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J R Bamburg

Publications and source records attributed to J R Bamburg.

At least 19 recordsLinked to original sources
PubMed1992

Actin depolymerizing factor is a component of slow axonal transport.

We examined the low molecular weight proteins transported with actin in the chicken sciatic nerve after injection of [35S]methionine into the lumbar spinal cord. A prominent component of slow axonal transport with apparent molecular mass 19 kDa comigrated on two-dimensional gels with chicken actin depolymerizing factor (ADF), previously shown to be a major actin-binding protein in brain. There was comparatively little radioactivity associated with the actin monomer sequestering proteins, profilin or cofilin, and examination of the rapid component of axonal transport failed to reveal appreciable quantities of actin, ADF, profilin, or cofilin. These results show that both actin and ADF are carried by slow axonal transport and raise the possibility that actin travels within the axon in an unpolymerized form in a complex with ADF.

Actin Depolymerizing Factors↗
PubMed1991

Actin-depolymerizing factor (ADF) in the cerebellum of the developing rat: a quantitative and immunocytochemical study.

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.

Actin Depolymerizing Factors↗
PubMed1991

Responses of growth cones to changes in osmolality of the surrounding medium.

The possible involvement of osmotically generated hydrostatic pressure in driving actin-rich extensions of the cell surface was examined using cultures of chick neurons. Estimation of the excess internal osmotic pressure of chick neural tissue by vapor pressure deficit osmometry, and of the excess internal hydrostatic pressure in cultured chick neurons using a calibrated pressure pipette, gave upper limits of 10 mosM and 0.1 atmosphere (1 atmosphere = 101325 Pa), respectively. Increases in the osmolality of the medium surrounding cultured neurons by addition of sucrose, mannitol or polyethylene glycol by amounts that should eliminate any internal pressure not only failed to arrest the growth of filopodia but caused them to increase in length up to twofold in 3-5 min. Lamellipodia remained unchanged following hyperosmotic shifts of 20 mosM, but higher levels caused a small decrease in area. Reduction of osmolality by the addition of water to the culture fluid down to 50% of its normal value failed to show any detectable change in either filopodial length or lamellipodia area. These observations argue against an osmotic mechanism for growth cone extension and show that the growth of filopodia, in particular, is unlikely to be driven by osmotically generated hydrostatic pressure. In contrast to the short-term effects on growth cone morphology, the slower elongation of the neuritic cylinder showed a consistent osmotic response. Growth rates were reduced following addition of osmolytes and increased in rate (as much as sixfold) following addition of water to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor.

Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.

Actin Depolymerizing Factors↗
PubMed1990

A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents.

A method is described for quantitation of protein in the presence of reducing agents, detergents, and other substances which often interfere with assays of protein in solution. The proteins are applied to Whatman No. 1 filter paper, air-dried, washed with methanol, and then stained with Coomassie brilliant blue G. Following destaining, the paper is air-dried and the protein-bound dye is extracted. Sample absorbance measurements are made in a 96-well plate using an automated microplate reader (600-405 nm) or in a cuvette at 610 nm. This filter paper assay is useful for determining 100 ng to 20 micrograms of protein in the presence of ammonium sulfate, urea, thiol-reducing agents, amino acids, DNA, ionic and nonionic detergents, and acid or base.

Animals↗
PubMed1989

Cycling of actin assembly in synaptosomes and neurotransmitter release.

We have investigated the regulation of actin assembly in whole mouse brain synaptosomes and how that regulation modulates neurotransmitter release. During a 30 s depolarization with high K+, filamentous actin (F-actin) levels, monitored by staining with rhodamine phalloidin, increase dramatically (up to 300% in 3 s), decrease, and increase once again. This F-actin cycling is regulated by pathways both dependent and independent of Ca2+ influx and is markedly affected by exposing synaptosomes to Li+, tetrodotoxin, and diacylglycerol. Measurement of [3H]norepinephrine release from synaptosomes containing entrapped agents that modulate actin assembly (DNAase I or phalloidin) indicates that actin depolymerization is necessary for normal release and that repolymerization limits release.

Actins↗
PubMed1988

Properties of purified actin depolymerizing factor from chick brain.

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Depolymerizing Factors↗
PubMed1988

Inhibition of deoxyribonuclease I activity by actin covalently cross-linked to chick brain actin depolymerizing factor through exposed sulfhydryls.

