Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives.

Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 microM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 microM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy.

Haplotypic divergence coupled with lack of diversity at the Arabidopsis thaliana alcohol dehydrogenase locus: roles for both balancing and directional selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as "hypervariable" on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana's tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.

Formation of tyrosine O-sulfate by mitochondria and chloroplasts of Euglena.

Mitochondria that have been purified from cells of light-grown wild-type Euglena gracilis Klebs var. bacillaris Cori or dark-grown mutant W10BSmL and incubated with 35SO4(2-) and ATP accumulate a labeled compound in the surrounding medium. This compound is also labeled when mitochondria are incubated with [14C]tyrosine and nonradioactive sulfate under the same conditions. This compound shows exact coelectrophoresis with synthetic tyrosine O-sulfate at pH 2.0, 5.8, and 8.0, and yields sulfate and tyrosine on acid hydrolysis. Treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester; no hydrolysis of tyrosine methyl ester to tyrosine is observed under identical conditions, ruling out methyl esterase activity in the aryl sulfatase preparation. Thus the compound is identified as tyrosine O-sulfate. No tyrosine O-sulfate is found outside purified developing chloroplasts of Euglena incubated with 35SO4(2-) and ATP, but both chloroplasts and mitochondria accumulate labeled tyrosine-O-sulfate externally when incubated with adenosine 3'-phosphate 5'-phospho[35S]-sulfate (PAP35S). Since tyrosine does not need to be added, it must be provided from endogenous sources. Labeled tyrosine O-sulfate is found in the free pools of light-grown Euglena cells grown on 35SO4(2-) or in dark-grown cells incubated with 35SO4(2-) in light, but none is found in the medium after cell growth. No labeled tyrosine O-sulfate is found in Euglena proteins (including those in extracellular mucus) after growth or incubation of cells with 35SO4(2-) or after incubation of organelles with 35SO4(2-) and ATP or PAP35S, ruling out sulfation of the tyrosine in protein or incorporation of free-pool tyrosine O-sulfate into protein. The system forming tyrosine O-sulfate is membrane-bound and may be involved in transporting tyrosine out of the organelles.