Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

Biosynthesis in the Albumen Gland-Capsule Gland Complex Limits Reproductive Effort in the Invasive Apple Snail Pomacea canaliculata.

High fecundity often contributes to successful invasives. In molluscs, this may be facilitated by the albumen gland-capsule gland complex, which in gastropods secretes the egg perivitelline fluid that nourishes and protects embryos. The biochemistry of the albumen gland-capsule gland complex and its relationship with fecundity remain largely unknown. We addressed these issues in Pomacea canaliculata (Lamarck, 1822), a highly invasive gastropod whose fecundity and reproductive effort exceed those of ecologically similar gastropods. We evaluated the dynamics of its major secretion compounds (calcium, polysaccharides, and total proteins) as well as the gene expression and stored levels of perivitellins during key moments of the reproductive cycle, that is, before and after first copulation and at low, medium, and high reproductive output. Copulation and first oviposition do not trigger the onset of albumen gland-capsule gland complex biosynthesis. On the contrary, soon after an intermediate reproductive effort, genes encoding perivitellins overexpressed. A high reproductive effort caused a decrease in all albumen gland-capsule gland complex secretion components. Right after a high reproductive output, the albumen gland-capsule gland complex restored the main secretion components, and calcium recovered baseline reserves; but proteins and polysaccharides did not. These metabolic changes in the albumen gland-capsule gland complex after multiple ovipositions were reflected in a reduction in egg mass but did not compromise egg quality. At the end of the cycle, egg dry weight almost doubled the initial albumen gland-capsule gland complex weight. Results indicate that albumen gland-capsule gland complex biosynthesis limits a constantly high reproductive output. Therefore, lowering fecundity by targeting biosynthesis could effectively reduce the rate of this species' spread.

Distinct functions of the ATP binding cassettes of transporters associated with antigen processing: a mutational analysis of Walker A and B sequences.

The transporters associated with antigen processing (TAP1/TAP2) provide peptides to MHC class I molecules in the endoplasmic reticulum. Like other ATP-binding cassette proteins, TAP uses ATP hydrolysis to power transport. We have studied peptide binding to as well as translocation by TAP proteins with mutations in the Walker A and B sequences that are known to mediate ATP binding and hydrolysis. We show that a mutation in the TAP1 Walker B sequence reported to abrogate class I expression by a lung tumor does not affect ATP binding affinity, suggesting a defect restricted to ATP hydrolysis. This mutation reduces peptide transport by only 50%, suggesting that TAP function can be highly limiting for antigen presentation in non-lymphoid cells. Single substitutions in Walker A sequences (TAP1K544A, TAP2K509A), or their complete replacements, abrogate nucleotide binding to each subunit. Although all of these mutations abrogate peptide transport, they reveal distinct roles for nucleotide binding to the two transporter subunits in TAP folding and in regulation of peptide substrate affinity, respectively. Alteration of the TAP1 Walker A motif can have strong effects on TAP1 and thereby TAP complex folding. However, TAP1 Walker A mutations compatible with correct folding do not affect peptide binding. In contrast, abrogation of the TAP2 nucleotide binding capacity has little or no effect on TAP folding but eliminates peptide binding to TAP at 37 degrees C in the presence of nucleotides. Thus, nucleotide binding to TAP2 but not to TAP1 is a prerequisite for peptide binding to TAP. Based on these results, we propose a model in which nucleotide and peptide release from TAP are coupled and followed by ATP binding to TAP2, which induces high peptide affinity and initiates the transport cycle.