Pronounced uptake and metabolism of organic substrates by diatoms revealed by pulse-labeling metabolomics

Diatoms contribute as a dominant group of microalgae to approximately 20 per cent of the global carbon fixation. In the plankton, these photosynthetic algae are exposed to a plethora of metabolites, especially when competing algae are lysed. It is well established that diatoms can take up specific metabolites, such as vitamins, amino acids as nitrogen source, or dimethylsulfoniopropoionate to compensate for changes in water salinity. It is, however, unclear to which extent diatoms take up other organic resources and if these are incorporated into the cells metabolism. Here, we ask about the general scope of uptake of metabolites from competitors. Using labeled metabolites released during lysis of algae grown under a 13CO2 atmosphere, we show that the cosmopolitan diatom Chaetoceros didymus takes up organic substrates with little bias and remarkable efficiency. The newly developed pulse label/ mass spectrometry metabolomics approach reveals that polarity and molecular weight has no detectable influence on uptake efficiency. We also reveal that the taken-up pool of metabolites is partly maintained unaltered within the cells but is also subject to catabolic and anabolic transformation. One of the most dominant phytoplankton groups is thus substantially competing with other heterotrophs for organic material, suggesting that the observed absorbotrophy may substantially impact organic material fluxes in the oceans. Our findings call for the refinement of our understanding of competition in the plankton.

Visualization of giant virus particles using BONCAT labeling and STED microscopy

Giant DNA viruses of the phylum Nucleocytoviricota are being increasingly recognized as important regulators of natural protist populations. However, our knowledge of their infection cycles is still very limited due to a lack of cultured virus-host systems and molecular tools to study them. Here, we apply bioorthogonal noncanonical amino acid tagging (BONCAT) to pulse label the marine heterotrophic flagellate Cafeteria burkhardae during infection with the lytic giant virus CroV. In absence of CroV, we report efficient incorporation of the L-methionine analog L-azidohomoalanine (AHA) into newly synthesized proteins of the methionine prototrophic C. burkhardae. During CroV infection, AHA was predominantly found in viral proteins, and single CroV virions were imaged with stimulated emission depletion (STED) super-resolution microscopy. CroV particles incorporated AHA with 95-100% efficiency while retaining their infectivity, which makes BONCAT/STED a powerful tool to study viral replication cycles in this ecologically relevant marine bacterivore.SignificanceGiant DNA viruses are the dominant class of protist-infecting viruses, yet the vast majority of described giant virus-protist systems remain uncultured. One of the better studied cultured systems is composed of the stramenopile Cafeteria burkhardae (previously C. roenbergensis), the giant Cafeteria roenbergensis virus (CroV) and the virophage mavirus. C. burkhardae is a widespread marine phagotrophic protist that plays an important role in regulating bacterial populations. In addition to being grazed upon by larger zooplankton, C. burkhardae populations are controlled by the lytic giant virus CroV. In turn, CroV is parasitized by the virophage mavirus that increases host population survival in the presence of CroV and forms a mutualistic symbiosis with its host. Despite being of fundamental ecological and evolutionary interest, this tripartite host-virus-virophage system suffers from a lack of molecular tools. Here, we show that CroV particles can be fluorescently labeled and imaged by super-resolution microscopy. To achieve this we established robust procedures for analyzing protist and viral populations and implemented the use of bioorthogonal noncanonical amino acid tagging (BONCAT) in a marine unicellular flagellate.

Distinct neural progenitor pools in the ventral telencephalon generate diversity in striatal spiny projection neurons

Heterogeneous populations of neural progenitors in the embryonic lateral ganglionic eminence (LGE) generate all GABAergic spiny projection neurons (SPNs) found in the striatum. Here we investigate how this diversity in neural progenitors relates to diversity of adult striatal neurons and circuits. Using a combination of in utero electroporation to fluorescently pulse-label striatal neural progenitors in the LGE, brain slice electrophysiology, electrical and optogenetic circuit mapping and immunohistochemistry, we characterise a population of neural progenitors enriched for apical intermediate progenitors (aIPs) and a distinct population of other progenitors (OPs) and their neural offspring. We find that neural progenitor origin has subtle but significant effects on the properties of striatal SPNs. Although aIP and OP progenitors can both generate D1-expressing direct pathway as well as D2-expressing indirect pathway SPNs found intermingled in the striatum, the aIP derived SPNs are found in more medial aspects of the striatum, exhibit more complex dendritic arbors with higher spine density and differentially sample cortical input. Moreover, optogenetic circuit mapping of the aIP derived neurons show that they further integrate within striatal circuits and innervate both local D1 and D2 SPNs. These results show that it is possible to fluorescently pulse-label distinct neural progenitor pools within the LGE and provide the first evidence that neural progenitor heterogeneity can contribute to the diversity of striatal SPNs.

Apical progenitors remain multipotent throughout cortical neurogenesis

The diverse subtypes of excitatory neurons that populate the neocortex are born from progenitors located in the ventricular zone (apical progenitors, APs). During corticogenesis, APs progress through successive temporal states to sequentially generate deep- followed by superficial-layer neurons directly or via the generation of intermediate progenitors (IPs). Yet little is known about the plasticity of AP temporal identity and whether individual progenitor subtypes remain multipotent throughout corticogenesis. To address this question, we used FlashTag (FT), a method to pulse-label and isolate APs in the mouse neocortex with high temporal resolution to fate-map neuronal progeny following heterochronic transplantation of APs into younger embryos. We find that unlike daughter IPs, which lose the ability to generate deep layer neurons when transplanted into a younger host, APs are temporally uncommitted and become molecularly respecified to generate normally earlier-born neuron types. These results indicate that APs are multipotent cells that are able to revert their temporal identity and re-enter past molecular and neurogenic states. AP fate progression thus occurs without detectable fate restriction during the neurogenic period of corticogenesis. These findings identify unforeseen cell-type specific differences in cortical progenitor fate plasticity, which could be exploited for neuroregenerative purposes.

Mitotic transcription and waves of gene reactivation during mitotic exit

Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and found that many genes exhibit transcription during mitosis, as confirmed with fluorescein isothiocyanate–uridine 5′-triphosphate labeling, RNA fluorescence in situ hybridization, and quantitative reverse transcription polymerase chain reaction. The first round of transcription immediately after mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type–specific functions. We propose that the cell’s transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is reestablished during mitotic exit.

New markers for murine memory B cells that define mutated and unmutated subsets

The study of murine memory B cells has been limited by small cell numbers and the lack of a definitive marker. We have addressed some of these difficulties with hapten-specific transgenic (Tg) mouse models that yield relatively large numbers of antigen-specific memory B cells upon immunization. Using these models, along with a 5-bromo-2′-deoxyuridine (BrdU) pulse-label strategy, we compared memory cells to their naive precursors in a comprehensive flow cytometric survey, thus revealing several new murine memory B cell markers. Most interestingly, memory cells were phenotypically heterogeneous. Particularly surprising was the finding of an unmutated memory B cell subset identified by the expression of CD80 and CD35. We confirmed these findings in an analogous V region knock-in mouse and/or in non-Tg mice. There also was anatomic heterogeneity, with BrdU+ memory cells residing not just in the marginal zone, as had been thought, but also in splenic follicles. These studies impact the current understanding of murine memory B cells by identifying new phenotypes and by challenging assumptions about the location and V region mutation status of memory cells. The apparent heterogeneity in the memory compartment implies either different origins and/or different functions, which we discuss.

Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation

During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96–120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83 delta C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83 delta C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83 delta C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells.

Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte.

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, we injected transcripts encoding a myc-tagged C protein into amphibian oocytes. The distribution of protein translated from the injected RNA was essentially the same in continuous and pulse-label experiments. In both cases the C protein localized within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that caused truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localized normally in the germinal vesicle and could be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own and there associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localized within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs.

Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum

The 47,000-D collagen-binding glycoprotein, heat shock protein 47 (HSP47), is a stress-inducible protein localized in the ER of collagen-secreting cells. The location and collagen-binding activity of this protein led to speculation that HSP47 might participate in collagen processing. Chemical crosslinking studies were used to test this hypothesis both before and after the perturbation of procollagen processing. The association of procollagen with HSP47 was demonstrated using cleavable bifunctional crosslinking reagents. HSP47 and procollagen were shown to be coprecipitated by the treatment of intact cells with anti-HSP47 or with anticollagen antibodies. Furthermore, several proteins residing in the ER were noted to be crosslinked to and coprecipitated with HSP47, suggesting that these ER-resident proteins may form a large complex in the ER. When cells were heat shocked, or when stable triple-helix formation was inhibited by treatment with alpha,alpha'-dipyridyl, coprecipitation of procollagen with HSP47 was increased. This increase was due to the inhibition of procollagen secretion and to the accumulation of procollagen in the ER. Pulse label and chase experiments revealed that coprecipitated procollagen was detectable as long as procollagen was present in the endoplasmic reticulum of alpha,alpha'-dipyridyl-treated cells. Under normal growth conditions, coprecipitated procollagen was observed to decrease after a chase period of 10-15 min, whereas total procollagen decreased only after 20-25 min. In addition, the intracellular association between HSP47 and procollagen was shown to be disrupted by a change in physiological pH, suggesting that the dissociation of procollagen from HSP47 is pH dependent. These findings support a specific role for HSP47 in the intracellular processing of procollagen, and provide evidence of a new category of "molecular chaperones" in terms of its substrate specificity and the dissociation mechanism.

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.