The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the ...discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher (around 95,000 lacunae per mm.sup.3) than in the mastoid of the temporal bone (around 28,000 lacunae per mm.sup.3 ), as well as the cortical bone of the femur (around 27,000 lacunae per mm.sup.3). The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also show that the pig petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore confirm and significantly expand upon previous observations of osteocytic lacuna concentrations in the petrous bone, supporting the notion that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains at least three times more DNA than other bones. Thus during diagenesis more DNA is likely to be preserved in the petrous bone compared to other bones.
Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional ...changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
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•Resource of SG spatiotemporal proteomic landscape by multi-bait proximity labeling•Distinct substructures and >100 novel SG proteins•Disassembly-engaged proteins (DEPs) coordinate SG disassembly•SUMOylation controls SG dynamics and is dysregulated in models of C9orf72-ALS
Marmor-Kollet et al. utilize proximity proteomics to identify stress granule composition, internal organization, and mechanisms of regulated disassembly in health and disease. Disassembly-engaged proteins (DEPs), including SUMO-conjugating enzymes, are critical for normal stress granule disassembly and dysregulated in ALS-like conditions.
The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D ...Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.
Germinal centers (GCs) are sites wherein B cells proliferate and mutate their immunoglobulins in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). Here, we mapped the ...location of single B cells in the context of intact lymph nodes (LNs) throughout the GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we demonstrated that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response.
Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and ...subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.
Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after ...completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.
Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are ...patterned is unknown. Here, we identify components of the genetic program that control the patterning of
/
superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the
/
progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified
as a global regulator of superstructure patterning, whereas
,
,
and
act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in
and
compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.
Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules ...required for axon pruning of mushroom body (MB) γ neurons in Drosophila, we profiled astrocytes before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, we identified 12 astrocytic genes that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factor β (TGF-β) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, our findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting our understanding of neuron-glia interactions during remodeling.
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•Larval and adult astrocytic transcriptome reveals potential neuro-glia interactions•Astrocytic F-actin regulating genes are required for neuronal remodeling•Arpc1-KD astrocytes show reduced infiltration into the axon bundle before pruning•Interplay between astrocytic infiltration and axonal adhesion regulate axon pruning
Marmor-Kollet et al. profile Drosophila larval and adult astrocytes to identify astrocytic genes potentially important for neuronal remodeling of mushroom body neurons. Astrocytic Arpc1, a branched F-actin nucleator, is required for infiltration into the axon bundle prior to pruning. The interplay between astrocytic infiltration and axonal adhesion governs axon pruning.
The p53 tumor suppressor exerts a variety of cell-autonomous effects that are aimed to thwart tumor development. In addition, however, there is growing evidence for cell nonautonomous tumor ...suppressor effects of p53. In the present study, we investigated the impact of stromal p53 on tumor growth. Specifically, we found that ablation of p53 in fibroblasts enabled them to promote more efficiently the growth of tumors initiated by PC3 prostate cancer-derived cells. This stimulatory effect was dependent on the increased expression of the chemokine SDF-1 in the p53-deficient fibroblasts. Notably, fibroblasts harboring mutant p53 protein were more effective than p53-null fibroblasts in promoting tumor growth. The presence of either p53-null or p53-mutant fibroblasts led also to a markedly elevated rate of metastatic spread of the PC3 tumors. These findings implicate p53 in a cell nonautonomous tumor suppressor role within stromal fibroblasts, through suppressing the production of tumor stimulatory factors by these cells. Moreover, expression of mutant p53 by tumor stroma fibroblasts might exert a gain of function effect, further accelerating tumor development.
Breast tumors and their derived circulating cancer cells express the leukocyte β
2
integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast ...cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct
in vivo
contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The
in vivo
elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice.
Ex vivo
, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells
via
ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.
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