All but one of the six free sulfhydryl groups of chick brain actin depolymerizing factor (ADF) are protected from modification when ADF forms a 1:1 complex with actin. This exposed sulfhydryl can be cross-linked to cys 374 of actin with N,N'-phenylenedimaleimide. The cross-linked complex inhibits the hydrolytic activity of pancreatic deoxyribonuclease (DNase I) to an identical extent as both the untreated complex and an equivalent amount of free actin. These data indicate that ADF binds to actin at a site which does not overlap with the DNase I binding site.

Actin Depolymerizing Factors↗
PubMed1988

Identification of two species of actin depolymerizing factor in cultures of BHK cells.

High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

Actin Depolymerizing Factors↗
PubMed1987

Distribution and cellular localization of actin depolymerizing factor.

Actin depolymerizing factor (ADF) is a low molecular mass (19 kD) protein that forms a tightly bound dimeric complex with actin. We have raised a rabbit antiserum to chick brain ADF and used it to analyze the distribution and cellular localization of ADF. We find that ADF is a major constituent of all chick embryonic and most adult tissues examined, accounting for 0.1-0.4% of the total protein. Some tissues have as much as 0.6 mol ADF per mole actin. Adult heart and skeletal muscle are unusual in having very low levels of ADF: less than 0.02% of the soluble protein. During the development of skeletal muscle, ADF levels are maximal up to approximately 11 d in ovo and then decline to reach their adult levels by 14 d posthatching. Brain tissue and cultured cell lines from several other vertebrates, including mammals, all possess proteins of identical size to ADF that are recognized by the ADF antiserum. No proteins are specifically recognized by the ADF antiserum in extracts from Acanthamoeba castellanii or from nerve tissue of several invertebrates. Indirect immunofluorescence shows that ADF is present throughout the cytosol of most cells and at the leading edge of ruffled membranes and in the neuronal growth cone. Its abundance and widespread distribution together with its ability to sequester actin molecules, even those in an already polymerized state, suggest that ADF is a major factor in the regulation of actin filaments in many vertebrate cells.

Actin Depolymerizing Factors↗
PubMed1985

Reorganization of actin in depolarized synaptosomes.

Depolarization of whole brain synaptosomes, which stimulates transmitter release, also affects regulation of the assembly of actin microfilaments. Lysates of depolarized synaptosomes contain 20% less cytoskeletal actin than lysates of unstimulated synaptosomes. Parameters affecting the assembly of actin are modified before lysis, but release of actin from the Triton-insoluble cytoskeleton does not occur until after lysis. Actin released from the cytoskeleton is not precipitated with myosin, indicating that it consists of monomers and/or short oligomers. Synaptosomes were incubated for 12 sec in one of three solutions of identical ionic strength but of different salt mixtures: 75 mM KCl-2 mM CaCl2, 5 mM KCl-2mM CaCl2, or 75 mM KCl-0.1 mM EGTA. Synaptosomes were then lysed in an F-actin stabilizing buffer containing 1% Triton X-100. Control synaptosomes (no incubation) were lysed directly into the same lysis buffer containing one of the three different salt mixtures. The cytoskeletal and noncytoskeletal actin pools were separated 25 sec after lysis by centrifugation at 10(4) X g for 1 min, and the actin in each pool was quantitated by the DNase I inhibition assay. The drop in cytoskeletal actin induced by depolarization is maximized by including Ca2+ in the depolarizing buffer, and it is blocked completely by adding a neutral thiol protease inhibitor, leupeptin, to either the pre- or post-lysis buffer. The drop is also completely reversed by repolarizing the synaptosomes.

Actins↗
PubMed1979

Effects of hyperthermia (41.5 degrees) on Chinese hamster ovary cells analyzed in motisis.

The mitotic cells from an asynchronous population of Chinese hamster ovary cells exposed to 41.5 degrees for 7 hr were examined by light and electron microscopy to determine if there were any morphological abnormalities related to cell death or lengthening of metaphase induced by hyperthermia. All components of the mitotic apparatus were formed during exposure to heat, and the mitotic apparatus was functional as demonstrated by eventual cell division. However, heat caused the nuclear envelope to reform precociously around the chromosomes except in the region of the kinetochores, and the nuclear envelope remained associated with the chromatids during segregation. The precocious reformation of the nuclear envelope may be responsible for the lengthening of metaphase. Cells undergoing mitosis during the heat treatment possessed large evaginations of the plasma membrane, and the ubiquitous cortical microfilaments were absent in the region of these evaginations. Possibly related to the membrane damage were osmotic changes resulting in swollen mitochondria observed in heated cells entering mitosis. Since hyperthermic damage to the plasma membrane-microfilament complex was not observed in interphase cells or in cells completing division but was morphologically expressed during mitosis, the thermal lability of the plasma membrane must increase as the cells enter mitosis.

Animals↗