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ABSTRACT: Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may
mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments
that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion
and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing
factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from
neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated
rods are stable in dithiothreitol, EGTA, Ca2+, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods
formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence
staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly
of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation.
Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant
small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from
22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated
F-actin.
Journal of Biological Chemistry 02/2010; 285(8):5450-5460. · 4.77 Impact Factor
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ABSTRACT: Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.
Journal of Biological Chemistry 12/2009; 285(8):5450-60. · 4.77 Impact Factor
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ABSTRACT: When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid beta-peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (approximately 60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (Delta Psi(m)) and ATP in neurites with rods is slower than in neurites without them. The Delta Psi(m) was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because Delta Psi(m) depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (approximately 50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis.
AJP Cell Physiology 12/2006; 291(5):C828-39. · 3.54 Impact Factor
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ABSTRACT: Rod-like inclusions (rods), composed of actin saturated with actin depolymerizing factor (ADF)/cofilin, are induced in hippocampal neurons by ATP depletion, oxidative stress, and excess glutamate and occur in close proximity to senile plaques in human Alzheimer's disease (AD) brain (Minamide et al., 2000). Here, we show rods are found in brains from transgenic AD mice. Soluble forms of amyloid beta (Abeta(1-42)) induce the formation of rods in a maximum of 19% of cultured hippocampal neurons in a time- and concentration-dependent manner. Approximately one-half of the responding neurons develop rods within 6 h or with as little as 10 nM Abeta(1-42). Abeta(1-42) induces the activation (dephosphorylation) of ADF/cofilin in neurons that form rods. Vesicles containing amyloid precursor protein (APP), beta-amyloid cleavage enzyme, and presenilin-1, a component of the gamma-secretase complex, accumulate at rods. The beta-secretase-cleaved APP (either beta-C-terminal fragment of APP or Abeta) also accumulates at rods. These results suggest that rods, formed in response to either Abeta or some other stress, block the transport of APP and enzymes involved in its processing to Abeta. These stalled vesicles may provide a site for producing Abeta(1-42), which may in turn induce more rods in surrounding neurons, and expand the degenerative zone resulting in plaque formation.
Journal of Neuroscience 01/2006; 25(49):11313-21. · 7.11 Impact Factor
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ABSTRACT: 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.
Biochemistry 07/2004; 43(22):7127-42. · 3.42 Impact Factor
