A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning. Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.Open in a separate windowClick here to view.^{(46M, flv)}     

Transendothelial transport (transcytosis) of iron-transferrin complex in the rat liver

To determine the transport pathway of iron-transferrin complex (Fe-TF) into the hepatocyte, we labeled Fe-TF with colloidal gold and perfused rat liver through the portal vein with this probe under different conditions. The tissue was then studied by transmission electron microscopy. At a cold temperature (approximately 4 degrees C), the probe bound to the luminal surface of sinusoidal endothelium without internalization. The binding was limited to the endothelium and there was no binding to Kupffer cells or hepatocytes. The binding was inhibitable in the presence of excess soluble Fe-TF, indicating the specificity of the bindings. At 37 degrees C the probe was internalized via a system of coated pits and vesicles. Morphometric analyses of the temporal sequence of events suggested that the probe was transported, in part, across the endothelium via a system of tubules and vesicles and externalized on the abluminal side via a system of coated pits. The probe was then taken up by hepatocytes. Only minimal uptake was noted when gold-labeled bovine serum albumin was used either at the low temperature or at 37 degrees C. The findings suggest that the liver uptake of Fe-TF complex by hepatocytes is a transendothelial phenomenon (transcytosis).     

Liver endothelium mediates the hepatocyte''s uptake of ceruloplasmin

The mode of transport of ceruloplasmin (CP) into the liver was investigated in fractionated liver cell suspensions. Incubation of 125I-CP at 4 degrees C with these different fractions led to its binding only to endothelial cells but not Kupffer cells and hepatocytes. Incubation at 37 degrees C led to rapid uptake of 125I-CP by endothelium, but cell-associated radioactivity declined after 15 min, which suggests the release of the labeled substance. Internalization was confirmed by fractionation of surface-bound and internalized ligand. The released label now acquired binding potential for fresh target hepatocytes, and the binding was inhibitable with asialoceruloplasmin but not native CP. This suggested that the released molecule was modified in the endothelium by desialation. Desialation was confirmed by incubation of endothelium with double-labeled CP (3H label on sialic acid and 125I on the protein part). We conclude that in the liver, CP is first recognized and taken up by endothelial cells that are endowed with appropriate surface receptors for the protein. Endothelium then modifies the molecule by desialation to expose the penultimate galactosyl residues. The modified molecule is then released, recognized, and taken up by hepatocytes through their membrane galactosyl-recognition system. These findings are consistent with the role of endothelium as an active mediator of molecular transport between blood and tissue, and further assign a biological role for the galactosyl-recognition system in hepatocytes.     

Retention of hemopoiesis in tail vertebrae of newborn rats

Summary In newborn rats, the marrow cavity of tail vertebrae is hemopoietic and contains no adipose tissue. The latter develops soon after birth to replace the hemopoietic tissue within the nonexpansile volume of the marrow cavity. By transposing the tail into the warmer environment of the abdomen, hemopoiesis was retained, and the development of adipose tissue was prevented in the transposed segment, when the operation was done in preweanling but not in adult animals. Systemic stimuli of erythropoiesis (phlebotomy, induced hemolysis) acted synergistically with the temperature increment to retain hemopoiesis. The findings support the concept that adipose cells in the yellow marrow are relatively stable and once developed, they are not readily mobilized. The findings may also explain the discrepancies in the results obtained by rat tail transposition.Supported by ERDA Contract EP-78-S-03-0899, NASA Grant NSG 9061 and RCDA AM16125 awarded to Mehdi Tavassoli     

Characterisation of Schizosaccharomyces pombe rad31, a UBA-related gene required for DNA damage tolerance.

The fission yeast rad31-1 mutant is sensitive to both UV and ionising radiation and exhibits a growth defect at 35 degrees C. In addition, the mutant displays defects in cell morphology and nuclear division at 26 degrees C which are exaggerated at 35 degrees C. We have cloned the rad31 gene and have shown that it is not essential for viability, although cells containing a disrupted rad31 gene grow slowly. The null allele has similar cell and nuclear morphologies to the original allele and displays an extremely high frequency of loss of minichromosomes. rad31 is not required for either the S/M or G2/M checkpoint, however double mutant analysis indicates that rad31 acts in a process which is defective in the checkpoint rad mutants and which involves hus5 . Sequence analysis indicates that rad31 encodes a protein which is related to ubiquitin activating proteins and more particularly to an ORF in Saccharomyces cerevisiae and to the Arabidopsis thaliana AXR1 and human APP-BP1 genes. We have isolated the S.cerevisiae sequence, which we have named RHC31 ( ad31homologue in S. erevisiae), since we show that it can complement the slow growth phenotype and radiation sensitivity of S.pombe rad31.     

Localization of megakaryocytes in the bone marrow

In the bone marrow, megakaryocytes are located in the extravascular space, applied to the abluminal surface of endothelium. In this position, they send cytoplasmic projections into the lumen. Some of these projections are organelle free and may serve to anchor the cell to the endothelium. They could also serve to monitor the circulation and to receive information as to the requirement of the body for platelet formation. Megakaryocytes also send organelle containing projections into the lumen. This may be an early step in the migration of these cells into the lumen or, alternatively, part of the proplatelet formation. These proplatelets are 2.5 x 120 microns elongated structures that penetrate the lumen and can each subsequently make 1000 platelets. Each megakaryocyte can make six to eight proplatelets. In the perisinal position, megakaryocytes may subserve an adventitial function as well; many blood cells can then take a transmegakaryocytic route to reach the endothelium and enter the circulation.     

Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination.

A new Schizosaccharomyces pombe mutant (rad32) which is sensitive to gamma and UV irradiation is described. Pulsed field gel electrophoresis of DNA from irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks. The mutant also undergoes decreased meiotic recombination and displays reduced stability of minichromosomes. The rad32 gene has been cloned by complementation of the UV sensitive phenotype. The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae. Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevisiae RAD51) and that cells deleted for the rad32 gene in conjunction with either the rad3 deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable.     

Synthesis and biological evaluation of N-[4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of N-[4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid (2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The inhibitor 2 was prepared in a convergent synthesis involving C-alkylation of methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)benzoate with 1-chloro-3-iodopropane followed by construction of the pyrimidinone ring. Compound 2 was found to be an effective inhibitor of recombinant human GAR Tfase (K(i) = 0.50 microM), whereas it was inactive (K(i) > 100 microM) against E. coli GAR Tfase as well as recombinant human AICAR Tfase. Compound 2 exhibited modest, purine-sensitive growth inhibitory activity against the CCRF-CEM cell line (IC50 = 6.0 microM).     

Rational design,synthesis, evaluation,and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.