SUMMARY We present a modified diaminobenzidine (DAB) photoconversion method that enables staining... more SUMMARY We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punc- tate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural re- cipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells
A single type of high density lipophorin (HDLp) binding sites is present at intact fat body tissu... more A single type of high density lipophorin (HDLp) binding sites is present at intact fat body tissue and in fat body membranes of larval and adult locusts. HDLp is bound with high affinity (Kd = 107 M). This interaction does not require divalent cations and is heat-labile because heat-treatment of fat body membranes results in a substantial reduction of the
The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics b... more The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a n...
A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expr... more A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P...
The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDL... more The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by insect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and diaminobenzidine photoconversion, respectively. Internalization of HDLp seems to conflict with the selective process by which the lipids are transported between HDLp and fat body cells. The pathway followed by the internalized lipoproteins was investigated. In addition, the localizations of HDLp in fat body cells of young and older adult locusts were compared because of the previously reported age-related differences in distribution of cell-associated and internalized HDLp. In the present study, internalized labeled HDLp was observed in early endosomes, late endosomes, and putative lysosomes. In older adults, these labeled structures were much less abundant than in young adults. Moreover, in these animals, the ...
Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces... more Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces. In the present study, the lipoprotein binding site was identified as an endocytotic receptor involved in receptor-mediated uptake of its lipoprotein ligand, high density lipophorin. After an initial period of high endocytotic uptake of high density lipophorin in the adult stage, this process strongly diminished. In the same period, a dramatic increase in cell surface-associated lipoproteins was observed. When animals were starved, however, internalization of lipoproteins was maintained. The pathway followed by the internalized lipoproteins appears to be different from the endosomal/lysosomal pathway, as the vast majority of apolipoproteins seemed to escape from lysosomal hydrolysis. In addition, no substantial intracellular accumulation of apolipoproteins was observed, suggesting that internalized lipoproteins were resecreted. It is unlikely that internalization is required for transpo...
A single type of high density lipoprotein (HDLp) binding sites is present at intact fat body tiss... more A single type of high density lipoprotein (HDLp) binding sites is present at intact fat body tissue and in fat body membranes of larval and adult locusts. HDLp is bound with high affinity (Kd approximately 10(-7) M). This interaction does not require divalent cations and is heat-labile because heat-treatment of fat body membranes results in a substantial reduction of the maximal binding capacity. In addition to unlabeled HDLp and low density lipophorin (LDLp), human low density lipoprotein also seems to compete with radiolabeled HDLp for this binding site, suggesting a relaxed specificity. Induction of lipid mobilization with adipokinetic hormone did not change the binding characteristics of the fat body. An increase in the binding capacity of intact fat body tissue in the adult stage suggests that the number of cell surface binding sites is upregulated during development. However, the total number of HDLp binding sites appears to be constant, because larval and adult fat body membr...
The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDL... more The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by in- sect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and di- aminobenzidine photoconversion, respectively. Internaliza- tion of HDLp seems to conflict with the selective process by which the lipids are transported
The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation,... more The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.
SUMMARY We present a modified diaminobenzidine (DAB) photoconversion method that enables staining... more SUMMARY We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punc- tate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural re- cipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells
A single type of high density lipophorin (HDLp) binding sites is present at intact fat body tissu... more A single type of high density lipophorin (HDLp) binding sites is present at intact fat body tissue and in fat body membranes of larval and adult locusts. HDLp is bound with high affinity (Kd = 107 M). This interaction does not require divalent cations and is heat-labile because heat-treatment of fat body membranes results in a substantial reduction of the
The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics b... more The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a n...
A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expr... more A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P...
The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDL... more The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by insect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and diaminobenzidine photoconversion, respectively. Internalization of HDLp seems to conflict with the selective process by which the lipids are transported between HDLp and fat body cells. The pathway followed by the internalized lipoproteins was investigated. In addition, the localizations of HDLp in fat body cells of young and older adult locusts were compared because of the previously reported age-related differences in distribution of cell-associated and internalized HDLp. In the present study, internalized labeled HDLp was observed in early endosomes, late endosomes, and putative lysosomes. In older adults, these labeled structures were much less abundant than in young adults. Moreover, in these animals, the ...
Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces... more Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces. In the present study, the lipoprotein binding site was identified as an endocytotic receptor involved in receptor-mediated uptake of its lipoprotein ligand, high density lipophorin. After an initial period of high endocytotic uptake of high density lipophorin in the adult stage, this process strongly diminished. In the same period, a dramatic increase in cell surface-associated lipoproteins was observed. When animals were starved, however, internalization of lipoproteins was maintained. The pathway followed by the internalized lipoproteins appears to be different from the endosomal/lysosomal pathway, as the vast majority of apolipoproteins seemed to escape from lysosomal hydrolysis. In addition, no substantial intracellular accumulation of apolipoproteins was observed, suggesting that internalized lipoproteins were resecreted. It is unlikely that internalization is required for transpo...
A single type of high density lipoprotein (HDLp) binding sites is present at intact fat body tiss... more A single type of high density lipoprotein (HDLp) binding sites is present at intact fat body tissue and in fat body membranes of larval and adult locusts. HDLp is bound with high affinity (Kd approximately 10(-7) M). This interaction does not require divalent cations and is heat-labile because heat-treatment of fat body membranes results in a substantial reduction of the maximal binding capacity. In addition to unlabeled HDLp and low density lipophorin (LDLp), human low density lipoprotein also seems to compete with radiolabeled HDLp for this binding site, suggesting a relaxed specificity. Induction of lipid mobilization with adipokinetic hormone did not change the binding characteristics of the fat body. An increase in the binding capacity of intact fat body tissue in the adult stage suggests that the number of cell surface binding sites is upregulated during development. However, the total number of HDLp binding sites appears to be constant, because larval and adult fat body membr...
The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDL... more The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by in- sect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and di- aminobenzidine photoconversion, respectively. Internaliza- tion of HDLp seems to conflict with the selective process by which the lipids are transported
The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation,... more The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.
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