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bioRxiv2021

Two timescales control the creation of large protein aggregates in cells

Protein aggregation is of particular interest due to its connection with many diseases and disorders. Many factors can alter the dynamics and result of this process, one of them being the diffusivity of the monomers and aggregates in the system. Here, we study experimentally and theoretically an aggregation process in cells, and we identify two distinct physical timescales that set the number and size of aggregates. The first timescale involves fast aggregation of small clusters freely diffusing in the cytoplasm, while, in the second one, the aggregates are larger than the pore size of the cytoplasm and thus barely diffuse, and the aggregation process is slowed down. However, the process is not entirely halted, potentially reflecting a myriad of active but random forces forces that stir the aggregates. Such slow timescale is essential to account for the experimental results of the aggregation process. These results could also have implications in other processes of spatial organization in cell biology, such as phase-separated droplets. O_TEXTBOXSIGNIFICANCEProtein aggregation is a physico-chemical process that underlies many diseases and disorders, such as Alzheimers or Huntingtons disease. Here, we study experimental and theoretically the effect of a sharp decrease of diffusivity in the aggregation dynamics, such as the one that could happen in the cell due to the presence of obstacles. We find that two different timescales are important in setting the size of large aggregates and we give an estimate of the size of the aggregate at which this dramatic change in behaviour occurs, which could not be exclusive of protein aggregation but affect many other intracellular processes. C_TEXTBOX

biophysics↗

bioRxiv2022

The microtubule-associated protein SlMAP70 interacts with SlIQD21 and regulates fruit shape formation in tomato

The shape of tomato fruits is closely correlated to microtubule organization and the activity of microtubule associated proteins (MAP), but insights into the mechanism from a cell biology perspective are still largely elusive. Analysis of tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs are highly expressed during fruit development. Among these, several members of the plant-specific MAP70 family are preferably expressed at the initiation stage of fruit development. Transgenic tomato lines overexpressing SlMAP70 produced elongated fruits that show reduced cell circularity and microtubule anisotropy, while SlMAP70 loss-of-function mutant showed an opposite effect with flatter fruits. Microtubule anisotropy of fruit endodermis cells exhibited dramatic rearrangement during tomato fruit development, and SlMAP70-1 is likely implicated in cortical microtubule organization and fruit elongation throughout this stage by interacting with SUN10/SlIQD21a. The expression of SlMAP70 (or co-expression of SlMAP70 and SUN10/SlIQD21a) induces microtubule stabilization and prevents its dynamic rearrangement, both activities are essential for fruit shape establishment after anthesis. Together, our results identify SlMAP70 as a novel regulator of fruit elongation, and demonstrate that manipulating microtubule stability and organization at the early fruit developmental stage has a strong impact on fruit shape.

plant biology↗

bioRxiv2025

Multiple Xpa In vivo crystallization routes in HEK 293 human cells

Phase changes of macromolecules in living cells gained recently a major interest in cell biology as liquid liquid phase separation or liquid solid phase transition phenomenon being observed in increasing number of biological or pathological processes. Comprehensive characterization of these phases remains challenging and requires new methodological approaches for their complete description across spatial and temporal scales. We propose a combination of imaging methods applied to a model system and report the in vivo crystallization pathways of a macromolecule from its solution to crystalline states at the cell population level down to the meso scale. Combining various live fluorescence based techniques and a high resolution cryo imaging technique within unaltered cryopreserved cells, we could described the unexpectedly wide landscape of the in vivo crystalline states of the fluorescent coral derived protein xpa, gain information of crystal growth dynamics in cellulo, and provide hypothesis of the crystal nucleation requirements of this stochastic process.

biophysics↗

bioRxiv2025

Inverted Assembly of the Lens Within Ocular Organoids Reveals Alternate Paths to Ocular Morphogenesis

The eye is a complex organ composed of two main structures - the retina and the lens. It forms by the invagination of the lens forming head surface ectoderm embedding into the forming optic cup. This "outside-in" mode of morphogenesis ensures that the light focusing lens is positioned centrally inside of the eye in the highly constrained environment of the developing embryo. Advances in stem cell biology in the last decade introduced organoids as model to study organogenesis under normal and diseased conditions. However, even though strikingly similar at some points, it remained elusive to which extend the generation of individual structural features in organoids recapitulates in vivo organogenesis. Here we describe the generation of fish ocular organoids composed of both, lens and retina, using pluripotent embryonic cells from medaka (Oryzias latipes). Formation of the organoid lens followed the key molecular features of the process in vivo, including the establishment of lens progenitor cells and their subsequent differentiation into lens fiber cells. In a process dependent on the coordinated activity of BMP and FGF signaling, lens formation in ocular organoids was marked by the expression of key genes implicated in organismal lens development. Despite adhering to the basic molecular machinery of lens formation in vivo, the morphogenesis into a spherical lens followed an "inside-out" mode. Lens progenitor cells were initially established and differentiated into a spherical lens directly inside of the retina. Subsequent displacement of the lens from the center of the organoid towards its surface ultimately led to the formation of a cup-like shaped retina with a centrally positioned lens. Our study highlights that the self-organization of the organoid can favor routes that were not selected for in the developing embryo. Those routes can lead to an alternative, though highly similar outcome with the respect to achieving specific structural features in an unconstrained, embryo-free environment.

developmental biology↗

bioRxiv2022

Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line

The tumor suppressor BRCA1-BARD1 complex functions in many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity reveal both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is essential for BRCA1-BARD1 to concentrate at both DNA damage and recombination sites in meiotic germ cells, but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRC-1 serves a structural role for DNA damage signaling and repair while BRD-1 plays an accessory role to enhance BRC-1 function. Author SummaryBRCA1-BARD1 is a E3 ubiquitin ligase, which modifies proteins by the addition of the small protein ubiquitin. While mutations that disrupt E3 ligase activity and stability of the BRCA1-BARD1 complex lead to a predisposition for breast and ovarian cancer, the specific requirement for E3 ligase activity in tumor suppression is not known. Here we probe the function of E3 ligase activity and BARD1 in the maintenance of genome integrity by engineering point mutations that disrupt E3 ligase activity in C. elegans BRCA1 as well as a null mutation in BARD1. We find that while E3 ligase activity is important for genome integrity, the complex plays additional roles besides ubiquitylating proteins. Further, our data suggest that BRCA1 is the key functional unit of the complex while BARD1 is an accessory partner that enhances BRCA1s function. These findings may help explain why there is a higher prevalence of cancer-causing mutations in BRCA1 compared to BARD1.

genetics↗

bioRxiv2024

Altered Purinergic Signaling and CD8+ T Cell Dysregulation in STAT3 GOF Syndrome

Signal transduction downstream of activating stimuli controls CD8+ T cell biology, however these external inputs can become uncoupled from transcriptional regulation in Primary Immune Regulatory Disorders (PIRD). Gain-of-function (GOF) variants in STAT3 amplify cytokine signaling and cause a severe PIRD characterized by early onset autoimmunity, lymphoproliferation, recurrent infections, and immune dysregulation. In both primary human and mouse models of STAT3 GOF, CD8+ T cells have been implicated as pathogenic drivers of autoimmunity. The molecular mechanisms by which STAT3 GOF variants drive this pathology remain unclear. We found that naive CD8+ T cells have an increased capacity for IFN-{gamma} and TNF- secretion. Given this dysregulation of CD8+ T cell function, we evaluated changes in immunoregulatory pathways and found evidence of dysregulated purinergic signaling via high dimensional immune profiling, single-cell RNA sequencing, and functional assessment. Specifically, while expression of CD39, which transforms ATP to AMP, was increased on CD8+ T cells from patients with STAT3 GOF, downstream purinergic family members, CD73 and the adenosine receptor, A2AR, were downregulated, impairing the potential to produce or sense inhibitory adenosine. Patients with STAT3 GOF can be clinically treated with JAK inhibitors and this partially normalized naive CD8+ T cell dysregulation, including aberrant cytokine production. The extent of normalization of cytokine secretion scaled with normalization of CD73 and A2AR. This suggests that a dysregulated purinergic signaling axis plays an important role in CD8+ T cell dysregulation in STAT3 GOF, which may have implications for other inflammatory disorders with amplified STAT signaling. One Sentence SummaryAmplified STAT3 signaling in CD8+ T cells from STAT3 GOF patients alters purinergic signaling and dysregulates CD8+ T cell function.

immunology↗

bioRxiv2025

A Recessive oca2 Mutation Underlies Albinism in Xiphophorus fish

Oculocutaneous albinism (OCA) is a group of genetic disorders characterized by impaired melanin production, leading to reduced pigmentation in the skin, hair, and eyes. Xiphophorus, a genus of small freshwater fish, has been a pivotal model organism in pigmentation disorder research, providing key findings in the genetic pathways governing physiological and pathological pigment cell biology. Leveraging the well-established research framework provided by Xiphophorus, we have identified a spontaneously occurring albinism phenotype in swordtail fish Xiphophorus hellerii. Genetic mapping of albino fish showed that albinism is associated with a recessive mutation in the oca2 gene. This discovery provides a novel opportunity to explore functions of oca2 gene in pigment cell differentiation, pigment synthesis, melanosome assembly and transportation function and amelanotic melanoma development.

genetics↗

bioRxiv2025

SIMPROV : Provenance capturing for simulation studies

Improving interpretability and reusability has become paramount for modeling and simulation studies. Provenance, which encompasses information about the entities, activities, and agents involved in producing a model, experiment, or data, is pivotal in achieving this goal. However, capturing provenance in simulation studies presents a tremendous challenge due to the diverse software systems employed by modelers and the various entities and activities to be considered. Existing methods only automatically capture partial provenance from individual software systems, leaving gaps in the overall story of a simulation study. To address this limitation, we introduce a lightweight method that can record the provenance of complete simulation studies by monitoring the modeler in their familiar yet heterogeneous work environment, posing as few restrictions as possible. The approach emphasizes a clear separation of concerns between provenance capturers, which collect data from the diverse software systems used, and a provenance builder, which assembles this information into a coherent provenance graph. Furthermore, we provide a web interface that enables modelers to enhance and explore their provenance graphs. We showcase the practicality of SIMPROV through two cell biological case studies. Author summaryWith the importance of simulation studies in understanding and managing complex dynamic systems, the need to support the interpretation and (re-)use of their results increases. Provenance documents how the products of a simulation study were created and what other products, agents, and activities have been involved in this process. For example, the information based on which data from which cell line a simulation model has been calibrated and validated is central to interpreting the results and assessing how the results can be reused. Therefore, some software tools offer to record provenance information. However, for complete provenance information, the tool must offer all functionalities required for a simulation study. In practice, various tools are typically used. To accommodate this situation, we propose a flexible, decentralized approach: SIMPROV. A provenance capturer - a small piece of software designed to record the modelers actions within a software tool - observes each tool used by the modeler. A central provenance builder then combines the recorded information from all captures. A capturer has to be programmed only once for each software tool used in systems biology, and modelers can work as before with minimal effort needed to record the provenance of their simulation studies automatically.

bioinformatics↗

bioRxiv2020

Trade-off between plasticity and velocity in mycelial growth

Tip-growing fungal cells maintain the cell polarity at the apical regions and elongate by de novo synthesis of cell wall. Cell polarity and growth rate affect the mycelial morphogenesis, however, it remains unclear how they act cooperatively to determine cell shape. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 m-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, the hyphae must change its morphology when they grow through the channels. Live imaging analysis revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels or lost the cell polarity after emerging from the channels. Fluorescence live imaging analysis of the Spitzenkorper, a collection of secretory vesicles and polarity-related proteins at hyphal tips, in Neurospora crassa hyphae indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, which also include phytopathogenic fungi. This comparative cell biology showed that the growth defects in the channels were not correlated with their taxonomic classification nor with the width of hyphae, but, correlated with the hyphal elongation rate. This is the first report indicating a trade-off between plasticity and velocity in mycelial growth, and serves to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

microbiology↗

bioRxiv2019

Horizontal gene transfer by natural transformation promotes both genetic and epigenetic inheritance of traits

Natural transformation (NT) is a major mechanism of horizontal gene transfer in microbial species that promotes the spread of antibiotic resistance determinants and virulence factors. Here, we develop a cell biological approach to characterize the spatial and temporal dynamics of homologous recombination during NT in Vibrio cholerae. Our results directly demonstrate (1) that transforming DNA efficiently integrates into the genome as single-stranded DNA, (2) that the resulting heteroduplexes are resolved by chromosome replication and segregation, and (3) that integrated DNA is rapidly expressed prior to cell division. We show that the combination of these properties results in the epigenetic transfer of gene products within transformed populations, which can support the transgenerational epigenetic inheritance of antibiotic resistance in both V. cholerae and Streptococcus pneumoniae. Thus, beyond the genetic acquisition of novel DNA sequences, NT can also promote the epigenetic inheritance of traits during this conserved mechanism of horizontal gene transfer.

microbiology↗

bioRxiv2023

Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cells biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD+, but not {beta}-NAD+, consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCESARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. -NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze {beta}-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a -NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1s mechanisms of action is important to provide possible therapeutic targets for COVID-19.

biochemistry↗

bioRxiv2024

IGF-1 Peptide Mimetic-functionalized Hydrogels Enhance MSC Survival and Immunomodulatory Activity

Human mesenchymal stem cells (MSCs) have demonstrated promise when delivered to damaged tissue or tissue defects for their cytokine secretion and inflammation modulation behaviors that can promote repair. Insulin-like growth factor 1 (IGF-1) has been shown to augment MSCs viability and survival and promote their secretion of cytokines that signal to endogenous cells, in the treatment of myocardial infarction, wound healing, and age-related diseases. Biomaterial cell carriers can be functionalized with growth factor-mimetic peptides (i.e. IGF-1 mimicking peptides) to enhance MSC function while promoting cell retention and minimizing off-target effects seen with direct administration of soluble growth factors. Here, we functionalized alginate hydrogels with three distinct IGF-1 peptide mimetics and the integrin-binding peptide, cyclic RGD. One IGF-1 peptide mimetic (IGM-3) in combination with integrin ligand was found to activate Akt and ERK1/2 signaling and support survival of serum-deprived MSCs. MSCs encapsulated in alginate hydrogels that presented both IGM-3 and cRGD showed a significant reduction in pro-inflammatory cytokine secretion when challenged with interleukin-1{beta}. Finally, MSCs cultured within the cRGD/IGM-3 hydrogels were able to blunt pro-inflammatory gene expression of human primary cells from degenerated intervertebral discs. These studies indicate the potential to leverage cell adhesive and IGF-1 growth factor peptide mimetics together to control therapeutic secretory behavior of MSCs. Significance StatementInsulin-like growth factor 1 (IGF-1) plays a multifaceted role in stem cell biology and may promote proliferation, survival, migration, and immunomodulation for MSCs. In this study, we functionalized alginate hydrogels with integrin-binding and IGF-1 peptide mimetics to investigate their impact on MSC function. Encapsulating MSCs in the dual peptide (cRGD/IGM-3) hydrogels enhanced their ability to reduce inflammatory cytokine production and promote anti-inflammatory gene expression in cells from degenerative human intervertebral discs exposed to proteins secreted by the MSC. This approach suggests a new way to retain and augment MSC functionality using IGF-1 peptide mimetics, offering an alternative to co-delivery of cells and high dose soluble growth factors for tissue repair and immune-system modulation.

bioengineering↗

bioRxiv2019

The Manifold Actions of Signaling Peptides on Subcellular Dynamics of a Receptor Specify Stomatal Cell Fate

Receptor endocytosis is important for signal activation and transduction. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. During plant development, the family of EPIDERMAL PATTERNING FACTOR (EPF) peptides fine-tunes stomatal patterning through ERECTA-family receptor kinases. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting stomatal differentiation, undergoes dynamic subcellular behaviors in response to different signal inputs. ERL1 is constitutively recycled, whereas its activation by EPF1 peptide induces rapid internalization to multivesicular bodies (MVB). In contrast, the dominant-negative ERL1 resides predominantly in plasma membrane. The co-receptor, TOO MANY MOUTHS (TMM), is essential for EPF1-induced ERL1 internalization but dispensable for EPFL6-induced ERL1 internalization. The peptide antagonist of EPF1, Stomagen/EPFL9, triggers retention of ERL1 in the endoplasmic reticulum. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

plant biology↗

bioRxiv2021

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology - including the tendency to clump, and "differential" culturability - and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis BCG time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.

microbiology↗

bioRxiv2023

Divergent evolution of sleep functions

Most living organisms have evolved to synchronize their biological activities with the earths rotation, a daily regulation of biology and behaviour controlled by an evolutionary conserved molecular machinery known as the circadian clock. For most animals, circadian mechanisms are meant to maximize their exposure to positive activities (e.g.: social interactions, mating, feeding - generally during the day) and minimize their exposure to peril (e.g.: predation, weather, darkness - generally during the night1). On top of circadian regulation, some behaviours also feature a second layer of homeostatic control acting as a fail-safe to ensure important activities are not ignored. Sleep is one of these behaviours: largely controlled by the circadian clock for its baseline appearance, it is at the same time modulated by a - poorly understood - homeostatic regulator ensuring animals obey their species-specific amount of daily sleep2. An evolutionary conserved homeostatic control is often considered the main evidence for a core biological function of sleep beyond the trivial one (that is: keeping us out of trouble by limiting our energy expenditure and exposure to danger3,4) and it is hypothesized that sleep evolved around this mysterious basic biological function. Here we characterize sleep regulation in a group of seven species of the Drosophila genus at key evolutionary distances and representing a variety of ecological niche adaptations. We show that the spontaneous circadian-driven aspects of sleep are conserved among all species but the homeostatic regulation, unexpectedly, is not. We uncover differences in the behavioural, cell-biological and neuro-pharmacological aspects of sleep and suggest that, in Drosophilids, sleep primarily evolved to satisfy a circadian role, keeping animals immobile during dangerous hours of the day. The homeostatic functions of sleep evolved independently, in a species-specific fashion, and are not conserved.

evolutionary biology↗

bioRxiv2024

The Roboscope: Smart and Fast Microscopy for Generic Event-Driven Acquisition

Automation of fluorescence microscopy is a challenge for capturing rare or transient events in biology and medicine. It relies on smart devices that integrate and interpret the observed data, and react to the targeted biological event. We report on the Roboscope, a novel autonomous microscope combining sequence interruption and deep learning integration, allowing generic event-driven acquisitions. This system distinguishes itself by its adaptability to various experiments, quick capture of dynamic events, and minimal data greediness - training with less than 100 images per class. The Roboscopes capability is demonstrated in non-synchronized cells by capturing the metaphase, a 20-minute event happening once per day or less. Conversely, double thymidine-block synchronisation, despite occurring during DNA replication, may perturb mitotic-spindle mechanics. The Roboscopes versatility and efficiency offer significant advancements to tackle the current challenges of cell biology, spreading out advanced microscopy methods to fundamental research as well as high content screening and precision medicine.

bioengineering↗

bioRxiv2024

Phenotypic plasticity in bacterial elongation among closely related species

Cell elongation is a fundamental component of the bacterial cell cycle and has been studied over many decades, in part owing to its mechanisms being a target of numerous antibiotic classes. While several distinct modes of cell elongation have been described, these studies have largely relied on a handful of model bacterial species. Therefore, we have a limited view of the diversity of cell elongation approaches that are employed by bacteria, and how these vary in response to evolutionary and environmental influences. Here, by employing fluorescent D-amino acids (FDAAs) to track the spatiotemporal dynamics of elongation, we reveal previously unsuspected diversity of elongation modes among closely related species of the Caulobacteraceae, with species-specific combinations of dispersed, midcell and polar elongation that can be either unidirectional or bidirectional. Using genetic, cell biology, and phylogenetic approaches, we demonstrate that evolution of unidirectional-midcell elongation is accompanied by changes in the localization pattern of the peptidoglycan synthase PBP2 and infer that elongation complexes display a high degree of phenotypic plasticity, both among the Caulobacteraceae and more widely among the Alphaproteobacteria. Demonstration that even closely related bacterial species employ highly distinct mechanisms of cell elongation reshapes our understanding of the evolution and regulation of bacterial cell growth, with broad implications for bacterial morphology, adaptation, and antibiotic resistance.

microbiology↗

bioRxiv2025

Effective Tubulin Degradation by Rationally Designed Proteolysis Targeting Chimeras

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that induce the degradation of proteins of interest (POIs) via the ubiquitin-proteasome pathway by recruiting E3 ligases to form a ternary complex with the POI. In this study, we rationally designed and synthesized PROTACs targeting the {beta}-tubulin heterodimer, the building block of microtubules (MTs) that are essential for numerous cellular functions and represent important therapeutic targets in cancer and neurodegenerative diseases. Maytansinol, a known MT-destabilising agent, was selected as the POI ligand, functionalised and conjugated to linkers bearing cereblon or Von Hippel-Lindau ligands as E3 ligase recruiters. Four compounds were synthesized and characterized through structural, biophysical and cell biology studies to evaluate their ability to form degradation-prone tubulin-PROTAC-E3 ligase ternary complexes. We confirmed that the PROTACs effectively bind tubulin and recruit E3 ligases. Remarkably, two PROTACs exhibited cellular degradation activity, representing an important advancement in chemically inducing tubulin-E3-ligase interactions. This work integrates rational design, biophysical and structural validation, and cell-based studies to establish a robust framework for developing tubulin-targeting PROTACs, offering significant implications for basic research and therapeutic developments.

biochemistry↗