Metabolism regulates cell fate in lymphocytes and progenitor cells

Self-renewal mediates homeostasis across mammalian organ systems as the cellular components of mature tissues are continually replaced in the face of wear and tear, injury, infection, and malignancy. The hematopoietic and immune systems are crucial for organismal longevity and rely on the ability of progenitor cells to bifurcate in fate to produce mature terminally differentiated progeny while self-renewing to maintain more quiescent progenitors. Asymmetric cell division is associated with self-renewal of lymphocytes and hematopoietic progenitors, but the mechanisms underlying the cell biology of these processes remain incompletely understood. Here we show that metabolic signals in the form of differential anabolism and catabolism regulate asymmetric division and cell fate bifurcations. Key transcription factors, including TCF1 and IRF4 in lymphocytes and IRF8 in hematopoietic progenitors, occupy regulatory nodes where signals associated with metabolism and traditional cell fate determinants converge. Notably, anabolic PI3K/mTOR signaling was required for terminal differentiation of both lymphocytes and hematopoietic progenitors through the regulation of a constellation of nutrient uptake, mitochondrial turnover, reactive oxygen species production, and autophagy. Further, we found that antigen receptor signaling in lymphocytes organizes a cell-intrinsic polarity pathway of asymmetric intracellular membrane trafficking that is regulated by PI3K activity and associated with terminal differentiation. These results support a model wherein cell fate bifurcations are organized by metabolic signaling at the population and subcellular level to ensure self- renewal of progenitor and memory populations

Metabolic Control of Dendritic Cell Functions: Digesting Information

Dendritic cells (DCs) control innate and adaptive immunity by patrolling tissues to gather antigens and danger signals derived from microbes and tissue. Subsequently, DCs integrate those environmental cues, orchestrate immunity or tolerance, and regulate tissue homeostasis. Recent advances in the field of immunometabolism highlight the notion that immune cells markedly alter cellular metabolic pathways during differentiation or upon activation, which has important implications on their functionality. Previous studies showed that active oxidative phosphorylation in mitochondria is associated with immature or tolerogenic DCs, while increased glycolysis upon pathogen sensing can promote immunogenic DC functions. However, new results in the last years suggest that regulation of DC metabolism in steady state, after immunogenic activation and during tolerance in different pathophysiological settings, may be more complex. Moreover, ontogenically distinct DC subsets show different functional specializations to control T cell responses. It is, thus, relevant how metabolism influences DC differentiation and plasticity, and what potential metabolic differences exist among DC subsets. Better understanding of the emerging connection between metabolic adaptions and functional DC specification will likely allow the development of therapeutic strategies to manipulate immune responses

Molecular mechanisms of Us9-mediated anterograde transport of alphaherpes viruses

Alphaherpes viruses infect the nervous system of their hosts and undergo long-distance transport in neuronal axons during different steps of the life cycle. Active viral replication at the neuronal soma produces progeny virions, which must undergo anterograde transport down the axon to facilitate anterograde spread within the host. Throughout this work, the molecular mechanisms underlying anterograde transport are explored using pseudorabies virus infection as a model system. Several functional domains in the viral protein Us9, which is essential for anterograde transport, were characterized through a convergence of methodologies including live-cell imaging of fluorescent viruses as well as in vitro spread and biochemical assays. In Chapter 1, GFP-Us9 fusion proteins are employed to characterize the role of a dityrosine motif in the protein in anterograde transport of virion structural components. The dityrosine motif was required for anterograde neuron-to-cell spread in vitro as well as for axonal targeting of virion structural components. GFP-Us9 fusion proteins are then employed further in Chapter 2 to characterize the Us9 diserine motif. Interestingly, unlike the dityrosine motif, mutagenesis of the diserine motif resulted in only a modest defect in spread and was found to modulate the efficiency of anterograde transport. In Chapter 3, the requirement of Us9 for transport of viral membrane proteins is assessed for particles that do not constitute mature virions. Interestingly, we established that the viral glycoprotein M is capable of undergoing anterograde transport independently of Us9. A summation of our current understanding of the anterograde transport mechanism, known as the Married Model, is then presented in Chapter 4 through a critical analysis of experimental techniques in published works. Finally, Appendix A contains a preliminary investigation of glycoprotein E functionality with respect to Us9-Kif1A interactions and anterograde transport of virions. Together, these results expand our understanding of Us9 functionality and biochemical properties

Live cell imaging of alphaherpes virus anterograde transport and spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread

Distribution of Us9 and phospho-Us9 across membrane microdomains.

<p>A.) WB analysis following lipid raft flotation from differentiated PC12 cells at 14 hours post-infection with PRV Becker. Samples were collected from a discontinuous 5%–30%–40% Optiprep gradient. DRMs localize to the 5%–30% interface, while solubilized membrane proteins remain at the 30%–40% interface. Each 1 mL fraction from this gradient was run and probed with polyclonal anti-Us9 antibody to detect total Us9 protein content and phospho-specific monoclonal antibody to detect only phosphorylated Us9. B.) Quantitation of total Us9 and phospho-Us9 in insoluble raft membrane fraction and soluble fraction by WB for PC12 cells 14 hours post-infection with PRV Becker; values are reported as arbitrary chemiluminescence units from WB. Curves are representative of two independent lipid raft flotation experiments and show detection of each sample across a 2-fold dilution series. Dilution series covers detection of the respective Us9 protein from point of saturation, through the linear range of detection, to undetectable levels.</p

Phosphorylation of Us9 is not essential for Kif1A binding.

<p>Differentiated PC12 cells were infected with the indicated PRV strains, lysed at 12 hours post-infection, and subject to co-immunoprecipitation analysis using anti-GFP rabbit polyclonal antibodies. The ability of these mutant GFP-Us9 variants to interact with KIf1A was specifically assessed through WB detection.</p

Role of Us9 Phosphorylation in Axonal Sorting and Anterograde Transport of Pseudorabies Virus

<div><p>Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread <i>in vivo</i>. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.</p> </div

Compilation of PRV strains expressing mutant Us9 variants employed in this study as well as in previous work.

<p>Data on <i>in vitro</i> anterograde spread is from infection of compartmentalized neuronal cultures. All <i>in vivo</i> spread data from the rodent eye model system has been reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058776#pone.0058776-Brideau2" target="_blank">[13]</a> and is presented here for comparison. +/− symbols denote phenotypes from robust spread (+++) to no spread (−).</p