Pulse-chase Research Articles

Tripartite motif 25 ameliorates doxorubicin-induced cardiotoxicity by degrading p85α

Doxorubicin (DOX)-based chemotherapy is widely used to treat malignant tumors; however, the cardiotoxicity induced by DOX restricts its clinical usage. A therapeutic dose of DOX can activate ubiquitin-proteasome system. However, whether and how ubiquitin-proteasome system brings out DOX-induced cardiotoxicity remains to be investigated. Here we conducted a proteomics analysis of a DOX-induced cardiotoxicity model to screen the potentially ubiquitination-related molecules. Dysregulated TRIM25 was found to contribute to the cardiotoxicity. In vivo and in vitro cardiotoxicity experiments revealed that TRIM25 ameliorated DOX-induced cardiotoxicity. Electron microscopy and endoplasmic reticulum stress markers revealed that TRIM25 mitigated endoplasmic reticulum stress and apoptosis in DOX-induced cardiomyocytes. Mechanistically, the Co-immunoprecipitation assays and CHX pulse-chase experiment determined that TRIM25 affected p85α stability and promoted its ubiquitination and degradation. This leads to increase of nuclear translocation of XBP-1s, which mitigates endoplasmic reticulum stress. These findings reveal that TRIM25 may have a therapeutic role for DOX-induced cardiotoxicity.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Radial Glia and Neuronal-like Ependymal Cells Are Present within the Spinal Cord of the Trunk (Body) in the Leopard Gecko (Eublepharis macularius).

As is the case for many lizards, leopard geckos (Eublepharis macularius) can self-detach a portion of their tail to escape predation, and then regenerate a replacement complete with a spinal cord. Previous research has shown that endogenous populations of neural stem/progenitor cells (NSPCs) reside within the spinal cord of the original tail. In response to tail loss, these NSPCs are activated and contribute to regeneration. Here, we investigate whether similar populations of NSPCs are found within the spinal cord of the trunk (body). Using a long-duration 5-bromo-2′-deoxyuridine pulse-chase experiment, we determined that a population of cells within the ependymal layer are label-retaining following a 20-week chase. Tail loss does not significantly alter rates of ependymal cell proliferation within the trunk spinal cord. Ependymal cells of the trunk spinal cord express SOX2 and represent at least two distinct cell populations: radial glial-like (glial fibrillary acidic protein- and Vimentin-expressing) cells; and neuronal-like (HuCD-expressing) cells. Taken together, these data demonstrate that NSPCs of the trunk spinal cord closely resemble those of the tail and support the use of the tail spinal cord as a less invasive proxy for body spinal cord injury investigations.

Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.

Characterization and application of a novel immortalized murine thick ascending limb (imTAL) cell line to investigate the role of myosin II motor proteins Myh9 and Myh10 in TAL cargo transport

