A Genetically Encoded Reporter for Real-Time Imaging of Cofilin-Actin Rods in Living Neurons

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30–60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.     

Metabolic Syndrome Components Are Associated with Intervertebral Disc Degeneration: The Wakayama Spine Study

Objective

The objective of the present study was to examine the associations between metabolic syndrome (MS) components, such as overweight (OW), hypertension (HT), dyslipidemia (DL), and impaired glucose tolerance (IGT), and intervertebral disc degeneration (DD).

Design

The present study included 928 participants (308 men, 620 women) of the 1,011 participants in the Wakayama Spine Study. DD on magnetic resonance imaging was classified according to the Pfirrmann system. OW, HT, DL, and IGT were assessed using the criteria of the Examination Committee of Criteria for MS in Japan.

Results

Multivariable logistic regression analysis revealed that OW was significantly associated with cervical, thoracic, and lumbar DD (cervical: odds ratio [OR], 1.28; 95% confidence interval [CI], 0.92–1.78; thoracic: OR, 1.75; 95% CI, 1.24–2.51; lumbar: OR, 1.87; 95% CI, 1.06–3.48). HT and IGT were significantly associated with thoracic DD (HT: OR, 1.54; 95% CI, 1.09–2.18; IGT: OR, 1.65; 95% CI, 1.12–2.48). Furthermore, subjects with 1 or more MS components had a higher OR for thoracic DD compared with those without MS components (vs. no component; 1 component: OR, 1.58; 95% CI, 1.03–2.42; 2 components: OR, 2.60; 95% CI, 1.62–4.20; ≥3 components: OR, 2.62; 95% CI, 1.42–5.00).

Conclusion

MS components were significantly associated with thoracic DD. Furthermore, accumulation of MS components significantly increased the OR for thoracic DD. These findings support the need for further studies of the effects of metabolic abnormality on DD.     

A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

Magnetosomes are membrane-enveloped bacterial organelles containing nano-sized magnetic particles, and function as a cellular magnetic sensor, which assist the cells to navigate and swim along the geomagnetic field. Localized with each magnetosome is a suite of proteins involved in the synthesis, maintenance and functionalization of the organelle, however the detailed molecular organization of the proteins in magnetosomes is unresolved. MamA is one of the most abundant magnetosome-associated proteins and is anchored to the magnetosome vesicles through protein-protein interactions, but the identity of the protein that interacts with MamA is undetermined. In this study, we found that MamA binds to a magnetosome membrane protein Mms6. Two different molecular masses of Mms6, 14.5-kDa and 6.0-kDa, were associated with the magnetosomes. Using affinity chromatography, we identified that the 14.5-kDa Mms6 interacts with MamA, and the interaction was further confirmed by pull-down, immunoprecipitation and size-exclusion chromatography assays. Prior to this, Mms6 was assumed to be strictly involved with biomineralizing magnetite; however, these results suggest that Mms6 has an additional responsibility, binding to MamA.     

ADF/cofilin controls cell polarity during fibroblast migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.     

Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function

Inclusions containing actin-depolymerizing factor (ADF) and cofilin, abundant proteins in adult human brain, are prominent in hippocampal and cortical neurites of the post-mortem brains of Alzheimer's patients, especially in neurites contacting amyloid deposits. The origin and role of these inclusions in neurodegeneration are, however, unknown. Here we show that mediators of neurodegeneration induce the rapid formation of transient or persistent rod-like inclusions containing ADF/cofilin and actin in axons and dendrites of cultured hippocampal neurons. Rods form spontaneously within neurons overexpressing active ADF/cofilin, suggesting that the activation (by dephosphorylation) of ADF/cofilin that occurs in response to neurodegenerative stimuli is sufficient to induce rod formation. Persistent rods that span the diameter of the neurite disrupt microtubules and cause degeneration of the distal neurite without killing the neuron. These findings suggest a common pathway that can lead to loss of synapses.     

Intracellular pH modulation of ADF/cofilin proteins

The ADF/cofilin (AC) proteins are necessary for the high rates of actin filament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 and pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation to alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intracellular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocalization analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increases. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), when overexpressed as a GFP chimera, uniquely produces aberrant cellular phenotypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate different phenotypes in response to pH changes. Phospho-AC levels increase with cell density, and in motile cells, phospho-AC increases with alkalization, suggesting a homeostatic mechanism that compensates for increased AC activity and filament turnover. These results show that the behavior of AC proteins with pH-sensitivity in vitro is affected by pH in vivo.     

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

In vitro activity differences between proteins of the ADF/cofilin family define two distinct subgroups

The actin depolymerizing factor (ADF)/cofilins are an essential group of proteins that are important regulators of actin filament turnover in vivo. Although protists and yeasts express only a single member of this family, metazoans express two or more members in many cell types. In cells expressing both ADF and cofilin, differences have been reported in the regulation of their expression, their pH sensitivity, and their intracellular distribution. Each member has qualitatively similar interactions with actin, but quantitative differences have been noted. Here we compared quantitative differences between chick ADF and chick cofilin using several assays that measure G-actin binding, actin filament length distribution, and assembly/disassembly dynamics. Quantitative differences were measured in the critical concentrations of the complexes required for assembly, in the effects of nucleotide and divalent metal on actin monomer binding, in pH-dependent severing, in enhancement of filament minus end off-rates, and in steady-state filament length distributions generated in similar mixtures. Some of these assays were used to compare the activities of several ADF/cofilins from across phylogeny, most of which fall into one of two groups based upon their behavior. The ADF-like group has higher affinities for Mg(2+)-ATP-G-actin than the cofilin-like group and a greater pH-dependent depolymerizing activity.     

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.