ARL13B controls male reproductive tract physiology through primary and Motile Cilia.

ARL13B is a small regulatory GTPase that controls ciliary membrane composition in both motile cilia and non-motile primary cilia. In this study, we investigated the role of ARL13B in the efferent ductules, tubules of the male reproductive tract essential to male fertility in which primary and motile cilia co-exist. We used a genetically engineered mouse model to delete Arl13b in efferent ductule epithelial cells, resulting in compromised primary and motile cilia architecture and functions. This deletion led to disturbances in reabsorptive/secretory processes and triggered an inflammatory response. The observed male reproductive phenotype showed significant variability linked to partial infertility, highlighting the importance of ARL13B in maintaining a proper physiological balance in these small ducts. These results emphasize the dual role of both motile and primary cilia functions in regulating efferent duct homeostasis, offering deeper insights into how cilia related diseases affect the male reproductive system.

Coordination of force-generating actin-based modules stabilizes and remodels membranes in vivo.

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

The axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish.

Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the ALO is readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.

Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observe that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cellular analyses, we discover that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr forms a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhances Plk4's functionality by promoting its relocalization to the procentriole assembly and induces centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogates Vpr's capacity to induce these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induces multiple centrosomes and aneuploidy in human primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling.

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

Airway Cells 3D Reconstruction via Manual and Machine-Learning Aided Segmentation of Volume EM Datasets.

Volume electron microscopy (vEM) is a high-resolution imaging technique capable of revealing the 3D structure of cells, tissues, and model organisms. This imaging modality is gaining prominence due to its ability to provide a comprehensive view of cells at the nanometer scale. The visualization and quantitative analysis of individual subcellular structures however requires segmentation of each 2D electron micrograph slice of the 3D vEM dataset; this process is extremely laborious de facto limiting its applications and throughput. To address these limitations, deep learning approaches have been recently developed including Empanada-Napari plugin, an open-source tool for automated segmentation based on a Panoptic-DeepLab (PDL) architecture. In this chapter, we provide a step-by-step protocol describing the process of manual segmentation using 3dMOD within the IMOD package and the process of automated segmentation using Empanada-Napari plugins for the 3D reconstruction of airway cellular structures.

CDK2 inhibition disorders centrosome stoichiometry and alters cellular outcomes in aneuploid cancer cells.

Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.

Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observed that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cell biology analyses, we discovered that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr formed a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhanced Plk4's functionality by promoting its relocalization to the procentriole assembly and induced centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogated Vpr's capacity to induce all these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induced multiple centrosomes and aneuploidy in primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling.

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal (PP) and pericentral (PC) axis. How these mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combined intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We found that PP and PC mitochondria are morphologically and functionally distinct; beta-oxidation was elevated in PP regions, while lipid synthesis was predominant in the PC mitochondria. In addition, comparative phosphoproteomics revealed spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifted mitochondrial phenotypes in the PP and PC regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

Instance segmentation of mitochondria in electron microscopy images with a generalist deep learning model trained on a diverse dataset.

Mitochondria are extremely pleomorphic organelles. Automatically annotating each one accurately and precisely in any 2D or volume electron microscopy (EM) image is an unsolved computational challenge. Current deep learning-based approaches train models on images that provide limited cellular contexts, precluding generality. To address this, we amassed a highly heterogeneous ∼1.5 × 106 image 2D unlabeled cellular EM dataset and segmented ∼135,000 mitochondrial instances therein. MitoNet, a model trained on these resources, performs well on challenging benchmarks and on previously unseen volume EM datasets containing tens of thousands of mitochondria. We release a Python package and napari plugin, empanada, to rapidly run inference, visualize, and proofread instance segmentations. A record of this paper's transparent peer review process is included in the supplemental information.

Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms.

The histone-like protein HU plays a diverse role in bacterial physiology from the maintenance of chromosome structure to the regulation of gene transcription. HU binds DNA in a sequence-non-specific manner via two distinct binding modes: (i) random binding to any DNA through ionic bonds between surface-exposed lysine residues (K3, K18, and K83) and phosphate backbone (non-specific); (ii) preferential binding to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that induce and/or stabilize the kinks. First, we show here that the P63-mediated structure-specific binding also requires the three lysine residues, which are needed for a non-specific binding. Second, we demonstrate that substituting P63 to alanine in HU had no impact on non-specific binding but caused differential transcription of diverse genes previously shown to be regulated by HU, such as those associated with the organonitrogen compound biosynthetic process, galactose metabolism, ribosome biogenesis, and cell adhesion. The structure-specific binding also helps create DNA supercoiling, which, in turn, may influence directly or indirectly the transcription of other genes. Our previous and current studies show that non-specific and structure-specific HU binding appear to have separate functions- nucleoid architecture and transcription regulation- which may be true in other DNA-binding proteins.

Spatial and Temporal Coordination of Force-generating Actin-based Modules Drives Membrane Remodeling In Vivo.

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that: a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane; and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

A versatile enhanced freeze-substitution protocol for volume electron microscopy.

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (Hordeum vulgare), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia.

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell's cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.

Embedding of HIV Egress within Cortical F-Actin.

F-Actin remodeling is important for the spread of HIV via cell-cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.