Conditional genetic inactivation of Myh9and Myh10, the genes which encode the heavy chains of nonmuscle myosin IIA and IIB, respectively, in adult murine renal epithelium leads to aberrant trafficking of the thick ascending limb‐specific GPI‐anchored glycoprotein uromodulin (UMOD) and its subcellular accumulation in cells of the thick ascending limb of Henle’s loop (Otterpohl et al. 2020). A novel immortalized murine thick ascending limb (imTAL) cell line harboring doxycycline inducible Myh9and Myh10conditional knockout (cKO) capability was established in our lab to facilitate long term in vitrostudies. We utilized biotinylated anti‐uromodulin antibody to isolate cells from collagenase digested Myh9&10cKO whole mouse kidneys. Characterization of these cells has revealed endogenous expression of TAL‐specific protein UMOD and NKCC2. RT‐qPCR detected mRNA expression of TAL specific proteins. We generated imTAL cells stably expressing GFP‐tagged UMOD using retroviral transduction. GFP‐UMOD expressing cells underwent 72‐hour doxycycline treatment and, along with untreated control cells, were fixed and analyzed via confocal microscope. While GFP‐UMOD is enriched along the plasma membrane in control cells, we observe accumulation of GFP‐UMOD within vesicular structures upon doxycycline‐induced loss of Myh9 and Myh10 proteins. We also utilized imTAL cells stably expressing an GFP‐tagged thermosensitive variant of vesicular stomatitis virus glycoprotein (VSV‐GtsO45‐GFP), which has been widely employed in studying the secretory pathway. The temperature sensitive nature of VSV‐GtsO45 enabled us to perform pulse‐chase type experiments, following its anterograde transport through the ER, Golgi, and ultimately to the plasma membrane. Following incubation at the restrictive temperature (40°C) overnight, cells were moved to permissive temperature (32°C) and followed until 45 minutes. VSV‐GtsO45‐GFP in control cells undergoes ER export immediately (5 minutes) after incubation at the permissive temperature and is transported along the secretory pathway, arriving at the plasma membrane 30‐40 minutes later. Inactivation of Myh9and Myh10 results in sequestration of VSV‐GtsO45‐GFP in perinuclear vesicular and tubular structures. Immunolocalization studies are being performed to identify the vesicular compartment(s) in which VSV‐GtsO45‐GFP is sequestered in the absence of Myh9 and Myh10 proteins. imTAL cells stably expressing NKCC2‐GFP are also being analyzed to assess the anterograde transport of NKCC2 in doxycycline‐treated cells. Taken together, we have established a novel, immortalized, inducible, Myh9&10 cKO murine TAL cell line to enable long term in vitro studies and have expanded the molecular toolbox to elucidate the mechanisms underlying NM2 motor mediated TAL cargo transport.

The Dynamics of Phosphorus Uptake and Remobilization during the Grain Development Period in Durum Wheat Plants.

Post-anthesis phosphorus (P) uptake and the remobilization of the previously acquired P are the principal sources of grain P nutrition in wheat. However, how the acquired P reaches the grains and its partitioning at the whole plant level remain poorly understood. Here, the temporal dynamics of the newly acquired P in durum wheat organs and its allocation to grain were examined using pulse-chase P-labeling experiments at 5 and 14 days after anthesis. Durum wheat plants were grown hydroponically under high and low P supplies. Each labeling experiment lasted for 24 h. Plants were harvested 24, 48, and 96 h after labeling. Low and high P treatments significantly affected the allocation of the newly acquired P at the whole plant level. Three days (96 h) after the first P-labeling, 8% and 4% of the newly acquired P from exogenous solution were allocated to grains, 73% and 55% to the remainder aboveground organs, and 19% and 41% to the roots at low and high P supplies, respectively. Three days after the second labeling, the corresponding values were 48% and 20% in grains, 44% and 53% in the remainder aboveground organs, and 8% and 27% in roots at low and high P supplies, respectively. These results reveal that the dynamics of P allocation to grain was faster in plants grown under low P supply than under high supply. However, the obtained results also indicate that the origin of P accumulated in durum wheat grains was mainly from P remobilization with little contribution from post-anthesis P uptake. The present study emphasizes the role of vegetative organs as temporary storage of P taken up during the grain filling period before its final allocation to grains.

De novo Mutation Enables NOTCH3ECD Aggregation and Mitochondrial Dysfunction via Interactions with BAX and BCL-2.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most common monogenic hereditary pattern of cerebral small vessel disease. The aggregation of the mutant NOTCH3 may play a cytotoxic role in CADASIL. However, the main mechanism of this process remains unclear. We aimed to investigate the possible pathogenesis of the mutant NOTCH3 in CADASIL. The clinical information of two pedigrees were collected and analyzed. Furthermore, we constructed cell lines corresponding to this mutation in vitro. The degradation of the extracellular domain of NOTCH3 (NOTCH3ECD) was analyzed by Cycloheximide Pulse-Chase Experiment. Flow cytometry and cell counting kit-8 assay were performed to observe the effects of the NOTCH3 mutation on mitochondrial function and apoptosis. We confirmed a de novo heterozygous missense NOTCH3 mutation (c.1690G > A, p.A564T) in two pedigrees. In vitro, the NOTCH3ECD aggregation of A564T mutant may be related to their more difficult to degrade. The mitochondrial membrane potential was attenuated, and cell viability was significant decreased in NOTCH3ECD A564T group. Interestingly, BAX and cytochrome c were significantly increased, which are closely related to the mitochondrial-mediated pathway to apoptosis. In our study, the aggregation of NOTCH3ECD A564T mutation may be associated with more difficult degradation of the mutant, and the aggregation may produce toxic effects to induce apoptosis through the mitochondrial-mediated pathway. Therefore, we speculated that mitochondrial dysfunction may hopefully become a new breakthrough point to explain the pathogenesis of cysteine-sparing NOTCH3 mutations.

Root Absorption and Xylem Movement of Ethephon in Tomato

Although ethephon is commonly used as a plant growth regulator during commercial production of horticultural crops, information on its movement within plants is limited. In this study, we developed a method to detect ethephon in plant tissues, and determined ethephon localization and movement using tomato (Solanum lycopersicum L.) as a model system. Tissues were ground in an acidic buffer that preserved ethephon intact. Ethylene was released from the extracts by adding sodium hydroxide and was determined subsequently by gas chromatography. Ethephon was detected in leaves within 1 hour of application to peat-based root zones and within 10 minutes in hydroponics. In a pulse–chase experiment, ethephon levels increased initially, then decreased after the plants were returned to ethephon-free solutions. Ethephon was present in directly collected xylem fluid; fluid collected from petiole stumps (after leaf blade excision) had similar ethephon levels between the different petioles. Stem girdling had no effect on ethephon accumulation in leaves. Together, these data indicate ethephon is readily mobile in the xylem stream and provides insight into the commercial use of ethephon as a root zone-applied growth regulator.

Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases

Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, has brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with bioorthogonal click chemistry. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells make click labeling unique. Here, we establish click labeling in living primary neurons and use it for fixed-cell, live-cell, dual-color pulse–chase, and super-resolution microscopy of neurofilament light chain (NFL). We also show that click labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced multicolor microscopy techniques, we anticipate that click labeling will contribute to novel discoveries in the neurobiology field.

Limited fatty-acid supply from the plastid and active catabolism of triacylglycerol prevent the accumulation of triacylglycerol in Coccomyxa sp. strain Obi grown under nitrogen-replete conditions

The Coccomyxa sp. strain Obi is a unicellular green alga that accumulates >40% triacylglycerol (TAG) per cell dry weight under nitrogen (N)-limiting conditions. In this study, we analyzed the de novo synthesis of TAG under N-replete and N-limiting conditions using 14C tracers and quantitative reverse transcription PCR (RT-qPCR) approaches. In cells grown under N-replete conditions, the incorporation of photosynthetically fixed 14C into TAG was <5% of that incorporated into polar lipids, whereas the incorporation of exogenous 14C-fatty acids into TAG was approximately 50% of that incorporated into polar lipids. These results demonstrated that enzyme activities that convert fatty acids to TAG are appreciably expressed under N-replete conditions; however low fatty acid supply limits the TAG synthesis in N-replete cells. In agreement with this inference, the expression of FAT1 that encodes acyl-ACP thioesterase, which releases free fatty acids from acyl-ACP, was poorly expressed under N-replete conditions but induced under N-limiting conditions. Meanwhile, pulse-chase labeling experiments demonstrated a significant turnover of TAG under N-replete conditions. Taken together, we propose that both the limited carbon flux into TAG and active catabolism of TAG prevented the accumulation of TAG under N-replete conditions, whereas the increased carbon flux into TAG and possibly decreased catabolism of TAG contributed to the enhanced accumulation of TAG under N-limiting conditions. Furthermore, pulse-chase experiments demonstrated that the rate of TAG synthesis was much slower than that of polar lipid synthesis under N-limiting conditions, partly because of the slow conversion of diacylglycerol to TAG.

Advances in stable isotope labeling: dynamic labeling for spatial and temporal proteomic analysis.

The field of proteomics is continually improving, requiring the development of new quantitative methods. Stable isotope labeling in cell culture (SILAC) is a metabolic labeling technique originating in the early 2000s. By incorporating isotopically labeled amino acids into the media used for cell culture, unlabeled versus labeled cells can be differentiated by the mass spectrometer. Traditional SILAC labeling has been expanded to pulsed applications allowing for a new quantitative dimension of proteomics - temporal analysis. The complete introduction of Heavy SILAC labeling chased with surplus unlabeled medium mimics traditional pulse-chase experiments and allows for the loss of heavy signal to track proteomic changes over time. In a similar fashion, pulsed SILAC (pSILAC) monitors the initial incorporation of a heavy label across a period of time, which allows for the rate of protein label integration to be assessed. These innovative techniques have aided in inspiring numerous SILAC-based temporal and spatial labeling applications, including super SILAC, spike-in SILAC, spatial SILAC, and a revival in label multiplexing. This review reflects upon the evolution of SILAC and the pulsed SILAC application, introduces advances in SILAC labeling, and proposes future perspectives for this novel and exciting field.

Broken teeth? Fish dentists hate this one simple trick

Many fishes like sharks are known for their ability to regrow sets of teeth. Regrowing teeth comes in handy if a tooth breaks or is worn down over time. Fishes also often have a second set of jaws – pharyngeal jaws – which hide in their throats. These jaws and the teeth upon them are used to crush food, in contrast to oral jaws, which grab morsels. Previous tooth replacement studies have focused on fishes with tooth shapes specialized to the specific food they consume. But little was known about tooth replacement in fish that eat most anything, such as Pacific lingcod (Ophiodon elongatus) with simple cone-shaped teeth. What are the tooth replacement rates in either set of jaws? Is a new tooth fated to replace another of the same size? And does feeding influence tooth replacement rates? To study these questions, Emily Carr from the University of South Florida, USA, and colleagues from the University of Washington, USA, compared tooth replacement rates and positions in the oral and pharyngeal jaws of Pacific lingcod.The scientists used special fluorescent staining techniques, called pulse–chase, to monitor new versus old teeth so that new teeth would fluoresce as green and old teeth would fluoresce as red. First, the team separated the fish into two groups – one that was fed and a second that was fed nothing to determine whether feeding affects tooth replacement rates. The scientists then used a microscope and fluorescent lamp to monitor the replacement rates of the fishes’ teeth, as well as the size and location of new teeth, on both the oral and pharyngeal jaws.It turns out that the size and locations of replacement teeth are fated. New teeth erupted as small cone-shaped structures next to the teeth that they were destined to replace, before growing to the size of the original teeth. In addition, small teeth were replaced more often than large teeth, as they broke more often. The team also found that the tooth replacement rate was mostly similar across the pharyngeal and oral jaws, except in the lower pharyngeal jaw, where the replacement rate was nearly twice as fast, possibly because food hits these teeth at many different angles thanks to the mobility of the jaw. Lastly, the team realised that whether a fish is actively feeding or not does not influence how often they replace their teeth.So, it seems likely that all fishes with single simplified tooth shapes like the lingcod follow the replacement pattern that Carr and colleagues have found. Interestingly, neither wear nor the high cost of tooth replacement determines the replacement rate. Instead, the team suggests that replacement rates may be driven by a developmental network which controls when and where teeth are replaced. Future studies can investigate the signals that determine replacement tooth size and whether size is decided by the cells that surround the tooth. This work will help us to understand the diversity of tooth development and shape in fishes, which are the largest group of vertebrate animals on the planet.

The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses.

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

Osmotrophy of dissolved organic carbon by coccolithophores in darkness.

Summary The evolutionary and ecological story of coccolithophores poses questions about their heterotrophy, surviving darkness after the end‐Cretaceous asteroid impact as well as survival in the deep ocean twilight zone. Uptake of dissolved organic carbon might be an alternative nutritional strategy for supply of energy and carbon molecules.Using long‐term batch culture experiments, we examined coccolithophore growth and maintenance on organic compounds in darkness. Radiolabelled experiments were performed to study the uptake kinetics. Pulse–chase experiments were used to examine the uptake into unassimilated, exchangeable pools vs assimilated, nonexchangeable pools.We found that coccolithophores were able to survive and maintain their metabolism for up to 30 d in darkness, accomplishing about one cell division. The concentration dependence for uptake was similar to the concentration dependence for growth in Cruciplacolithus neohelis, suggesting that it was taking up carbon compounds and immediately incorporating them into biomass. We recorded net incorporation of radioactivity into the particulate inorganic fraction.We conclude that osmotrophy provides nutritional flexibility and supports long‐term survival in light intensities well below threshold for photosynthesis. The incorporation of dissolved organic matter into particulate inorganic carbon, raises fundamental questions about the role of the alkalinity pump and the alkalinity balance in the sea.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

Assembly Apparatus of Light-Harvesting Complexes: Identification of Alb3.1–cpSRP–LHCP Complexes in the Green Alga Chlamydomonas reinhardtii

The unicellular green alga, Chlamydomonas reinhardtii, contains many light-harvesting complexes (LHCs) associating chlorophylls a/b and carotenoids; the major LHCIIs (types I, II, III and IV) and minor light-harvesting complexes, CP26 and CP29, for photosystem II, as well as nine LHCIs (LHCA1-9), for photosystem I. A pale green mutant BF4 exhibited impaired accumulation of LHCs due to deficiency in the Alb3.1 gene, which encodes the insertase involved in insertion, folding and assembly of LHC proteins in the thylakoid membranes. To elucidate the molecular mechanism by which ALB3.1 assists LHC assembly, we complemented BF4 to express ALB3.1 fused with no, single or triple Human influenza hemagglutinin (HA) tag at its C-terminus (cAlb3.1, cAlb3.1-HA or cAlb3.1-3HA). The resulting complemented strains accumulated most LHC proteins comparable to wild-type (WT) levels. The affinity purification of Alb3.1-HA and Alb3.1-3HA preparations showed that ALB3.1 interacts with cpSRP43 and cpSRP54 proteins of the chloroplast signal recognition particle (cpSRP) and several LHC proteins; two major LHCII proteins (types I and III), two minor LHCII proteins (CP26 and CP29) and eight LHCI proteins (LHCA1, 2, 3, 4, 5, 6, 8 and 9). Pulse-chase labeling experiments revealed that the newly synthesized major LHCII proteins were transiently bound to the Alb3.1 complex. We propose that Alb3.1 interacts with cpSRP43 and cpSRP54 to form an assembly apparatus for most LHCs in the thylakoid membranes. Interestingly, photosystem I (PSI) proteins were also detected in the Alb3.1 preparations, suggesting that the integration of LHCIs to a PSI core complex to form a PSI-LHCI subcomplex occurs before assembled LHCIs dissociate from the Alb3.1-cpSRP complex.

Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

Cytomegalovirus (CMV) infection initiates massive rearrangement of cytoplasmic organelles to generate assembly compartment (AC). The earliest events, the establishment of the preAC, are initiated in the early phase as an extensive reorganization of early endosomes (EEs), endosomal recycling compartment (ERC), trans-Golgi network (TGN), and the Golgi. Here, we demonstrate that dynamin inhibitors (Dynasore, Dyngo-4a, MiTMAB, and Dynole-34-2) block the establishment of the preAC in murine CMV (MCMV) infected cells. In this study, we extensively analyzed the effect of Dynasore on the Golgi reorganization sequence into the outer preAC. We also monitored the development of the inner preAC using a set of markers that define EEs (Rab5, Vps34, EEA1, and Hrs), the EE-ERC interface (Rab10), the ERC (Rab11, Arf6), three layers of the Golgi (GRASP65, GM130, Golgin97), and late endosomes (Lamp1). Dynasore inhibited the pericentriolar accumulation of all markers that display EE-ERC-TGN interface in the inner preAC and prevented Golgi unlinking and dislocation to the outer preAC. Furthermore, in pulse-chase experiments, we demonstrated that the presence of dynasore only during the early phase of MCMV infection (4–14 hpi) is sufficient to prevent not only AC formation but also the synthesis of late-phase proteins and virion production. Therefore, our results indicate that dynamin-2 acts as a part of the machinery required for AC generation and rearrangement of EE/ERC/Golgi membranes in the early phase of CMV infection.