Oligomerization of the vesicular stomatitis virus phosphoprotein is dispensable for mRNA synthesis but facilitates RNA replication

Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the

Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification

Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone. © 2010 American Society for Bone and Mineral Research

Quiescence and Activation of Stem and Precursor Cell Populations in the Subependymal Zone of the Mammalian Brain Are Associated with Distinct Cellular and Extracellular Matrix Signals

The subependymal zone (SEZ) of the lateral ventricles is one of the areas of the adult brain where new neurons are continuously generated from neural stem cells (NSCs), via rapidly dividing precursors. This neurogenic niche is a complex cellular and extracellular microenvironment, highly vascularized compared to non-neurogenic periventricular areas, within which NSCs and precursors exhibit distinct behavior. Here, we investigate the possible mechanisms by which extracellular matrix molecules and their receptors might regulate this differential behavior. We show that NSCs and precursors proceed through mitosis in the same domains within the SEZ of adult male mice—albeit with NSCs nearer ependymal cells—and that distance from the ventricle is a stronger limiting factor for neurogenic activity than distance from blood vessels. Furthermore, we show that NSCs and precursors are embedded in a laminin-rich extracellular matrix, to which they can both contribute. Importantly, they express differential levels of extracellular matrix receptors, with NSCs expressing low levels of α6β1 integrin, syndecan-1, and lutheran, and in vivo blocking of β1 integrin selectively induced the proliferation and ectopic migration of precursors. Finally, when NSCs are activated to reconstitute the niche after depletion of precursors, expression of laminin receptors is upregulated. These results indicate that the distinct behavior of adult NSCs and precursors is not necessarily regulated via exposure to differential extracellular signals, but rather via intrinsic regulation of their interaction with their microenvironment

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma

Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAFs) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but co-injected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created co-culture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in 3-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration

Swelling-Activated Ca2+ Channels Trigger Ca2+ Signals in Merkel Cells

Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must efficiently remove their substrates throughout the cells in a timely manner as many of these proteins can be toxic. This study shows that proteasomes can do so efficiently because they are highly mobile. Furthermore this study uncovers that proteasome mobility requires functional Arc3, a subunit of the Arp2/3 complex

Regional blood flow in the SEZ is significantly less than in the striatum. (A–B

<p>) Deposited microspheres were imaged in SEZ flatmounts for measuring regional blood flow. DAPI (A) was used to locate the surface of the ependymal wall for determining microsphere depths. (<b>C</b>) Mean blood flow is not equal at all depths (p<0.05, Kruskal-Wallis test). (<b>D</b>) Mean blood flow in the SEZ is significantly different from the mean blood flow in the striatum (p<0.05, Wilcoxon Rank Sum test; n_mice = 4, n_{observations,SEZ} = 8, n_{observations,Str} = 8). (<b>C–D</b>) Plotted values represent mean ± standard error.</p

Vessels in the anterior SEZ and striatum are morphologically distinct.

<p>(<b>A</b>) Mean vessel density is not equal at all depths (p<0.01, Kruskal-Wallis test). (<b>B</b>) Mean vessel density in the SEZ is significantly different from the mean density in the striatum (p<0.05, Wilcoxon Rank Sum test; n_mice = 5, n_{observations,SEZ} = 5, n_{observations,Str} = 5). (<b>C</b>) Mean vessel tortuosity is not equal at all depths (p<0.001, Kruskal-Wallis test). (<b>D</b>) Mean vessel tortuosity in the SEZ is significantly different from the mean tortuosity in the striatum (p<0.001, Wilcoxon Rank Sum test; n_mice = 5, n_{observations,SEZ} = 395, n_{observations,Str} = 904). (<b>E</b>) The mean angle of the ependymal wall to vessels is not equal at all depths (p<0.001, Kruskal-Wallis test). (<b>F</b>) The mean angle of the ependymal wall to vessels in the SEZ is significantly different from the mean angle in the striatum (p<0.001, Wilcoxon Rank Sum test; n_mice = 5, n_{observations,SEZ} = 10,988, n_{observations,Str} = 20,159). (<b>A–F</b>) Plotted values represent mean ± standard error.</p

The neural stem cell niche exhibits functional hypoxia.

<p>(<b>A–B</b>) In mice treated with Hypoxyprobe-1 (hpi), hypoxia was detected in SEZ flatmounts throughout the ependymal cell layer (A). Bright Hypoxyprobe-1 staining was also detected in a subpopulation of cells immediately beneath the ependymal wall. Tissue treated with vehicle only (B) revealed minimal background staining, indicating that the staining seen in (A) is antigen specific. Immunostaining protocols and imaging settings were kept constant for (A) and (B). Representative images are shown. (<b>C–D</b>) In mice treated with Hypoxyprobe-1, hypoxia was also detected in the ependymal cell layer in coronal brain sections (C). Bright Hypoxyprobe-1 staining was detected in a distinct subpopulation of cells that was visible in the SEZ and striatum as well. Tissue treated with vehicle only (D) revealed minimal background staining, indicating that the staining seen in (C) is antigen specific. Immunostaining protocols and imaging settings were kept constant for (C) and (D). Representative images are shown.</p

A subpopulation of non-ependymal cells in the SEZ and striatum exhibits functional hypoxia.

<p>(<b>A</b>) Non-ependymal cells staining brightly for Hypoxyprobe-1 (hpi) were characterized by large cell bodies and long dendrite-like processes. (<b>B</b>) Coronal brain sections were imaged to search for these hypoxic cells in various brain regions. Insets, shown at higher magnification in (C–E), are representative of the striatum (C), SEZ (D), and cerebral cortex (E). All insets were imaged using the same settings. (<b>C</b>) Cells staining brightly for Hypoxyprobe-1 (arrows) were observed in coronal sections of the striatum. Cells were always found in isolation, and never in clusters. (<b>D</b>) Non-ependymal cells staining brightly for Hypoxyprobe-1 (arrows) were also observed in coronal sections of the SEZ. Cells were always found in isolation, and never in clusters. (<b>E</b>) In comparison, cells staining brightly for Hypoxyprobe-1 were observed in the cerebral cortex only rarely.</p