Radial Migration Assay and Analysis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: haab brian
PROVIDER: S-ECPF-GEOD-21566 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:When either Panc-1 or MiaPaCa-2 were plated as a discrete clump of cells in the middle of a plate, two morphologically distinct populations could be created. The cells on the periphery of the cell cluster migrated away from the region of high cell density and took on a classical mesenchymal phenotype with spindle like projections and loss of cell to cell adhesion. Cells that remained in the highly populated center showed greater intercellular adhesion and were more rounded in appearance. This model was included to provide information about whether glyco-gene expression changes observed in models of the epithelial to mesenchymal transition also are involved in migration.
2010-04-29 | GSE21566 | GEO
Project description:It is often desirable to evaluate the ability of cells to move in an unrestricted manner in multiple directions without chemical gradients. By combining the standard radial migration assay with injection-molded gaskets and a rigid fixture, we have developed a highly reliable and sensitive method for observing and measuring radial cell migration. This method is adapted for use on high-throughput automated imaging systems. The use of injection-molded gaskets enables low-cost replacement of cell-wetted components. Moreover, the design enables secondary placement of attractants and co-cultures. This device and its enhanced throughput permit the use of therapeutic screening to evaluate phenotypic responses, for example, cancer cell migration response due to drugs or chemical signals. This approach is orthogonal to other 2D cell migration applications, such as scratch wound assays, although here we offer a noninvasive, enhanced-throughput device, which currently is not commercially available but is easily constructed. The proposed device is a systematic, reliable, rapid application to monitor phenotypic responses to chemotherapeutic screens, genetic alterations (e.g., RNAi and CRISPR), supplemental regimens, and other approaches, offering a reliable methodology to survey unbiased and noninvasive cell migration.
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Project description:Neuronal migration is a crucial process in the organization of the developing cerebral cortex. Although a number of positive regulatory mechanisms of radial migration have been identified, negative cell-autonomous mechanisms have yet to be fully described. Here we report a newly identified Migration Inhibitory Protein (MINP, formerly known as 2900011O08Rik) that negatively regulates radial migration. MINP mRNA was specifically detected in the central and peripheral nervous system, and especially enriched in the cerebral cortex. MINP immunoreactivity co-localized with the neuronal marker Tuj1 and was detected in the cytoplasm of post-mitotic neurons. To elucidate the function of MINP in the developing brain, we performed in utero electroporation of MINP siRNA, MINP shRNA, or MINP-overexpressing vectors into mouse cortices and carried out in vivo migration assays. Whereas knockdown of MINP did not alter neuronal morphology, the radial migration was found accelerated by MINP knockdown, and reduced by MINP overexpression. This migration phenotype was also confirmed in vitro, indicating that MINP regulates neuronal migration in a cell-autonomous fashion. Furthermore, downregulation of MINP affected microtubule stability by interacting with tubulin that is a potential mechanism involved in the regulation of neuronal migration.
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Project description:Neocortical excitatory neurons migrate radially along the glial fibers of mother radial glial progenitors (RGPs) in a birth-date-dependent inside-out manner. However, the precise functional significance of this well-established orderly neuronal migration remains largely unclear. Here, we show that strong electrical synapses selectively form between RGPs and their newborn progeny and between sister excitatory neurons in ontogenetic radial clones at the embryonic stage. Interestingly, the preferential electrical coupling between sister excitatory neurons, but not that between RGP and newborn progeny, is eliminated in mice lacking REELIN or upon clonal depletion of DISABLED-1, which compromises the inside-out radial neuronal migration pattern in the developing neocortex. Moreover, increased levels of Ephrin-A ligand or receptor that laterally disperse sister excitatory neurons also disrupt preferential electrical coupling between radially aligned sister excitatory neurons. These results suggest that RGP-guided inside-out radial neuronal migration facilitates the initial assembly of lineage-dependent precise columnar microcircuits in the neocortex.
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Project description:Radial migration of cortical projection neurons is a prerequisite for shaping a distinct multilayered cerebral cortex during mammalian corticogenesis. Members of Rab GTPases family were reported to regulate radial migration. Here, in vivo conditional knockout or in utero knockdown (KD) of Rab23 in mice neocortex causes aberrant polarity and halted migration of cortical projection neurons. Further investigation of the underlying mechanism reveals down-regulation of N-cadherin in the Rab23-deficient neurons, which is a cell adhesion protein previously known to modulate radial migration. (Shikanai M, Nakajima K, Kawauchi T. 2011. N-cadherin regulates radial glial fiber-dependent migration of cortical locomoting neurons. Commun Integr Biol. 4:326-330.) Interestingly, pharmacological inhibition of extracellular signal-regulated kinases (ERK1/2) also decreases the expression of N-cadherin, implicating an upstream effect of ERK1/2 on N-cadherin and also suggesting a link between Rab23 and ERK1/2. Further biochemical studies show that silencing of Rab23 impedes activation of ERK1/2 via perturbed platelet-derived growth factor-alpha (PDGFRα) signaling. Restoration of the expression of Rab23 or N-cadherin in Rab23-KD neurons could reverse neuron migration defects, indicating that Rab23 modulates migration through N-cadherin. These studies suggest that cortical neuron migration is mediated by a molecular hierarchy downstream of Rab23 via N-cadherin.
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Project description:Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are critical for radial migration and lamination of cortical neurons. However, how this kinase is regulated by extracellular and intracellular signals during cortical morphogenesis remains unclear. Here, we show that PKCdelta, a member of novel PKC expressing in cortical neurons, could stabilize p35 by direct phosphorylation. PKCdelta attenuated the degradation of p35 but not its mutant derivative, which could not be phosphorylated by PKCdelta. Down-regulation of PKCdelta by in utero electroporation of specific small interference RNA (siRNA) severely impaired the radial migration of cortical neurons. This migration defect was similar to that caused by down-regulation of p35 and could be prevented by cotransfection with the wild-type but not the mutant p35. Furthermore, PKCdelta could be activated by the promigratory factor brain-derived neurotrophic factor (BDNF) and was required for the activation of Cdk5 by BDNF. Both PKCdelta and p35 were required for the promigratory effect of BDNF on cultured newborn neurons. Thus, PKCdelta may promote cortical radial migration through maintaining the proper level of p35 in newborn neurons.
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Project description:Proper brain function depends on correct neuronal migration during development, which is known to be regulated by cytoskeletal dynamics and cell-cell adhesion. Myosin X (Myo10), an uncharacteristic member of the myosin family, is an important regulator of cytoskeleton that modulates cell motilities in many different cellular contexts. We previously reported that Myo10 was required for neuronal migration in the developing cerebral cortex, but the underlying mechanism was still largely unknown. Here, we found that knockdown of Myo10 expression disturbed the adherence of migrating neurons to radial glial fibers through abolishing surface Neuronal cadherin (N-cadherin) expression, thereby impaired neuronal migration in the developmental cortex. Next, we found Myo10 interacted with N-cadherin cellular domain through its FERM domain. Furthermore, we found knockdown of Myo10 disrupted N-cadherin subcellular distribution and led to localization of N-cadherin into Golgi apparatus and endosomal sorting vesicle. Taking together, these results reveal a novel mechanism of Myo10 interacting with N-cadherin and regulating its cell-surface expression, which is required for neuronal adhesion and migration.
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