Expansion microscopy-based imaging of nuclear structures in cultured cells.

Expansion microscopy is a sample preparation technique in which fixed and immunostained cells or tissues are embedded in a cross-linked network of swellable polyelectrolyte hydrogel that expands isotropically upon addition of deionized water. We utilize the X10 method for tenfold expansion of U2OS cells with concurrent DNA staining. A custom 3D-printed gel cutter and chambered slides minimize gel drift, facilitating analysis of the components of nuclear structures at nanoscale resolution by conventional microscopy or Airyscan confocal imaging.

Cdx2 Regulates Gene Expression through Recruitment of Brg1-associated Switch-Sucrose Non-fermentable (SWI-SNF) Chromatin Remodeling Activity.

The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors.

Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid.

Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion.

Background: Unraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion.

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism.

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174.

Amyotrophic lateral sclerosis-immunoglobulins selectively interact with neuromuscular junctions expressing P/Q-type calcium channels.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a gradual loss of motoneurons. The majority of ALS cases are associated with a sporadic form whose etiology is unknown. Several pieces of evidence favor autoimmunity as a potential contributor to sporadic ALS pathology.

Ca(V)2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: implications for calcium channelopathies.

Alternative splicing is known to generate multiple functionally distinct calcium channel variants that exhibit unique spatial and temporal expression patterns. In humans, naturally occurring mutations in genes encoding calcium channel pore forming alpha(1)-subunits are associated with several severe hereditary disorders although it remains to be described whether there exists any relationship between the physiological effects of these mutations and calcium channel splice variation. In the present study, we systematically compare the biophysical effects of three type-1 familial hemiplegic migraine (FHM-1) mutations in two predominant splice variants of the neuronal Ca(V)2.

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors.

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.

Molecular typing of West Nile Virus, Dengue, and St. Louis encephalitis using multiplex sequencing.

We report the development of an assay to simultaneously identify three of the clinically important flaviviruses (West Nile Virus, Dengue, and St. Louis encephalitis). This assay is based on the nucleotide sequence variations within a 266-bp region of the non-structural protein 5.

Identification of the severe acute respiratory syndrome coronavirus by simultaneous multigene DNA sequencing.

The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases.

Nucleotide sequence-based multitarget identification.

MULTIGEN technology (T. Vinayagamoorthy, U.S.