Biology subjects

Pastor-Pareja, J. C.

Publications and source records attributed to Pastor-Pareja, J. C..

8 recordsLinked to original sources

bioRxiv2025

Optimized optogenetic anti-CRISPR for endogenous gene regulation in Drosophila

Optogenetic tools - whose engineering requires a structural understanding of the target proteins - are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. To build an optogenetic system for controlling endogenous gene expression, we first identified Anti-CRISPR (Acr) proteins that can inhibit CRISPRa-mediated transcriptional activation in Drosophila. Next, we inserted optogenetic protein LOV2 in these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation (EnGup) through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. Hence, we engineered an Acr-LOV2 fusion module with refined length of intrinsically disordered and ordered regions (IDR and IOR) and optimized LOV2. This optimized variant, whose application yielded new findings in vivo, was significantly more sensitive for EnGup under blue light than the prototypes. In short, not only does this work introduce the application of Acr proteins in an animal model, but it also provides insights for in vivo characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-sensitive endogenous gene regulation in Drosophila. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/651826v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@8ee937org.highwire.dtl.DTLVardef@71401eorg.highwire.dtl.DTLVardef@128a930org.highwire.dtl.DTLVardef@7bcf88_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Mechanical coupling between dorsal and ventral surfaces shapes the Drosophila haltere

The extracellular matrix is an essential determinant of animal form, enabling organization of cells and tissues into organs with complex 3D shapes. In contrast with the dorso-ventrally flat Drosophila wing, its serial homolog the haltere adopts a globular shape thought to arise from a lack of matrix-mediated adhesion between its dorsal and ventral surfaces. Contradicting this model, however, matrix manipulations are known to deform halteres. To understand haltere morphogenesis, we characterized matrix behavior and monitored metamorphic development of the haltere. We found that, similar to the wing, correct haltere morphogenesis requires Collagen IV degradation, which we show is mediated by ecdysone-controlled expression of Matrix metalloprotease 2 in both wing and haltere. After Collagen IV is degraded, similar again to the wing, dorsal and ventral haltere surfaces establish Laminin-mediated contact through long cytoskeletal projections. Furthermore, time-lapse analysis of shape changes in wild type and mutant halteres indicates that these projections couple the two surfaces through a central tensor, ensuring load distribution across the whole organ to create a globular shape against tissue-wide deforming forces. Our findings reveal an unexpected role for matrix-mediated adhesion in haltere morphogenesis and describe a novel type of matrix-based tensor structure building 3D shape from 2D epithelia.

developmental biology↗

bioRxiv2024

p24-Tango1 interactions ensure ER-Golgi interface stability and efficient transport

The eukaryotic p24 family, consisting of -, {beta}-, {gamma}- and {delta}-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion, and that their localizations between ER exit site (ERES) and Golgi are interdependent in an [->]{beta}{delta}[->]{gamma} sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface. SummaryYang et al. systematically analyze in Drosophila the function of the four p24 protein subfamilies and discover that interaction with Tango1 is essential for their concentration between ER and Golgi and for efficiency of COPII-mediated general secretory transport.

cell biology↗

bioRxiv2022

FGF signaling promotes precursor spreading for adult adipogenesis in Drosophila

Knowledge of adipogenetic mechanisms is essential to understand and treat conditions affecting organismal metabolism and adipose tissue health. In Drosophila, mature adipose tissue (fat body) exists in larvae and adults. In contrast to the well-known development of the larval fat body from the embryonic mesoderm, adult adipogenesis has remained mysterious. Furthermore, conclusive proof of its physiological significance is lacking. Here, we show that the adult fat body originates from a pool of undifferentiated mesodermal precursors that migrate from the thorax into the abdomen during metamorphosis. Through in vivo imaging, we found that these precursors spread from the ventral midline and cover the inner surface of the abdomen in a process strikingly reminiscent of embryonic mesoderm migration, requiring FGF signaling as well. FGF signaling guides migration dorsally and regulates adhesion to the substrate. After spreading is complete, precursor differentiation involves fat accumulation and cell fusion that produces mature binucleate and tetranucleate adipocytes. Finally, we show that flies where adult adipogenesis is impaired by knock down of FGF receptor Heartless or transcription factor Serpent display ectopic fat accumulation in oenocytes and decreased resistance to starvation. Our results reveal that adult adipogenesis occurs de novo during metamorphosis and demonstrate its crucial physiological role.

developmental biology↗

bioRxiv2021

Intrinsic and tumor-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland

Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental roles of JAK/STAT intersect is unknown. Here, we show that JAK/STAT is active during development of the prothoracic gland (PG), the organ that controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and slightly advanced metamorphosis. Conversely, JAK/STAT hyperactivation, achieved through overexpression of pathway components or SUMOylation loss, caused PG hypertrophy and metamorphosis delay. Interestingly, tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis. Finally, we show that expression of transcription factor Apontic, a JAK/STAT target in the PG, recapitulates PG hypertrophy and metamorphosis delay. JAK/STAT damage signaling, therefore, regulates metamorphosis onset at least in part by coopting its developmental role in the PG. Summary statementDamage signaling from tumors mediated by JAK/STAT-activating Upd cytokines delays the Drosophila larva-pupa transition through cooption of a JAK/STAT developmental role in the prothoracic gland.

cancer biology↗

bioRxiv2021

Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to voluminous biological specimens such as tissues or particularly large mammalian cells, which usually require high-pressure freezing for vitrification. Here we show that adult mouse cardiomyocytes and dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of complex native specimens. Our data provides unanticipated insights into the molecular architecture of samples hitherto unexplored by cryo-ET.

cell biology↗

bioRxiv2021

ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers

Secretory cargos are collected at ER exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, we used transgenic tagging in Drosophila flies, 3D-SIM and FIB-SEM to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs and imaginal discs overexpressing ERES determinant Tango1. We found in front of ERES a pre-cis-Golgi region involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate intermediate compartment or collection of large carriers, for which we found no evidence. We found, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.

cell biology↗

bioRxiv2018

Analysis of the role of Nidogen/entactin in basement membrane assembly and morphogenesis in Drosophila

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen interlinked by the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg on morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from in vitro and cultured embryonic tissues experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival, strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.\n\nAuthor SummaryBasement membranes (BMs) are thin layers of specialized extracellular matrices present in every tissue of the human body. Its main constituents are two networks of Laminin and Type IV Collagen linked by Nidogen (Ndg) and proteoglycans. They form an organized scaffold that regulates organ morphogenesis and function. Mutations affecting BM components are associated with organ dysfunction and several congenital diseases. Thus, a better comprehension of BM assembly and maintenance will not only help to learn more about organogenesis but also to a better understanding and, hopefully, treatment of these diseases. Here, we have used Drosophila to analyse the role of Ndg in BM formation in vivo. Elimination of Ndg in worms and mice does not affect survival, strongly questioning its proposed linking role, derived from in vitro experiments. Here, we show that in the fly Ndg is dispensable for BM assembly and preservation in many tissues, but absolutely required in others. Furthermore, our functional study of the different Ndg domains challenges the significance of some interactions between BM components derived from in vitro experiments, while confirming others, and reveals a new key requirement for the Rod domain in Ndg function and incorporation into BMs.

developmental biology↗