Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, Microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacteria morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacteria images in TrackMate, (2) a TrackMate-Helper extension for parameter optimization, and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-24-INBS-0005 FBI BIOGEN, ANR-10-PATH-003 HELDIVPAT, ANR-10-LBX-62 IBEID, ANR-16-CONV-0005 INCEPTION, ANR-17-EURE-0012 EURIP European Research Council, Destop ERC, PGNfromSHAPEtoVIR, FP7-202283, IMI 2 Joint Undertaking (JU) under Grant Agreement No 853989 Fondation pour la Recherche Médicale, EQU202403018034, FDT202504020138 Gates Foundation, IMI 2 Joint Undertaking (JU) under Grant Agreement No 853989
The human cell cycle is a highly regulated process that integrates multiple signaling pathways and checkpoints to ensure faithful genome duplication and cell division. Disruptions in these regulatory networks contribute to a wide range of diseases. Here, we present a novel, updateable computational model of the full human cell cycle that shows sustained oscillations over time and reproduces experimental perturbations. We used a hybrid framework combining mass action and Michaelis–Menten kinetics, incorporating the synthesis, degradation, and regulation of key cell cycle proteins and protein complexes. It consists of 63 distinct biochemical species, interacting through 41 major reactions, and functioning through 63 ODEs. The model is built upon a modular framework, structured around the core regulatory networks of the G1, S, G2, and M phases. Due to its complexity, we determined parameter sets that met strict criteria, namely event timing, comparable concentrations, and continuous cycling. We validated the model’s behavior by reproducing canonical check-point responses, including mitogen dependence and the DNA damage response, both of which produced reversible and robust cell cycle arrests. Importantly, the model was trained and calibrated using in vitro data from human U251-MG glioma cells expressing the FastFUCCI cell cycle re-porter. We quantitatively aligned the simulated and experimentally determined phase durations and cell doubling times. Next, we experimentally tested and refined model parameters by using abemaciclib-mediated inhibition of CDK4 and volasertib-mediated inhibition of PLK1. In vitro and in silico data show dose-dependent G1 arrest by abemaciclib and dose-dependent mitotic arrest by volasertib. Finally, we demonstrated that the model predicts changes in cell proliferation over a wide range of drug concentrations and combinations. Overall, our work establishes a robust, data-driven computational model for systems-level analysis of the human cell cycle and its disruption by therapeutic perturbations. ### Competing Interest Statement The authors have declared no competing interest.
The meniscus is a fibrocartilaginous tissue critical for knee stability and load distribution, but its poor healing capacity makes regeneration after injury a major clinical challenge. Extracellular vesicles (EVs) are emerging as promising cell-free therapeutics for tissue regeneration. However, their clinical translation remains limited by low yield and variable bioactivity. Here, we investigated how dynamic mechanical loading regulates the production, composition, and function of meniscus fibrochondrocyte-derived EVs (MFC-EVs). Using a custom bioreactor system, cells were subjected to physiologically relevant cyclic tensile loading. Mechanical stimulation significantly increased EV production and secretion, likely through the ESCRT-independent pathway, without altering vesicle size or tetraspanin expression. Functionally, “mechanically primed” EVs enhanced aggrecan expression in recipient mesenchymal stromal cells (MSCs), mirroring the loading-response phenotype of their source cells. Proteomic profiling identified 380 unique proteins across all EV groups, with loaded EVs enriched in extracellular matrix- and cytoskeleton-associated proteins, as well as pathways related to tissue morphogenesis, cell migration, and cartilage development. Together, these findings demonstrate that physiologic mechanical loading enhances both the yield and regenerative potency of MFC-EVs by enriching their cargo with matrix- and development-associated proteins, providing a scalable and biologically inspired approach for engineering high-efficacy EV therapeutics for meniscus repair and musculoskeletal regeneration. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88 U.S. National Science Foundation, https://ror.org/021nxhr62
