scholarly journals Synapsin I: an actin-bundling protein under phosphorylation control.

1987 ◽  
Vol 105 (3) ◽  
pp. 1355-1363 ◽  
Author(s):  
T C Petrucci ◽  
J S Morrow
Keyword(s):  
Actin Filaments ◽  
Binding Activity ◽  
Brain Extract ◽  
Synapsin I ◽  
Peptide Fragments ◽  
Molecular Weights ◽  
Brain Extracts ◽  
The Brain ◽  
Precise Role

Synapsin I is a neuronal phosphoprotein comprised of two closely related polypeptides with apparent molecular weights of 78,000 and 76,000. It is found in association with the small vesicles clustered at the presynaptic junction. Its precise role is unknown, although it probably participates in vesicle clustering and/or release. Synapsin I is known to associate with vesicle membranes, microtubules, and neurofilaments. We have examined the interaction of purified phosphorylated and unphosphorylated bovine and human synapsin I with tubulin and actin filaments, using cosedimentation, viscometric, electrophoretic, and morphologic assays. As purified from brain homogenates, synapsin I decreases the steady-state viscosity of solutions containing F-actin, enhances the sedimentation of actin, and bundles actin filaments. Phosphorylation by cAMP-dependent kinase has minimal effect on this interaction, while phosphorylation by brain extracts or by purified calcium- and calmodulin-dependent kinase II reduces its actin-bundling and -binding activity. Synapsin's microtubule-binding activity, conversely, is stimulated after phosphorylation by the brain extract. Two complementary peptide fragments of synapsin generated by 2-nitro-5-thiocyanobenzoic cleavage and which map to opposite ends of the molecule participate in the bundling process, either by binding directly to actin or by binding to other synapsin I molecules. 2-Nitro-5-thiocyanobenzoic peptides arising from the central portion of the molecule demonstrate neither activity. In vivo, synapsin I may link small synaptic vesicles to the actin-based cortical cytoskeleton, and coordinate their availability for release in a Ca++-dependent fashion.

scholarly journals A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

2012 ◽  
Vol 23 (2) ◽  
pp. 324-336 ◽  
Author(s):  
Céline Revenu ◽  
Florent Ubelmann ◽  
Ilse Hurbain ◽  
Fatima El-Marjou ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Actin Filaments ◽  
Growth Delay ◽  
Actin Organization ◽  
Intestinal Physiology ◽  
Key Players ◽  
Membrane Protrusions ◽  
Endocytic Compartments ◽  
Precise Role

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.

scholarly journals Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends.

1996 ◽  
Vol 135 (5) ◽  
pp. 1309-1321 ◽  
Author(s):  
J W Murray ◽  
B T Edmonds ◽  
G Liu ◽  
J Condeelis
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Elongation Factor ◽  
Protein Translation ◽  
Binding Activity ◽  
Dependent Manner ◽  
Molar Ratios ◽  
Elongation Factor 1 Alpha ◽  
Elongation Factor 1

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.

scholarly journals Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

1981 ◽  
Vol 199 (2) ◽  
pp. 323-333 ◽  
Author(s):  
D G Williams ◽  
M K Johnson
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
The Other ◽  
Particulate Fraction ◽  
Molecular Weights ◽  
The Brain ◽  
Neurotoxic Esterase

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

2006 ◽  
Vol 281 (43) ◽  
pp. 32619-32629 ◽  
Author(s):  
Vasanthy Vigneswara ◽  
Jonathan D. Lowenson ◽  
Claire D. Powell ◽  
Matthew Thakur ◽  
Kevin Bailey ◽  
...  
Keyword(s):  
Repair Enzyme ◽  
Knock Out ◽  
Methyl Donor ◽  
Brain Extracts ◽  
Knock Out Mice ◽  
First Time ◽  
The Brain ◽  
Phosphatidylethanolamine Binding Protein ◽  
Mammalian Protein

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Effects of brain extracts on activity of the trigeminal motor and hypoglossal nuclei

1961 ◽  
Vol 201 (2) ◽  
pp. 341-346
Author(s):  
Yojiro Kawamura ◽  
Masaya Funakoshi ◽  
Mitsuru Takata
Keyword(s):  
Brain Tissue ◽  
Ethanol Extract ◽  
Tris Buffer ◽  
Brain Extract ◽  
Cortical Tissue ◽  
Saline Extract ◽  
Tissue Extracts ◽  
Lower Jaw ◽  
Brain Extracts ◽  
The Brain

