Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, here we show in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog's role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway.

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify Vmem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10-15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel KCa3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles.

A fundamental question is how proliferation and growth are timed during embryogenesis. Although it has been suggested that the cell cycle could be a timer, the underlying mechanisms remain elusive. Here we describe a cell cycle timer that operates in Sonic hedgehog (Shh)-expressing polarising region cells of the chick wing bud. Our data are consistent with Shh signalling stimulating polarising region cell proliferation via Cyclin D2, and then inhibiting proliferation via a Bmp2-p27kip1 pathway. When Shh signalling is blocked, polarising region cells over-proliferate and form an additional digit, which can be prevented by applying Bmp2 or by inhibiting D cyclin activity. In addition, Bmp2 also restores posterior digit identity in the absence of Shh signalling, thus indicating that it specifies antero-posterior (thumb to little finger) positional values. Our results reveal how an autoregulatory cell cycle timer integrates growth and specification and are widely applicable to many tissues.

Visual neuroscientists require accurate control of visual stimulation. However, few stimulator solutions simultaneously offer high spatio-temporal resolution and free control over the spectra of the light sources, because they rely on off-the-shelf technology developed for human trichromatic vision. Importantly, consumer displays fail to drive UV-shifted short wavelength-sensitive photoreceptors, which strongly contribute to visual behaviour in many animals, including mice, zebrafish and fruit flies. Moreover, many non-mammalian species feature more than three spectral photoreceptor types. Here, we present a flexible, spatial visual stimulator with up to six arbitrary spectrum chromatic channels. It combines a standard digital light processing engine with open source hard- and software that can be easily adapted to the experimentalist's needs. We demonstrate the capability of this general visual stimulator experimentally in the in vitro mouse retinal whole-mount and the in vivo zebrafish. With this work, we intend to start a community effort of sharing and developing a common stimulator design for vision research.

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG-Ctf43-1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy.

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.

The advent of high-yield electrophysiology using Neuropixels probes is now enabling researchers to simultaneously record hundreds of neurons with remarkably high signal to noise. However, these probes have not been well-suited to use in freely moving mice. It is critical to study neural activity in unrestricted animals for many reasons, such as leveraging ethological approaches to study neural circuits. We designed and implemented a novel device that allows Neuropixels probes to be customized for chronically implanted experiments in freely moving mice. We demonstrate the ease and utility of this approach in recording hundreds of neurons during an ethological behavior across weeks of experiments. We provide the technical drawings and procedures for other researchers to do the same. Importantly, our approach enables researchers to explant and reuse these valuable probes, a transformative step which has not been established for recordings with any type of chronically-implanted probe.

Trypanosomes are important disease agents of humans, livestock and cold-blooded species, including fish. The cellular morphology of trypanosomes is central to their motility, adaptation to the host's environments and pathogenesis. However, visualizing the behaviour of trypanosomes resident in a live vertebrate host has remained unexplored. In this study, we describe an infection model of zebrafish (Danio rerio) with Trypanosoma carassii. By combining high spatio-temporal resolution microscopy with the transparency of live zebrafish, we describe in detail the swimming behaviour of trypanosomes in blood and tissues of a vertebrate host. Besides the conventional tumbling and directional swimming, T. carassii can change direction through a 'whip-like' motion or by swimming backward. Further, the posterior end can act as an anchoring site in vivo. To our knowledge, this is the first report of a vertebrate infection model that allows detailed imaging of trypanosome swimming behaviour in vivo in a natural host environment.

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing.

Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a series of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide, and targeting of the mRNA for decay (No Go Decay or NGD). Using a reverse genetic screen in yeast, we identify Cue2 as the conserved endonuclease that is recruited to stalled ribosomes to promote NGD. Ribosome profiling and biochemistry provide strong evidence that Cue2 cleaves mRNA within the A site of the colliding ribosome. We demonstrate that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay and Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway. Finally, we show that the Cue2-dependent pathway becomes a major contributor to NGD in cells depleted of factors required for the resolution of stalled ribosome complexes. Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells.​.

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-to-volume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

Multiple sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord that are highly variable in terms of cellular content. Here, we used imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within staged MS lesions. To test the potential for IMC to discriminate between different types of lesions, we selected a case with severe rebound MS disease activity after natalizumab cessation. With post-acquisition analysis pipelines we were able to: (1) Discriminate demyelinating macrophages from the resident microglial pool; (2) Determine which types of lymphocytes reside closest to blood vessels; (3) Identify multiple subsets of T and B cells, and (4) Ascertain dynamics of T cell phenotypes vis-à-vis lesion type and location. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in MS patients.

Proper connectivity of the nervous system requires temporal and spatial control of axon guidance signaling. As commissural axons navigate across the CNS midline, ROBO-mediated repulsion has traditionally been thought to be repressed before crossing, and then to become upregulated after crossing. The regulation of the ROBO receptors involves multiple mechanisms that control protein expression, trafficking, and activity. Here, we report that mammalian ROBO1 and ROBO2 are not uniformly inhibited precrossing and are instead subject to additional temporal control via alternative splicing at a conserved microexon. The NOVA splicing factors regulate the developmental expression of ROBO1 and ROBO2 variants with small sequence differences and distinct guidance activities. As a result, ROBO-mediated axonal repulsion is activated early in development to prevent premature crossing and becomes inhibited later to allow crossing. Postcrossing, the ROBO1 and ROBO2 isoforms are disinhibited to prevent midline reentry and to guide postcrossing commissural axons to distinct mediolateral positions.

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.