Coordinated cell polarity and force-responsive protein localization are essential for tissue morphogenesis, yet how embryonic cells sense forces and respond to mechanical cues remains a challenging question. Afadin- and alpha-actinin-binding protein (ADIP) has been implicated in microtubule minus-end anchoring, centrosome maturation and ciliogenesis. ADIP is also proposed to associate with the actomyosin cortex and regulate collective cell migration. ADIP behaves as a mechanosensitive planar cell polarity (PCP) protein when overexpressed in Xenopus embryos, but the distribution and regulation of endogenous ADIP has been unknown. Here we show that ADIP is present in early ectoderm as randomly distributed puncta that rapidly reorganize and polarize during epithelial wound repair. Endogenous ADIP also becomes enriched and planar polarized in the anterior neural plate towards the midline, consistent with its regulation by mechanical forces that operate during neural tube closure. ADIP polarization is attenuated by depletion of the core PCP component Diversin/Ankrd6, in agreement with the proposed interaction between the two proteins during PCP establishment. Finally, pharmacological disruption of microtubules, F-actin, and nonmuscle myosin II eliminates ADIP polarization in the neuroectoderm, indicating roles for microtubules and actomyosin networks in PCP. Together, these findings suggest that endogenous ADIP senses mechanical cues via the cytoskeletal machinery and functions in the context-dependent manner to control collective cell behaviors during vertebrate morphogenesis. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R35GM122492
The unicellular parasite Trypanosoma brucei assembles a motile flagellum that is required for locomotion, cell division plane placement, and cell-cell communication. Inheritance of the flagellum during the cell cycle relies on the faithful duplication/segregation of multiple flagellum-associated cytoskeletal structures, including a centrin-marked, bar-shaped structure termed centrin arm, which also determines the biogenesis site for Golgi. Biogenesis of the centrin arm requires the Polo-like kinase homolog TbPLK and the orphan kinesin KIN-G, but the mechanistic role of TbPLK in centrin arm biogenesis remains elusive. Here we report that TbPLK phosphorylates KIN-G, disrupts its microtubule-binding activity, and negatively regulates its function in the procyclic form of T. brucei . TbPLK phosphorylates KIN-G in vitro at multiple residues, some of which are phosphorylated in vivo in T. brucei , including the Thr301 residue within one of the microtubule-binding motifs of the kinesin motor domain. Phosphorylation of Thr301 by TbPLK inhibits the microtubule-binding activity of KIN-G in vitro , and expression of a Thr301 phospho-mimic mutant in T. brucei disrupts centrin arm integrity, thereby impairing Golgi biogenesis, flagellum attachment zone elongation, flagellum positioning, and cell division plane placement. Therefore, TbPLK negatively regulates KIN-G activity by phosphorylating Thr301, and dephosphorylation of Thr301 is required for KIN-G to fulfill its cellular function in promoting centrin arm biogenesis. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, AI118736, AI101437
A complete transcriptome atlas of every cell type of a vertebrate could promote understanding of animal cell-type composition, organization, and evolution. The miniaturized, transparent, and regenerative teleost Danionella cerebrum brings whole-organism single-cell profiling experiments within experimental reach for adult vertebrate biology. We performed regionally stratified single-cell RNA sequencing experiments in adult Danionella to profile cells across the whole body and mapped cell types and gene expression spatially at single-cell resolution using whole-animal spatial transcriptomics. We delineated spatially distinct neural progenitor and neuronal cell types across the adult nervous system based on their regional gene expression signatures. The body-wide atlas uncovered paedomorphic features, allowed elucidation of cell types likely to harbor adult positional information, and revealed constitutive expression of conserved body region and appendage specification programs in adult connective tissue. Comparative analyses revealed conserved neural cell types over a large evolutionary distance, and neural regeneration datasets uncovered temporally resolved expression dynamics in neural progenitors for telencephalon regeneration. This whole-vertebrate transcriptome atlas yields a comprehensive resource for myriad questions in biology and neuroscience. ### Competing Interest Statement The authors have declared no competing interest. Howard Hughes Medical Institute, https://ror.org/006w34k90
Rxiv mechanobio