Biology subjects

Pavlovic, B.

Publications and source records attributed to Pavlovic, B..

5 recordsLinked to original sources

bioRxiv2026

NPEPPS segmental duplication drives position effect expression of TBC1D3 in the human brain

In humans, the TBC1D3 gene family is thought to play a critical role in the expansion of the frontal cortex by promoting neuronal proliferation during brain development. This gene family shows some of the greatest structural heterozygosity ([~]97%) with haplotype copy numbers ranging from 3-39 among different human haplotypes. This raises the question as to how a gene so crucial in the evolutionary expansion of the human frontal cortex can be so variable in the human population. Here, we characterize the regulatory architecture that explains this paradox. We show that 45-96% of TBC1D3 expression is attributable to a single paralog located at the most telomeric position at the edge of a cluster of TBC1D3 genes. We find that its >3-fold higher expression relative to other copies is driven by a 110 kbp segmental duplication that occurred [~]8.9 million years ago, relocating a partial duplication of the puromycin-sensitive aminopeptidase gene (NPEPPS), including its promoter, adjacent to this TBC1D3 locus. Using neurospheres and comparative transcriptomics of iPSC-derived cultures, we show that expression of NPEPPSP1-TBC1D3 increases as neurons differentiate as a result of alternative splicing and differential polyadenylation usage. While the fusion exists in other ape lineages, we show subsequent deletion of the NPEPPSP1 promoter in Gorilla and a separate, lineage-specific duplication in the Pan lineage ablated the production of this fusion product, rendering this position effect of TBC1D3 specific to humans.

genomics↗

bioRxiv2025

Loss of class 3 PI3K complex leads to retention of Wg at the apical membrane in polarized tissue

Wnt/Wingless (Wg) signalling is a key regulator of tissue patterning and morphogenesis in Drosophila, coordinating cell fate decisions and long-range morphogen signalling. In wing imaginal discs, Wg proteins rely on specialized trafficking machinery, including the cargo receptor Evi/Wls, and are secreted via multiple routes, comprising a glypican-dependent Wg pool and an endocytosis-dependent Wg pool. However, the cellular mechanisms controlling Wg secretion and post-endocytic trafficking in Drosophila remain incompletely understood. We performed an in vivo kinome- and phosphatome-wide CRISPR-Cas9 screen in Drosophila wing imaginal discs using endogenous fluorescently tagged Wg as a readout. Genetic perturbations were combined with super-resolution microscopy and ex vivo pharmacological treatments to resolve Wg and Evi/Wls secretion dynamics. We identified Vps15, a core subunit of the class III phosphatidylinositol 3-kinase (PI3K (III)) complex, as a critical factor of Wg secretion. Loss of Vps15 caused apical accumulation of Wg in Wg-secreting cells, selectively impairing an endocytosis-dependent Wg pool while leaving a glypican-mediated Wg pool intact. In contrast to Wg, Evi/Wls did not accumulate in PI3K (III) mutant cells, but instead was subject to proteasome-dependent degradation. Super-resolution imaging further revealed frequent spatial separation of Wg and Evi/Wls in Wg-secreting cells prior to the uptake of the endocytosis-dependent Wg pool. Our study establishes PI3K (III) perturbation as a powerful approach to dissect distinct Wg secretion routes in Drosophila wing imaginal discs. We uncovered divergent post-endocytic fates of Wg and Evi/Wls upon perturbation and provided new mechanistic insight into Wg-Evi/Wls dynamics.

developmental biology↗

bioRxiv2024

Restraining Wnt activation and intestinal tumorigenesis by a Rab35 dependent GTPase relay

Maintenance of homeostatic processes ensure curtailment of intestinal tumorigenesis. Inactivating mutations to Adenomatous Polyposis Coli (Apc) result in aberrantly activated Wnt signalling and initiates colorectal cancer (CRC) in approx. 80% of cases, yet our understanding of the subcellular mechanisms that modulate dysregulated pathway activity is limited. Here, using a conditional in vivo genetic screen, we identify Rab35 GTPase as a novel tumour suppressor that modulates regional Wnt activity after loss of Apc in progenitor cells. Single cell analysis revealed that progenitor cells respond to Apc depletion by increasing the expression of a GTPase activating protein, which we named blackbelt, and triggering Rab35 disassociation from the plasma membrane. Mechanistically, we demonstrate that Rab35 controls the localisation and activation of the Rho GTPase, Cdc42, which functions as a relay to regulate JNK signalling. This in turn tunes the Wnt pathway upstream of {beta}-catenin to direct proliferation and differentiation of progenitor cells. Importantly, we show that maintaining active JNK signalling is important for the propagation of Apc mutant mouse colon organoids. Our findings highlight a novel GTPase cascade that sustains aberrant Wnt activity in specific segments of the intestine and provides impetus to therapeutically exploit this pathway to target CRC.

cancer biology↗

bioRxiv2020

Systematic functional analysis of Rab GTPasesreveals limits of neuronal robustness in Drosophila

Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In addition, we created a transgenic fly collection for the acute, synchronous release system RUSH for all 26 Rabs. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous-system enriched Rabs yields viable and fertile flies without obvious morphological defects. However, 9 of these 13 affect either developmental timing when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. These defects are non-lethal under laboratory conditions, but represent sensitized genetic backgrounds that reveal limits of developmental and functional robustness to environmental challenges. Interestingly, the neuronal rab26 was previously proposed to function in synaptic maintenance by linking autophagy and synaptic vesicle recycling and we identified rab26 as one of six rab mutants with reduced synaptic function under continuous stimulation conditions. However, we found no changes to autophagy or synaptic vesicle markers in the rab26 mutant, but instead a cell-specific role in membrane receptor turnover associated with cholinergic synapses in the fly visual system. Our systematic functional analyses suggest that several Rabs ensure robust development and function under varying environmental conditions. The mutant and transgenic fly collections generated in this study provide a basis for further studies of Rabs during development and homeostasis in vivo.

neuroscience↗

bioRxiv2019

A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1600 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

genetics↗