Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation

The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity.     

Effects of high peak power microwaves on the retina of the rhesus monkey

We studied the retinal effects of 1.25 GHz high peak power microwaves in Rhesus monkeys. Preexposure fundus photographs, retinal angiograms, and electroretinograms (ERG) were obtained to screen for normal ocular structure and function and, after exposure, as endpoints of the study. Histopathology of the retina was an additional endpoint. Seventeen monkeys were randomly assigned to receive sham exposure or pulsed microwave exposures. Microwaves were delivered anteriorly to the face at 0, 4.3, 8.4, or 20.2 W/kg spatially and temporally averaged retinal specific absorption rates (R-SAR). The pulse characteristics were 1.04 MW ( approximately 1.30 MW/kg temporal peak R-SAR), 5.59 micros pulse length at 0, 0.59, 1. 18, and 2.79 Hz pulse repetition rates. Exposure was 4 h per day and 3 days per week for 3 weeks, for a total of nine exposures. The preexposure and postexposure fundus pictures and angiograms were all within normal limits. The response of cone photoreceptors to light flash was enhanced in monkeys exposed at 8.4 or 20.2 W/kg R-SAR, but not in monkeys exposed at 4.3 W/kg R-SAR. Scotopic (rod) response, maximum (combined cone and rod) response, and Naka-Rushton R(max) and log K of scotopic b-waves were all within normal range. Retinal histopathology revealed the presence of enhanced glycogen storage in photoreceptors among sham (2/5), 8.4 W/kg (3/3), and 20.2 W/kg (2/5) exposed monkeys, while enhanced glycogen storage was not observed in the 4.3 W/kg (0/4) exposed group. Supranormal cone photoreceptor b-wave was R-SAR dependent and may be an early indicator of mild injury. However no evidence of degenerative changes and ERG depression was seen. We concluded that retinal injury is very unlikely at 4 W/kg. Functional changes that occur at higher R-SAR are probably reversible since we saw no evidence of histopathologic correlation with ERG changes. Bioelectromagnetics 21:439-454, 2000. Published 2000 Wiley-Liss, Inc.     

Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.     

The Evolution of Enamel Microstructure: How Important Is Amelogenin?

The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes' process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure.     

What Is the Signal for the Posttranslational Arginylation of Proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [³H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.     

Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells

This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery.