10 research outputs found
The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1
The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein
Bioinspired Polymer Metal Complex Conjugates: [Co(NN)2Cl]2+ and [Cu(NN)2]2+ Moieties Graft in the Same Branched Polyethyleneimine (BPEI)—Synthesis, Biophysical and Biological Studies
Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells
DNA-based therapeutics and DNA delivery systems: A comprehensive review
The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated