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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Microvilli-derived Extracellular Vesicles Govern Morphogenesis in Drosophila wing epithelium

The regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo.

cell biology↗

bioRxiv2024

biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

cell biology↗

bioRxiv2021

Deformation-induced actuation of cells in asymmetric periodic flow fields

Analyzing and sorting particles and/or biological cells in microfluidic devices is a topical problem in soft-matter and biomedical physics. An easy and rapid screening of the deformation of individual cells in constricted microfluidic channels allows, for example, the identification of sick or aberrant cells with altered mechanical properties, even in vast cell ensembles. The subsequently desired softness-specific segregation of cells is, however, still a major challenge. Moreover, aiming at an intrinsic and unsupervised approach raises a very general question: How can one achieve a softnessdependent net migration of particles in a microfluidic channel? Here we show that this is possible by exploiting a deformation-induced actuation of soft cells in asymmetric periodic flow fields in which rigid beads show a vanishing net drift.

biophysics↗

bioRxiv2018

Quantitative Analysis of Interactive Behavior of Mitochondria and Lysosomes Using Structured Illumination Microscopy

Super-resolution optical microscopy has extended the spatial resolution of cell biology from the cellular level to the nanoscale, enabling the observation of the interactive behavior of single mitochondria and lysosomes. Quantitative parametrization of interaction between mitochondria and lysosomes under super-resolution optical microscopy, however, is currently unavailable, which has severely limited our understanding of the molecular machinery underlying mitochondrial functionality. Here, we introduce an M-value to quantitatively investigate mitochondria and lysosome contact (MLC) and mitophagy under structured illumination microscopy. We found that the M-value for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 to 1.0. This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes.

cell biology↗

bioRxiv2023

The image-based ultrasonic cell shaking test

Mechanical signals play a vital role in cell biology and is a vast area of research. Thus, there is motivation to understand cell deformation and mechanobiological responses. However, the ability to controllably deform cells in the ultrasonic regime and test their response is a noted challenge throughout the literature. Quantifying and eliciting an appropriate stimulus has proven to be difficult, resulting in methods that are either too aggressive or oversimplified. Furthermore, the ability to gain a real-time insight into cell deformation and link this with the biological response is yet to be achieved. One application of this understanding is in ultrasonic surgical cutting, which is a promising alternative to traditional methods, but with little understanding of its effect on cells. Here we present the image based ultrasonic cell shaking test, a novel method that enables controllable loading of cells and quantification of their response to ultrasonic vibrations. Practically, this involves seeding cells on a substrate that resonates at ultrasonic frequencies and transfers the deformation to the cells. This is then incorporated into microscopic imaging techniques to obtain high-speed images of ultrasonic cell deformation that can be analysed using digital image correlation techniques. Cells can then be extracted after excitation to undergo analysis to understand the biological response to the deformation. This method could aid in understanding the effects of ultrasonic stimulation on cells and how activated mechanobiological pathways result in physical and biochemical responses.

bioengineering↗

bioRxiv2018

A meta-analysis of Planarian: an animal model useful to understand the effect of magnetic field

The planarians research since the first description until nowadays has a broad spectrum of topics. The present paper deals on a meta-analysis which identified as a problem the lack of consolidate information and data analysis about the use of planarian as animal model, and specifically the use of magnetic field in regeneration experiments. To conduct the meta-analysis 1255 papers published since 1955 to 2017 were analysed, identifying the Stem cell biology and regeneration as the most published topic, with 276 papers, followed by molecular and cellular analyses (238), genetics (175), inter and transdisciplinary research (107), ecotoxicological evaluations (102), animal model (67), ecological and biological studies (56), magnetic field (57), developmental biology (32) and RNA regulation (31).Other statistics and metrics indicators were taken in to account like total of papers and distribution per year, distribution of paper by journals and selection of main journals according to the number of published papers, most cited papers, authors and countries and distribution of papers by countries. Finally, were analysed those papers with Planarians research using magnetic field, all of them published during the last three years. It was an evidence that this topic is becoming a trending with rising interest, being the most reported species to study the magnetic field effects Dugesia spp. and Girardia sp.\n\nSummary statementThis work presents a meta-analysis which allows consolidating information and a better understanding of trends in Planarian researches, emphasizing in the use of magnetic field.

cell biology↗

bioRxiv2018

Identification and characterization of conserved and divergent genes encoding the nuclear envelope LINC complex in maize (Zea mays L.).

The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture, and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. Using bioinformatic, biochemical, and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with ZmSUN2 in vivo. FRAP experiments using domain-deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.\n\nSUMMARY STATEMENTGenes encoding maize candidates for the core LINC and associated complex proteins have been comprehensively identified with functional validation by one or more assays for several of the KASH genes.

cell biology↗

bioRxiv2025

Core Clock Protein Subcellular Dynamics Coordinate Local and Global Circadian Control in Syncytia.

Circadian rhythms are pervasive among eukaryotes, and the underlying clocks share a common regulatory architecture - a negative feedback loop. A wealth of genetic and biochemical data underpin current perceptions of circadian oscillators but aspects of their cell biology remain cryptic, especially in syncytial systems. We employed novel microfluidic systems and a blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles, revealing remarkable subcellular and subnuclear dynamics of clock proteins and providing insights into spatiotemporal regulation in a multinucleated system. Despite heterogeneity of clock gene (frq) expression, we find robust cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form distinct, small, highly dynamic nuclear bodies that persist throughout the cycle, occasionally co-localizing for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how separate nuclear clocks ensure synchronous regulation of cellular activities across a macroscopic syncytium.

cell biology↗

bioRxiv2026

Analysis of the assembly, stabilization and maturation of the multiphasic TAZ biomolecular condensates

Phase separation is an important mechanism ensuring efficient regulation and function in Hippo signaling. Particularly, phase separation of nuclear TAZ has been demonstrated to be essential for its activity. However, the mechanisms of TAZ condensate assembly and maturation are yet undefined. Here we explored these mechanisms using FRAP with two laser beam sizes complemented by microscopy and cell biology approaches. We show that TAZ condensates are multiphasic, with a more stable core and labile periphery. TAZ initially forms small nascent clusters, likely via self-nucleation through the CC domain. These gradually mature into larger condensates through interaction with additional proteins via the WW domain. The condensates are further stabilized/activated by interaction with transcription factors and complexes including TEAD4 and P-TEFb. Of note, the ability of TAZ to form mature condensates is essential for its activities in cellular morphogenesis and tumorigenesis. Our study presents detailed mechanistic analysis of TAZ phase separation, revealing a highly dynamic nature of TAZ condensate maturation and activation. TeaserTAZ condensates grow from nascent clusters into mature condensates by interactions with transcription factors and complexes.

cell biology↗

bioRxiv2022

Re-direction of phagosomes to the recycling expulsion pathway by a fungal pathogen

The analysis of host-pathogen interactions bears the potential to discover novel pathogenicity mechanisms and to obtain novel insights into basic mechanisms of cell biology. Here, we obtained unprecedented insight into both. We discovered that the HscA protein on the conidial surface of the clinically important human-pathogenic fungus Aspergillus fumigatus acts as an effector protein. It inhibits phagosome maturation and reprograms phagosomes for expulsion of conidia. HscA anchors the human p11 protein to phagosomes. p11 is a decisive factor for targeting phagosomes either to the degradative or secretory pathway. The relevance of our findings is indicated by the identification of an SNP in the non-coding region of the human p11 gene that affects its translation and is associated with heightened susceptibility to invasive pulmonary aspergillosis.

cell biology↗

bioRxiv2020

PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 controls the transport of E-cadherin

ADP-ribosylation is a post-translational modification involved in physiological and pathological events catalyzed by Poly-ADP-Ribosyl-Polymerase (PARP) enzymes. Substrates of this reaction have been identified by mass-spectrometry, but the definition of PARPs-regulated cellular functions remains scarce. Here, we have analyzed the control of intracellular membrane traffic by the mono-ADP-ribosyl-transferase PARP12, motivated by its localization at the trans-Golgi network. By using bioinformatics, mutagenesis and cell biology approaches we identified Golgin-97, a protein regulating exocytosis, as a PARP12-specific substrate. Mono-ADP-ribosylation of Golgin-97 residues E558-E559-E565 is required for supporting traffic from the trans-Golgi network to the plasma membrane. This step is halted when PARP12 is deleted or when the Golgin-97 ADP-ribosylation-defective mutant is expressed. Under these conditions E-cadherin, whose transport is controlled by Golgin-97, does not reach the plasma membrane but accumulates in a trans-Golgi proximal compartment. Thus, we demonstrate that the ADP-ribosylation of Golgin-97 is required for E-cadherin exocytosis and thus this event may regulate the sorting of exocytic carriers as well as epithelial-to-mesenchymal transition.

cell biology↗

bioRxiv2021

Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation

The general mechanisms by which ESCRTs are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin is critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane, and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT-assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions. One-Sentence SummaryCondensation of multivalent ESCRT-0/polyubiquitin assemblies organizes cargo sorting reactions at lysosomes

cell biology↗

bioRxiv2025

Dynamic Organellar Mapping in yeast reveals extensive protein localization changes during ER stress

