Cellular Motility Driven by Assembly and Disassembly of Actin Filaments

Isolation and Maintenance.- Isolation and Differentiation of Medaka Embryonic Stem Cells.- Maintenance of Chicken Embryonic Stem Cells In Vitro.- Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro.- Derivation, Maintenance, and Characterization of Rat Embryonic Stem Cells In Vitro.- Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells.- Generation and Characterization of Monkey Embryonic Stem Cells.- Derivation and Propagation of Embryonic Stem Cells in Serum- and Feeder-Free Culture.- Signaling in Embryonic Stem Cell Differentiation.- Internal Standards in Differentiating Embryonic Stem Cells In Vitro.- Matrix Assembly, Cell Polarization, and Cell Survival.- Phosphoinositides, Inositol Phosphates, and Phospholipase C in Embryonic Stem Cells.- Cripto Signaling in Differentiating Embryonic Stem Cells.- The Use of Embryonic Stem Cells to Study Hedgehog Signaling.- Transfection and Promoter Analysis in Embryonic Stem Cells.- SAGE Analysis to Identify Embryonic Stem Cell-Predominant Transcripts.- Utilization of Digital Differential Display to Identify Novel Targets of Oct3/4.- Gene Silencing Using RNA Interference in Embryonic Stem Cells.- Genetic Manipulation of Embryonic Stem Cells.- Efficient Transfer of HSV-1 Amplicon Vectors Into Embryonic Stem Cells and Their Derivatives.- Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells.- Use of the Cytomegalovirus Promoter for Transient and Stable Transgene Expression in Mouse Embryonic Stem Cells.- Use of Simian Immunodeficiency Virus Vectors for Simian Embryonic Stem Cells.- Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells.- DNA Damage Response and Mutagenesis in Mouse Embryonic Stem Cells.- Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro.- Use of Embryonic Stem Cells in Pharmacological and Toxicological Screens.- Use of Differentiating Embryonic Stem Cells in Pharmacological Studies.- Embryonic Stem Cells as a Source of Differentiated Neural Cells for Pharmacological Screens.- Use of Murine Embryonic Stem Cells in Embryotoxicity Assays.- Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells.- Epigenetic Analysis of Embryonic Stem Cells.- Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion.- Methylation in Embryonic Stem Cells In Vitro.- Tumor-Like Properties.- Identification of Genes Involved in Tumor-Like Properties of Embryonic Stem Cells.- In Vivo Tumor Formation From Primate Embryonic Stem Cells.- Animal Models and Therapy.- Directed Differentiation and Characterization of Genetically Modified Embryonic Stem Cells for Therapy.- Use of Differentiating Embryonic Stem Cells in the Parkinsonian Mouse Model.

Isolation and characterization

Slowly but surely, Alzheimer's disease (AD) patients lose their memory and their cognitive abilities, and even their personalities may change dramatically. These changes are due to the progressive dysfunction and death of nerve cells that are responsible for the storage and processing of information. Although drugs can temporarily improve memory, at present there are no treatments that can stop or reverse the inexorable neurodegenerative process. But rapid progress towards understanding the cellular and molecular alterations that are responsible for the neuron's demise may soon help in developing effective preventative and therapeutic strategies.

/pdf/pathways-towards-and-away-from-alzheimer-s-disease-1j591hguge.pdf

Pathways towards and away from Alzheimer's disease

■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

http://wolfson.huji.ac.il/expression/local/bacteria/c41-c43-1996.pdf

Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.

/pdf/cofilin-changes-the-twist-of-f-actin-implications-for-actin-3q9zcdsflt.pdf

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility.

ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2880%2980219-5

The ATPase activities of rat cardiac myosin isoenzymes

The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N- or C-terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N-terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C-terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C-terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100-fold weaker binding in its absence. Removal of an additional five residues from the C-terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C-terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1990.tb07632.x

Brian Pope

Papers

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility.

Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments

The ATPase activities of rat cardiac myosin isoenzymes

Identification of a region in segment 1 of gelsolin critical for actin binding.