Barbara W. Bernstein

Regulating actin-filament dynamics in vivo

The assembly and disassembly (i.e. turnover) of actin filaments in response to extracellular signals underlie a wide variety of basic cellular processes such as cell division, endocytosis and motility. The bulk turnover of subunits is 100-200 times faster in cells than with pure actin, suggesting a complex regulation in vivo. Significant progress has been made recently in identifying and clarifying the roles of several cellular proteins that coordinately regulate actin-filament turnover.

Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases.

Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.

Actin-ATP Hydrolysis Is a Major Energy Drain for Neurons

In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced ∼50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly–disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na+–K+-ATPase inhibitor, and jasplakinolide both prevent ∼50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na+–K+-ATPase, a major energy consumer.

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.

Roles of ADF/cofilin in actin polymerization and beyond.

In collaboration or competition with many other actin-binding proteins, the actin-depolymerizing factor/cofilins integrate transmembrane signals to coordinate the spatial and temporal organization of actin filament assembly/disassembly (dynamics). In addition, newly discovered effects of these proteins in lipid metabolism, gene regulation, and apoptosis suggest that their roles go well beyond regulating the cytoskeleton.

Caffeine Eliminates Gamma-Ray-Induced G2-Phase Delay in Human Tumor Cells but Not in Normal Cells

Abstract Jha, M. N., Bamburg, J. R., Bernstein, B. W. and Bedford, J. S. Caffeine Eliminates Gamma-Ray-Induced G2-Phase Delay in Human Tumor Cells but Not in Normal Cells. Radiat. Res. 157, 26–31 (2002). It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or γ rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to γ rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the γ-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca++ from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca++-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.

Incorporation of Cofilin into Rods Depends on Disulfide Intermolecular Bonds: Implications for Actin Regulation and Neurodegenerative Disease

Rod-shaped aggregates (“rods”), containing equimolar actin and the actin dynamizing protein cofilin, appear in neurons following a wide variety of potentially oxidative stress: simulated microischemia, cofilin overexpression, and exposure to peroxide, excess glutamate, or the dimer/trimer forms of amyloid-β peptide (Aβd/t), the most synaptotoxic Aβ species. These rods are initially reversible and neuroprotective, but if they persist in neurites, the synapses degenerate without neurons dying. Herein we report evidence that rod formation depends on the generation of intermolecular disulfide bonds in cofilin. Of four Cys-to-Ala cofilin mutations expressed in rat E18 hippocampal neurons, only the mutant incapable of forming intermolecular bonds (CC39,147AA) has significantly reduced ability to incorporate into rods. Rod regions show unusually high oxidation levels. Rods, isolated from stressed neurons, contain dithiothreitol-sensitive multimeric forms of cofilin, predominantly dimer. Oligomerization of cofilin in cells represents one more mechanism for regulating the actin dynamizing activity of cofilin and probably underlies synaptic loss.

The depolymerization of actin by specific proteins from plasma and brain: a quantitative assay.

Abstract Protein preparations that depolymerize actin filaments have been characterized from porcine serum and chick embryo brain. Methods used to detect actin depolymerization include changes in viscosity, sedimentation behavior, and direct visualization in the electron microscope. These methods, however, are slow and do not allow characterization of the depolymerized products. The brain and serum actin-depolymerizing factors produce monomeric actin complexed with the particular protein concerned, and these complexes, like G-actin itself, will combine with DNase I and inhibit its activity. This property provides a rapid and highly sensitive assay which has been correlated with the more classical methods for measuring actin depolymerization. The optimal conditions of pH and ionic strength have been determined for DNase inhibition by G-actin. Denatured actin does not inhibit DNase I activity nor does heat-inactivated DNase I interfere with the measurement of actin monomer. Under the conditions used, F-actin does not depolymerize or “treadmill” spontaneously, even at subcritical concentrations. The two different factors bring about actin depolymerization under conditions where the F-actin is not depolymerized by DNase I. In addition to its use to measure ADF in cell free plasma and partially purified tissue extracts, the assay provides a valuable means of investigating biological mechanisms for actin filament assembly and disassembly.

A phosphatase for cofilin to be HAD

Regulating actin dynamics through cofilin dephosphorylation is the role of a newly identified phosphatase, chronophin, a member of the haloacid dehalogenase (HAD) superfamily. Chronophin, the second cofilin phosphatase to be identified, has high phosphoprotein specificity towards cofilin, but curiously it was also discovered independently as a pyridoxal phosphate (vitamin B6) phosphatase.

Actin dynamics and cofilin‐actin rods in alzheimer disease

Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid‐β (Aβ) peptides. Extracellular Aβ‐containing plaques and intracellular phospho‐tau‐containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod‐shaped bundles of filaments composed of ADF/cofilin‐actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP‐actin subunits in F‐actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide‐linked dimers, required for bundling cofilin‐actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein‐dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target.