Sophisticated techniques are available for systematic studies of yeast cell biology. However, it remains challenging to investigate protein subcellular localization changes on a proteome-wide scale. Here, we apply Dynamic Organellar Maps (DOMs) by label-free mass spectrometry to detect localization changes of native, untagged proteins during endoplasmic reticulum (ER) stress. We find that hundreds of proteins shift between cellular compartments. For example, we show that numerous secretory pathway proteins accumulate in the ER, thus defining the extent and selectivity of ER retention of misfolded proteins. Furthermore, we identify candidate cargo proteins of the ER reflux pathway, determine constituents of reticulon clusters that segregate from the remainder of the ER and provide evidence for altered nuclear pore complex composition and nuclear import. These findings uncover protein relocalization as a major aspect of cellular reorganization during ER stress and establish DOMs as a powerful discovery tool in yeast.

cell biology↗

bioRxiv2017

Local Control of Intracellular Microtubule Dynamics by End Binding Protein 1 (EB1) Photo-Dissociation

Dynamic remodelling of the microtubule cytoskeleton and local interactions with intracellular targets are central to many polarized cell biological processes, an idea first formalized as search-and-capture hypothesis three decades ago1. However, because of the rapid timescale of microtubule polymerization dynamics, it is difficult to directly ask how, when and where dynamic microtubules participate in specific biological processes. Here, we employ a blue light-sensitive interaction with the oat phototropin LOV2 domain2 to generate a photo-inactivated variant of the microtubule end-binding protein EB1, a small adaptor that is central to the interaction of functionally and structurally diverse proteins with growing microtubule ends3,4, that can replace endogenous EB1 function. Acute and reversible blue light-mediated n-EB1 photo-dissociation allows spatially and temporally precise control of intracellular microtubule polymerization dynamics. In addition to demonstrating that neither the GTP cap nor the MT polymerase CKAP5 are sufficient to sustain persistent MT polymerization at physiological growth rates, our data illustrate accurate subcellular control of a freely diffusible, cytoplasmic protein at the second and micrometer scale. This novel design may serve as a template for precise control of many other intracellular protein activities.

cell biology↗

bioRxiv2024

Improved whole-mount immunofluorescence protocol for consistent and robust labeling of adult Drosophila melanogaster adipose tissue

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophilas robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body. SUMMARY STATEMENTOptimization of adult Drosophila fat body fluorescence microscopy via modifications of tissue handling, fixation, and permeabilization steps in a whole mount immunolabeling protocol.

cell biology↗

bioRxiv2023

Identification of potential selective autophagy receptors from protein-content profiling of autophagosomes

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts e.g., under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR-docking site (LDS) and Ubiquitin-interacting motif (UIM)-UIM-docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.

cell biology↗

bioRxiv2024

Mosquito Tissue Ultrastructure-Expansion Microscopy (MoTissU-ExM) enables ultrastructural and anatomical analysis of malaria parasites and their mosquito.

Study of malaria parasite cell biology is challenged by their small size, which can make visualisation of individual organelles difficult or impossible using conventional light microscopy. In recent years, the [fi]eld has attempted to overcome this challenge through the application of ultrastructure expansion microscopy (U-ExM), which physically expands a biological sample approximately 4.5-fold. To date, U-ExM has mostly been used to visualise blood-stage parasites and used exclusively on parasites in vitro. Here we develop Mosquito Tissue U-ExM (MoTissU-ExM), a method for preparing dissected mosquito salivary glands and midguts by U-ExM. MoTissU-ExM preserves both host and parasite ultrastructure, enabling visualisation of oocysts and sporozoites in situ. We validate that MoTissU-ExM samples expand as expected, provide a direct comparison of the same dissected tissues before and after MoTissU-ExM, and highlight some of the key host and parasite structures that can be visualised following MoTissU-ExM. Finally, we provide a point-by-point protocol for how to perform MoTissU-ExM, along with details on how best to image the expanded tissues, and how to troubleshoot common issues.

cell biology↗

bioRxiv2019

Amyloid-like aggregates cause lysosomal defects in neurons via gain-of-function toxicity

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like {beta}-sheet proteins ({beta} proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, {beta} proteins interact with and sequester AP-31, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-31 expression ameliorates neurotoxicity caused by {beta} proteins. Altogether, our results highlight the link between protein aggregation and neurotoxicity, pointing to lysosomes as particularly vulnerable organelles.

cell biology↗