Single-cell protein analysis reveals metastable states during the transition to a sensory organ fate

Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make to undergo sensory organ differentiation has been sucessfully described as such. We have extended these studies by focusing on the Senseless protein, which orchestrates the sensory fate transition. Wing cells contain intermediate Senseless numbers prior to their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are not consistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the four states are stable over time, the number of molecules in each state vary with time. Remarkably, the fold-change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.

Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26

The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila. Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression, however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. Here, we present the crystal structure of the Mei-P26 NHL domain at 1.6 Å resolution and identify key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, we identify mRNA targets of Mei-P26 in cultured Drosophila cells and show that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. Our work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins.

Recruitment of Peroxin14 to lipid droplets affects triglyceride storage in Drosophila

The activity of multiple organelles must be coordinated to ensure cellular lipid homeostasis. This includes the peroxisomes which metabolise certain lipids and lipid droplets which act as neutral lipid storage centres. Direct organellar contact between peroxisomes and lipid droplets has been observed, and interaction between proteins associated with the membranes of these organelles has been shown, but the functional role of these interactions is not clear. In Drosophila cells, we identified a novel localization of a subset of three transmembrane Peroxin proteins (Peroxin3, Peroxin13, and Peroxin14), normally required for peroxisome biogenesis, to newly formed lipid droplets. This event was not linked to significant changes in peroxisome size or number, nor was recruitment of other Peroxin proteins or mature peroxisomes observed. The presence of these Peroxin proteins at lipid droplets influences their function as changes in the relative levels of Peroxin14 associated with the lipid droplet surface directly affected the presence of regulatory perilipin and lipases with corresponding effects on triglyceride storage.

Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status

Cell competition induces the elimination of less-fit loser cells by fitter winner cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are eliminated via cell competition by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also relies on the activities of the transcription factors Irbp18 and Xrp1, however how these contribute to the loser status is partially understood. Here, we show that Irbp18 and Xrp1 induce the loser status by promoting proteotoxic stress. We find that Xrp1 is necessary for Rp/+ -induced proteotoxic stress and is sufficient to induce proteotoxic stress in otherwise wild-type cells. Xrp1 is also induced downstream of proteotoxic stress and required for the competitive elimination of cells suffering from proteotoxic stress. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

Visual4DTracker: a tool to interact with 3D + t image stacks

Background Biological phenomena usually evolves over time and recent advances in high-throughput microscopy have made possible to collect multiple 3D images over time, generating $$3D+t$$ 3 D + t (or 4D) datasets. To extract useful information there is the need to extract spatial and temporal data on the particles that are in the images, but particle tracking and feature extraction need some kind of assistance. Results This manuscript introduces our new freely downloadable toolbox, the Visual4DTracker. It is a MATLAB package implementing several useful functionalities to navigate, analyse and proof-read the track of each particle detected in any $$3D+t$$ 3 D + t stack. Furthermore, it allows users to proof-read and to evaluate the traces with respect to a given gold standard. The Visual4DTracker toolbox permits the users to visualize and save all the generated results through a user-friendly graphical user interface. This tool has been successfully used in three applicative examples. The first processes synthetic data to show all the software functionalities. The second shows how to process a 4D image stack showing the time-lapse growth of Drosophila cells in an embryo. The third example presents the quantitative analysis of insulin granules in living beta-cells, showing that such particles have two main dynamics that coexist inside the cells. Conclusions Visual4DTracker is a software package for MATLAB to visualize, handle and manually track $$3D+t$$ 3 D + t stacks of microscopy images containing objects such cells, granules, etc.. With its unique set of functions, it remarkably permits the user to analyze and proof-read 4D data in a friendly 3D fashion. The tool is freely available at https://drive.google.com/drive/folders/19AEn0TqP-2B8Z10kOavEAopTUxsKUV73?usp=sharing