Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 —| RegA —| [cAMP] → PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.     

Isolation and characterization of a mucin-glycoprotein from rat submandibular glands

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.     

Comparative genomics of the social amoebae <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> and <Emphasis Type="Italic">Dictyostelium purpureum</Emphasis>

Background

The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.

Results

We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.

Conclusions

The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

     

Long-term preservation of dried phosphofructokinase by sugars and sugar/zinc mixtures

We have demonstrated that sugars and suger/zinc mixtures can be used to preserve the activity of dried phosphofructokinase (PFK) during long-term storage over CaSO4. After 9 weeks in the presence of either 200 mM sucrose or 200 mM trehalose little loss of PFK activity was noted, with almost 60% of the original prefreeze-dry activity recovered when samples were rehydrated. Even reducing sugars protected the dried enzyme throughout the entire storage period. Of the sugars tested, 200 mM lactose provided the most stability to PFK; at the end of the dry storage, over 80% of the initial activity was recovered. With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity was recovered at the end of the experiment. With all the sugars tested, the addition of 0.6 mM Zn2+ to sugar/PFK mixtures enhanced the stability of the enzyme, and no long-term adverse effects of the metal ion on enzyme activity were noted.     

SEED DORMANCY IN UNIOLA PANICULATA

Seeds of Uniola paniculata L., a sand-dune grass of the Southeast coast, showed an increasing dormancy under laboratory germination conditions that reached 98–100% six months after maturity. The embryos were viable and nondormant and the seeds absorbed water readily. Chemical treatments were only moderately effective, but leaching the seeds after cutting into the endosperm gave 100% germination. The presence of a diffusible growth inhibitor was demonstrated by this response and by tests of endosperm leachate. Temperature, particularly sand temperature above 30 C, appears to be the major factor in normal germination of the seed.     

Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.The acyl coenzyme A (CoA) binding protein (ACBP) has been well characterized for its role in intracellular lipid trafficking, but it also serves as the precursor of peptides that function as intercellular signals. ACBPs are involved in the transport and metabolism of long-chain acyl-CoA esters and steroid biosynthesis (15, 16, 32). In the mammalian brain ACBP is also secreted and processed to generate a diazepam binding inhibitor (DBI) peptide that regulates γ-aminobutyric acid A (GABA_A) ionotropic receptors in neurons (12). Qian and colleagues recently demonstrated the secretion of ACBP from Muller glial cells of the retina (36). In Dictyostelium discoideum the ACBP homolog AcbA is secreted and processed extracellularly into spore differentiation factor 2 (SDF-2), a 34-amino-acid peptide that is highly similar to DBI (2). However, neither ACBP nor AcbA carries a signal sequence that is necessary for entering the endoplasmic reticulum/Golgi pathway. Alternative, unconventional pathways for the secretion of proteins, including ACBPs, have been proposed over the past 20 years, involving the direct membrane transport of proteins, novel membrane trafficking, or autophagy (29, 39).In Dictyostelium, SDF-2 signaling controls the terminal cell differentiation of prespore cells into encapsulated spores during fruiting body formation. Prespore cells within the nascent sorus climb the elongating stalk in a process that requires their active motility (8). The extracellular processing of AcbA into SDF-2 by prestalk cells is thought to coordinate spore encapsulation with fruiting body morphogenesis such that immobile spores are not produced before the stalk begins to form. Approximately halfway through this process of culmination, sporulation occurs as a wave from the top to the bottom of the nascent sorus (38).An understanding of the regulation of SDF-2 signaling is now emerging. During culmination, prespore cells respond to a steroid signal by rapidly releasing GABA, which binds to the GABA_B-like receptor GrlE and stimulates a signal transduction pathway leading to the release of AcbA by prespore cells (3, 5). AcbA is processed into SDF-2 by TagC protease, which is displayed on the surface of prestalk cells in response to GABA. The 34-amino-acid peptide SDF-2 binds to the receptor histidine kinase DhkA, leading to elevated levels of intracellular cyclic AMP (cAMP), which induces spore encapsulation (2, 47). Low levels of SDF-2 also trigger the release of additional AcbA proteins, forming a positive-feedback loop (2, 6). Although only 1 to 3% of the total AcbA is secreted, the levels of SDF-2 in the sorus are far above that required to rapidly induce sporulation (5, 19).The release of AcbA is a critical step in this cascade, but the mechanism of its secretion is largely unknown. The Golgi-associated protein GRASP (Golgi reassembly stacking protein) appears to play an essential role in the process since grpA-null mutants lacking GRASP fail to produce SDF-2 (19). To further explore the role of GRASP and understand the regulation of AcbA secretion, we have determined the subcellular localization of AcbA before and after stimulating its release. Secreted AcbA appears to be localized within membrane-bound vesicles, which accumulate in the cortex of prespore cells during culmination. When AcbA secretion is stimulated by GABA or SDF-2, the cortical vesicles containing AcbA are lost from wild-type cells but remain in cells lacking GRASP. It appears that GRASP is not involved in the production or positioning of AcbA within the cortical vesicles, but it is essential for events leading to their regulated release.