Alterations in trigeminal motor and hypoglossal nuclei discharges were noted following microinjections of ethanol and saline brain tissue extracts, ACh and γ-aminobutyric acid (GABA). Background activities of these nuclei were not affected by microinjection of 0.9% saline solution or tris buffer [tris (hydroxymethyl) amino methane] of pH 7.2. Solutions of pH 8.5 (tris buffer) or pH 5.8 (glycine buffer) gradually inhibited these discharges. Spontaneous discharges of the trigeminal motor nuclei were accelerated by the saline extract of the brain tissue and ACh, and they were inhibited by the ethanol extract of the brain tissue and GABA. Discharge of this nucleus accelerated by lower jaw depression was also inhibited by the ethanol brain extract and GABA. Background activity of the hypoglossal nuclei was mildly accelerated by the saline brain extracts and ACh, greatly accelerated by the ethanol brain extracts, and strongly inhibited by GABA. The saline extract of the brain tissue extracted from brain stem had a stronger activator than that extracted from cortical tissue.

scholarly journals Capping protein binding to actin in yeast

2004 ◽  
Vol 164 (4) ◽  
pp. 567-580 ◽  
Author(s):  
Kyoungtae Kim ◽  
Atsuko Yamashita ◽  
Martin A. Wear ◽  
Yuichiro Maéda ◽  
John A. Cooper
Keyword(s):  
Actin Filaments ◽  
Physiological Function ◽  
Binding Constants ◽  
Binding Activity ◽  
Actin Binding ◽  
Null Mutant ◽  
Capping Protein ◽  
Mutant Phenotypes ◽  
Actin Capping

The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.

scholarly journals N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Kerstin Schlegel ◽  
Khader Awwad ◽  
Roland G. Heym ◽  
David Holzinger ◽  
Annika Doell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Minor Component ◽  
Cleavage Sites ◽  
Brain Extracts ◽  
And Control ◽  
Progressive Accumulation ◽  
The Brain ◽  
Tissue Fractionation

Abstract Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions. Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau. Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

scholarly journals Association of brain γ-tubulins with αβ-tubulin dimers

2002 ◽  
Vol 365 (3) ◽  
pp. 889-895 ◽  
Author(s):  
Vadym SULIMENKO ◽  
Tetyana SULIMENKO ◽  
Slobodan POZNANOVIC ◽  
Volodymyr NECHIPORUK-ZLOY ◽  
Konrad J. BÖHM ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
Microtubule Nucleation ◽  
Two Dimensional Electrophoresis ◽  
Microtubule Protein ◽  
Brain Extracts ◽  
Polar Orientation ◽  
The Brain ◽  
Dimensional Electrophoresis ◽  
Multiple Charge

γ-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by γ-tubulin and the regulation of this process are not fully understood. Here we show that there are two γ-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both γ-tubulins co-polymerized with tubulin dimers, and multiple γ-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against γ-tubulin and α-tubulin revealed interactions of both γ-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large γ-tubulin complexes, other molecular γ-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of γ-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of γ-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.

scholarly journals The Effect of Brain Homogenates on Directly Measured Water Fluxes Through The Pronotum of Periplaneta Americana

1992 ◽  
Vol 171 (1) ◽  
pp. 395-408
Author(s):  
J. MACHIN ◽  
P. KESTLER ◽  
G. J. LAMPERT
Keyword(s):  
Periplaneta Americana ◽  
Tritiated Water ◽  
Brain Homogenate ◽  
Permeability Change ◽  
Water Fluxes ◽  
Brain Extracts ◽  
Order Of Magnitude ◽  
The Brain ◽  
Measured Water

Measurements of tritiated water (THO) efflux using a cup, ventilated with dry air, attached to the pronotum of Periplaneta, have been used to obtain unambiguous measurements of the permeability of this structure. Permeability values were 53% of those determined gravimetrically. Our results support the proposal that cockroach cuticle permeability is hormonally controlled, to the extent that compounds extracted from the brain have been shown to be capable of inducing permeability change over relatively short periods. Fresh brain homogenate injections from hydrated donors produced a 28% increase in pronotal permeability in 5 h, rising to 46% the day after. Injections of saline or of fresh brain extracts from dehydrated and normally hydrated cockroaches had no effect. However, previously frozen brain homogenates, from donors at all hydration levels, significantly increased pronotal permeability the day following injection. The capacity of the pronotum to undergo increases in permeability over relatively short periods was also evident in other experiments. THO permeabilities of excised pronotal discs (5.16×10−10±0.31×10−10ms−1) were an order of magnitude higher than those of intact pronota (0.49×10−10±0.06×10−10 ms−1). In vivo permeability of pronota of accidentally injured cockroaches was significantly higher than that of uninjured animals, even though the pronota themselves were not damaged. We argue that the observed changes in cuticle permeability are too small primarily to serve osmoregulatory functions. We suggest, instead, that the changes might be associated with the control of secondary processes in which the cuticle is involved and which unavoidably promote water loss.

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