Daily Eastern News: March 03, 2017

https://thekeep.eiu.edu/den_2017_mar/1002/thumbnail.jp

What makes cells move: requirements and obstacles for spontaneous cell motility.

International audienceMovement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts

Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation

Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway

International audienc

The effect of p130Cas on spreading and migration of oligodendroglial cells.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 30 min after re-plating they were subjected to immunofluorescence analysis of cell spreading. Cells were classified as either not spread (arrowhead), showing lamellipodia (asterisk) or lamella (arrow). (B) Statistical evaluation of A. Data represents the mean ± SEM from 3 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Oli-<i>neu</i> cells were treated as in A. Here, immunofluorescence analysis of process thickness was carried out 4 hours after re-plating. Three examples per condition are shown. Areas of measurement are outlined by white dashed rectangles. (D) Statistical evaluation of C. The thickest process for each individual cell was measured and plotted on a chart (from high to low diameters) and the average ratio ± SEM from 3 independent experiments is presented; ** p<0.01 (Student’s <i>t</i> test). (E) Oli-<i>neu</i> cells were treated with siRNA as in A. After 24 hours, they were re-plated in Boyden chambers and allowed to migrate for 6 hours in the presence of 5 ng/ml bFGF before fixation and migration analysis were carried out. Data are expressed as a percentage of basal migration, i.e. the migration of Oli-<i>neu</i> without chemoattractant. Data represent the mean ± SEM from 3 independent experiments; * p<0.05 (Student’s <i>t</i> test). (F) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A-E were lysed and p130Cas levels were assessed by Western blotting. GAPDH serves as loading control.</p

The effect of p130Cas knockdown on spreading and migration in Oli-<i>neu</i> does not result from changes in cellular viability.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 6 h after re-plating a TUNEL assay was performed to test for apoptosis. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (B) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control. (C) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 h later, the cells were detached from their culture vessel and 0.5 h and 6 h after re-plating MTT and LDH assays were carried out to test for cell viability and membrane integrity, respectively. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (D) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in C were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control.</p

p130Cas is involved in oligodendroglial apoptosis.

<p>(A) Primary OPCs were treated with control or p130Cas siRNA. 48 hours later, cells were lysed and tested for levels of p130Cas and cleaved Caspase 3 (indicative of apoptosis) by Western analysis. GAPDH serves as loading control. (B) Primary OPCs were treated with siRNA as in A. Additionally, some cells were treated with the apoptosis inducing agent staurosporine as positive control. After 42 hours, the cells were subjected to a TUNEL assay to test for apoptotic cells. Data show the mean ± SEM from 11 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Relative cell numbers from B were counted as additional indication of cell death. Data show the mean ± SEM from 11 independent experiments; ** p<0.01, *** p<0.001 (Student’s <i>t</i> test).</p

Identification of p130Cas as an oligodendroglial tyrosine-phosphorylated protein in the presence of active Fyn.

<p>(A) Tyrosine-phosphorylated proteins were immunoprecipitated from Oli-<i>neu</i> cells overexpressing active Fyn. A Coomassie-stained SDS-Polyacrylamide gel with first and second elution (E1 and E2), antibody control (control) and MW marker (M) is shown. The indicated band (p130Cas) was excised and analyzed by mass spectrometry. Molecular weights in kilodalton are indicated on the right. (B) Primary structure of p130Cas. The identified peptides are shown in bold red and cover 20% of the protein sequence. (C) p130Cas expression at different developmental stages in cultured oligodendrocytes (DIV, days in vitro, 1, 2, 4, 6, 8) was analyzed by Western blots with antibodies indicated on the left. CNP and MOG represent early and late maturation markers, respectively, and GAPDH serves as a loading control.</p

Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2

<div><p>The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses.</p></div

NG2 is cleaved by α- and γ-secretases.

<p>(A, B) Immunoblot analysis of endogenous NG2 FL protein of an OPC cell line using an antibody against the ICD of NG2 (cyto AB) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001993#pbio.1001993.s001" target="_blank">Figure S1</a>). (A) Reduction of NG2 FL protein levels after overexpression of ADAM10 or 17. (B) NG2 FL levels were increased if endogenous ADAM10 was inhibited by GI. (C) Immunoblot analysis of the Flag_NG2_del construct after expression in an OPC cell line using an antibody against the Flag tag within the ectodomain. Major protein bands were detected at 66 kD (FL protein) and 54 kD (largest ectodomain). (D) Schematic illustration of the murine NG2 FL protein and NG2_del expression constructs used. Cleavage sites, fragments, and epitopes recognized by antibodies (AB) are indicated. (E) Immunoblot analysis of endogenous compared to the NG2_del derived CTF. NG2 CTF levels are increased after expression of the NG2_del construct in an OPC cell line, both CTF run at the same molecular weight of 12 kD. (F) Immunoblot analysis of NG2_del_Myc expression in three different cell lines. Application of the γ-secretase inhibitor DAPT highly increased Myc-tagged CTF in OPC and HEK cells. In MEF cells that are lacking presenilin 1 and 2, DAPT application was without effect. CTF levels of APP were used as a control to show the inhibition of γ-secretase activity by DAPT. (G) Immunoblot analysis of endogenous NG2 CTF levels in an OPC cell line after inhibition of the γ-secretase with DAPT. CTF levels were increased when DAPT was present and normalized against the FL protein levels. (In (A), <i>n</i> represents four experiments each performed in duplicate, the WB shows one experiment. In (B) and (G) each experiment consists of triplicates. Data represent the mean ± SEM (paired Student's <i>t</i> test).</p