Biology subjects

Francescutti, C. M.

Publications and source records attributed to Francescutti, C. M..

3 recordsLinked to original sources

bioRxiv2023

Enhanced in Vivo Blood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody

BackgroundThe blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. MethodsA mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H -neurotensin, caused >8{degrees}C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M156,96,102His-13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H -13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1R56H, P96H, Y102H -13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

neuroscience↗

bioRxiv2023

Genetics of continuous colour variation in a pair of sympatric sulphur butterflies

Continuous colour polymorphisms can serve as a tractable model for the genetic and developmental architecture of traits, but identification of the causative genetic loci is complex due to the number of individuals needed, and the challenges of scoring continuously varying traits. Here we investigated continuous colour variation in Colias eurytheme and C. philodice, two sister species of sulphur butterflies that hybridise in sympatry. Using Quantitative Trait Locus (QTL) analysis of 483 individuals from interspecific crosses and an high-throughput method of colour quantification, we found that two interacting large effect loci explain around 70% of the heritable variation in orange-to-yellow chromaticity. Knockouts of red Malphighian tubules (red), a candidate gene at the primary QTL likely involved in endosomal maturation, resulted in depigmented wing scales showing disorganised pterin granules. The Z sex chromosome contains a large secondary colour QTL that includes the transcription factor bric-a-brac (bab), which we show can act as a modulator of orange pigmentation in addition to its previously-described role in specifying UV-iridescence. We also describe the QTL architecture of other continuously varying traits, and that wing size maps to the Z chromosome, supporting a Large-X effect model where the genetic control of species-defining traits is enriched on sex chromosomes. This study sheds light on the genetic architecture of a continuously varying trait, and illustrates the power of using automated measurement to score phenotypes that are not always conspicuous to the human eye. ForewordThe colour phenotypes in this article involve nuanced gradations of yellow and orange that may be difficult to perceive for people who are colour vision deficient. Hue-shifted versions of all main figures are accessible online for dichromat readers (BioRxiv preprint: Supplementary Material).

genetics↗

bioRxiv2021

A genetic switch for male UV-iridescence in an incipient species pair of sulphur butterflies

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of UV iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern US with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knock-out, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation. Significance statementIncipient species are at an intermediate stage of speciation where reproductive isolation is counteracted by the homogenizing effects of gene flow. Human activity sometimes leads such species to reunite, as seen in the Orange Sulphur butterfly, which forms large hybridizing populations with the Clouded Sulphur in alfalfa fields. Here we show that the sex chromosome maintains these species as distinct, while the rest of their genome is admixed. Sex chromosomes notably determine which males display to females a bright, iridescent ultraviolet signal on their wings. Genetic mapping, antibody stainings, and CRISPR knock-outs collectively indicate that the gene bric a brac controls whether UV-iridescent nanostructures develop in each species, elucidating how a master switch gene modulates a male courtship signal.

evolutionary biology↗