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F-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
The prefrontal cortex (PFC) is usually defined as the frontal cortical area receiving a mediodorsal thalamic (MD) innervation. Certain areas in the medial wall of the rat frontal area receive a MD innervation. A second frontal area that is the target of MD projections is located dorsal to the rhinal sulcus and often referred to as the orbitofrontal cortex (OFC). Both the medial PFC and OFC are comprised of a large number of cytoarchitectonic regions. We assessed the afferent innervation of the different areas of the OFC, with a focus on projections arising from the mediodorsal thalamic nucleus, the basolateral nucleus of the amygdala, and the midbrain dopamine neurons. Although there are specific inputs to various OFC areas, a simplified organizational scheme could be defined, with the medial areas of the OFC receiving thalamic inputs, the lateral areas of the OFC being the recipient of amygdala afferents, and a central zone that was the target of midbrain dopamine neurons. Anterograde tracer data were consistent with this organization of afferents, and revealed that the OFC inputs from these three subcortical sites were largely spatially segregated. This spatial segregation suggests that the central portion of the OFC (pregenual agranular insular cortex) is the only OFC region that is a prefrontal cortical area, analogous to the prelimbic cortex in the medial prefrontal cortex. These findings highlight the heterogeneity of the OFC, and suggest possible functional attributes of the three different OFC areas.
© 2018 Wiley Periodicals, Inc.
Neurodegeneration is a central aspect of the early stages of diabetic retinopathy, the primary ocular complication associated with diabetes. While progress has been made to improve the vascular perturbations associated with diabetic retinopathy, there are still no treatment options to counteract the neuroretinal degeneration associated with diabetes. Our previous work suggested that the molecular chaperones α-crystallins could be involved in the pathophysiology of diabetic retinopathy; however, the role and regulation of α-crystallins remained unknown. In the present study, we demonstrated the neuroprotective role of αA-crystallin during diabetes and its regulation by its phosphorylation on residue 148. We further characterized the dual role of αA-crystallin in neurons and glia, its essential role for neuronal survival, and its direct dependence on phosphorylation on this residue. These findings support further evaluation of αA-crystallin as a treatment option to promote neuron survival in diabetic retinopathy and neurodegenerative diseases in general.
Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.
Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.
© 2017 Erdogan et al.
RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.
Copyright © 2017 Elsevier Inc. All rights reserved.
The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.
The IL-17/IL-17R axis has controversial roles in cancer, which may be explained by tumor-specific results. Here, we describe a novel molecular mechanism underlying IL-17RC-controlled tumor-specific proliferation. Triggered by IL-17RC knockdown (KD), B16 melanoma and 4T1 carcinoma cells inversely altered homeostatic tumor proliferation and tumor growth in vitro and in vivo. In contrast to the existing dogma that IL-17RC-dependent signaling activates the JNK pathway, IL-17RC KD in both tumor cell lines caused aberrant expression and activation of different JNK isoforms along with markedly diminished levels of the ubiquitin-editing enzyme A20. We demonstrated that differential up-regulation of JNK1 and JNK2 in the two tumor cell lines was responsible for the reciprocal regulation of c-Jun activity and tumor-specific proliferation. Furthermore, we showed that A20 reconstitution of IL-17RCKD clones with expression of full-length A20, but not a truncation-mutant, reversed aberrant JNK1/JNK2 activities and tumor-specific proliferation. Collectively, our study reveals a critical role of IL-17RC in maintaining baseline A20 production and a novel role of the IL-17RC-A20 axis in controlling JNK isoform-dependent tumor-specific homeostatic proliferation.
OBJECTIVE - Endoglin, a transforming growth factor-β superfamily coreceptor, is predominantly expressed in endothelial cells and has essential roles in vascular development. However, whether endoglin is also expressed in vascular smooth muscle cells (VSMCs), especially in vivo, remains controversial. Furthermore, the roles of endoglin in VSMC biology remain largely unknown. Our objective was to examine the expression and determine the function of endoglin in VSMCs during angiogenesis.
APPROACH AND RESULTS - Here, we determine that endoglin is robustly expressed in VSMCs. Using CRISPR/CAS9 knockout and short hairpin RNA knockdown in the VSMC/endothelial coculture model system, we determine that endoglin in VSMCs, but not in endothelial cells, promotes VSMCs recruitment by the endothelial cells both in vitro and in vivo. Using an unbiased bioinformatics analysis of RNA sequencing data and further study, we determine that, mechanistically, endoglin mediates VSMC recruitment by promoting VSMC migration and spreading on endothelial cells via increasing integrin/FAK pathway signaling, whereas endoglin has minimal effects on VSMC adhesion to endothelial cells. In addition, we further determine that loss of endoglin in VSMCs inhibits VSMC recruitment in vivo.
CONCLUSIONS - These studies demonstrate that endoglin has an important role in VSMC recruitment and blood vessel maturation during angiogenesis and also provide novel insights into how discordant endoglin function in endothelial and VSMCs may regulate vascular maturation and angiogenesis.
© 2017 The Authors.
Mutant-selective EGFR tyrosine kinase inhibitors (TKI), such as osimertinib, are active agents for the treatment of -mutant lung cancer. Specifically, these agents can overcome the effects of the T790M mutation, which mediates resistance to first- and second-generation EGFR TKI, and recent clinical trials have documented their efficacy in patients with -mutant lung cancer. Despite promising results, therapeutic efficacy is limited by the development of acquired resistance. Here we report that Src family kinases (SFK) and focal adhesion kinase (FAK) sustain AKT and MAPK pathway signaling under continuous EGFR inhibition in osimertinib-sensitive cells. Inhibiting either the MAPK pathway or the AKT pathway enhanced the effects of osimertinib. Combined SFK/FAK inhibition exhibited the most potent effects on growth inhibition, induction of apoptosis, and delay of acquired resistance. SFK family member was amplified in osimertinib-resistant -mutant tumor cells, the effects of which were overcome by combined treatment with osimertinib and SFK inhibitors. In conclusion, our data suggest that the concomitant inhibition of both SFK/FAK and EGFR may be a promising therapeutic strategy for -mutant lung cancer. .
©2017 American Association for Cancer Research.