Improper organization of the actin cytoskeleton affects protein synthesis at initiation

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

Activity-regulated cytoskeleton-associated protein controls AMPAR endocytosis through a direct interaction with clathrin-adaptor protein 2

The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc-AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2.

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.

An investigation of the effects of antitumour and other drugs on cell morphology and the cytoskeleton of erythrocytes

Human arythrocytes were used as a model system for an investigation of the mechanism of action of the antiproliferative drug Adriamycin. Erythrocytes were induced to undergo a change in morphology by elevation of intracellular calcium. It was revealed that the widely used media employed for the study of morphological change were unsuitable; a new incubation medium was developed so that cells were metabolically replete. In this medium echinocytosis took place both in a calcium concentration- and time-dependent manner. Pretreatment of erythrocytes with Adriamycin (10 M for 10 mins) protected the erythrocytes against calcium-induced echinocytosis at calcium concentrations < 150M. SDS-PAGE analysis of the cytoskeletal proteins prepared from erythrocytes revealed the calcium-induced proteolysis of two main cytoskeletal proteins: band 2:1 and band 4:1. Only the rate of the proteolysis of band 2.1 correlated with the onset of echinocytosis. Adriamycin inhibited the breakdown of band 2.1 even when the cells formed echinocytes; this raises doubts concerning the importance of band 2.1 in the maintenance of discocyte morphology. Adriamycin only marginally inhibited the purified calcium-activated thio protease (calpain). Calcium-loading of human erythrocytes increased the phosphorylation of several major cytoskeletal proteins including pp120, band 3, band 4.1 and band 4.9. The pattern of increase resembled that induced by 12-0-tetradecanoyl-phorbol-13-acetate. Pre-treatment with Adriamycin prior to calcium loading caused a general lowering of basal phosphorylation. Adriamycin had no effect on the activity of the calcium-activated phospholipid-dependent protein kinase (protein kinase C). A hypothesis is put forward that the morphological transition of erythrocytes might be dependent upon the activity of a contractile system.