A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

An α5 GABAA Receptor Inverse Agonist, α5IA, Attenuates Amyloid Beta-Induced Neuronal Death in Mouse Hippocampal Cultures

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder for which no cognition-restoring therapies exist. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. Increasing evidence suggests a remodeling of the GABAergic system in AD, which might represent an important therapeutic target. An inverse agonist of α5 subunit-containing GABAA receptors (α5GABAARs), 3-(5-Methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3–a]phthalazine (α5IA) has cognition-enhancing properties. This study aimed to characterize the effects of α5IA on amyloid beta (Aβ1–42)-induced molecular and cellular changes. Mouse primary hippocampal cultures were exposed to either Aβ1-42 alone, or α5IA alone, α5IA with Aβ1–42 or vehicle alone, and changes in cell viability and mRNA expression of several GABAergic signaling components were assessed. Treatment with 100 nM of α5IA reduced Aβ1–42-induced cell loss by 23.8% (p < 0.0001) after 6 h and by 17.3% after 5 days of treatment (p < 0.0001). Furthermore, we observed an Aβ1-42-induced increase in ambient GABA levels, as well as upregulated mRNA expression of the GABAAR α2,α5,β2/3 subunits and the GABABR R1 and R2 subunits. Such changes in GABARs expression could potentially disrupt inhibitory neurotransmission and normal network activity. Treatment with α5IA restored Aβ1-42-induced changes in the expression of α5GABAARs. In summary, this compound might hold neuroprotective potential and represent a new therapeutic avenue for AD

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow

peer reviewedIn situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

peer reviewedIn situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue