Division ◽  
Host Cell ◽  
Daughter Cell ◽  
Control Cell ◽  
Culture Conditions ◽  
Digestive Cells ◽  
Density Dependent ◽  
Green Hydra ◽  
The Mean ◽  
Control Cell Division

Although there is much evidence that green hydra digestive cells control cell division of their Chlorella symbionts, so that the symbionts divide only at host cell division, it is not clear how the population size of symbionts (numbers per cell) is regulated. In constant culture conditions the mean number of symbionts per cell also remains constant, but with a very large variance about the mean. The way in which symbionts are partitioned at host cell division appears to account for that variation. By counting numbers of Chlorella in daughter cells at late telophase it was found that partitioning of Chlorella symbionts was not symmetrical, but at random, closely following that predicted by the binomial distribution if it is assumed that each symbiont had an equal probability of entering either host daughter cell. A better fit to the predicted distribution was obtained from observations of partition in digestive cells from excised regenerating peduncles than in those from recently fed gastric regions, possibly because in the former, algae have completed their division before the host cell divides, while in the latter algal and host cell division takes place at the same time. There was only a small effect of differences in daughter cell volume on numbers of symbionts received, but comparison of variance and coefficient of variation of numbers of algae in mother (post-algal division, pre-partition) and daughter telophase digestive cells (pre-division, post-partition) suggested that algal division at host mitosis was density dependent. Random partitioning of algae at host cell telophase would account for the wide variation in numbers of algae per cell, and compensatory density-dependent algal division at the next host cell mitosis would ensure stability of the mean algal population.

Cytotoxicity Assessment of SH-SY5Y Cells Grown on Graphene Sheet

2015 ◽  
Vol 1110 ◽  
pp. 311-314
Author(s):  
Hong Gi Oh ◽  
Hyo Geun Nam ◽  
Kwang Hwan Jhee ◽  
Joon Mook Lim ◽  
Kwang Soup Song
Keyword(s):  
Cell Culture ◽  
Control Cell ◽  
Culture Conditions ◽  
Graphene Sheets ◽  
Pristine Graphene ◽  
Biological Applications ◽  
Calcein Am ◽  
Cytotoxicity Assessment ◽  
Human Nerve ◽  
Cell Culture Conditions

We investigate the effect of cell culture conditions, using pristine graphene sheets as growth substrate, on the human nerve cell line (SH-SY5Y). In order to evaluate cell viability and morphology, we applied the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and fluorescence microscopy of cells stained with Hochest 33342 and Calcein AM. Human nerve cells exhibited 84% viability on pristine graphene sheets compared with control (cell culture polystyrene) after 3 days culturing. Fluorescence data showed that the presence of graphene did not influence cell morphology. These results suggest that graphene sheets may be used for biological applications.

scholarly journals Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

2016 ◽  
Vol 213 (3) ◽  
pp. 355-369 ◽  
Author(s):  
Paulina S. Mrozowska ◽  
Mitsunori Fukuda
Keyword(s):  
Cell Cultures ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Small Gtpases ◽  
Cell Polarization ◽  
Rab Gtpases ◽  
3D Cell Cultures ◽  
Polarized Epithelia ◽  
3D Environments ◽  
2D And 3D

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures.

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