Live-cell Time-lapse Microscopy Research Articles

Tip extension and simultaneous multiple fission in a filamentous bacterium

Organisms display an immense variety of shapes, sizes, and reproductive strategies. At microscopic scales, bacterial cell morphology and growth dynamics are adaptive traits that influence the spatial organization of microbial communities. In one such community-the human dental plaque biofilm-a network of filamentous Corynebacterium matruchotii cells forms the core of bacterial consortia known as hedgehogs, but the processes that generate these structures are unclear. Here, using live-cell time-lapse microscopy and fluorescent D-amino acids to track peptidoglycan biosynthesis, we report an extraordinary example of simultaneous multiple division within the domain Bacteria. We show that C. matruchotii cells elongate at one pole through tip extension, similar to the growth strategy of soil-dwelling Streptomyces bacteria. Filaments elongate rapidly, at rates more than five times greater than other closely related bacterial species. Following elongation, many septa form simultaneously, and each cell divides into 3 to 14 daughter cells, depending on the length of the mother filament. The daughter cells then nucleate outgrowth of new thinner vegetative filaments, generating the classic "whip handle" morphology of this taxon. Our results expand the known diversity of bacterial cell cycles and help explain how this filamentous bacterium can compete for space, access nutrients, and form important interspecies interactions within dental plaque.

Downregulation of Fidgetin-Like 2 Increases Microglial Function: The Relationship Between Microtubules, Morphology, and Activity

The microtubule cytoskeleton regulates microglial morphology, motility, and effector functions. The microtubule-severing enzyme, fidgetin-like 2 (FL2), negatively regulates cell motility and nerve regeneration, making it a promising therapeutic target for central nervous system injury. Microglia perform important functions in response to inflammation and injury, but how FL2 affects microglia is unclear. In this study, we investigated the role of FL2 in microglial morphology and injury responses in vitro. We first determined that the pro-inflammatory stimulus, lipopolysaccharide (LPS), induced a dose- and time-dependent reduction in FL2 expression associated with reduced microglial ramification. We then administered nanoparticle-encapuslated FL2 siRNA to knockdown FL2 and assess microglial functions compared to negative control siRNA and vehicle controls. Time-lapse live-cell microscopy showed that FL2 knockdown increased the velocity of microglial motility. After incubation with fluorescently labeled IgG-opsonized beads, FL2 knockdown increased phagocytosis. Microglia were exposed to low-dose LPS after nanoparticle treatment to model injury-induced cytokine secretion. FL2 knockdown enhanced LPS-induced cytokine secretion of IL-1α, IL-1β, and TNFα. These results identify FL2 as a regulator of microglial morphology and suggest that FL2 can be targeted to increase or accelerate microglial injury responses.Graphical

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes

The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes.

The nuclei of human adult stem cells can move within the cell and generate cellular protrusions to contact other cells

BackgroundThe neuronal transdifferentiation of adult bone marrow cells (BMCs) is still considered an artifact based on an alternative explanation of experimental results supporting this phenomenon obtained over decades. However, recent studies have shown that following neural induction, BMCs enter an intermediate cellular state before adopting neural-like morphologies by active neurite extension and that binucleated BMCs can be formed independent of any cell fusion events. These findings provide evidence to reject the idea that BMC neural transdifferentiation is merely an experimental artifact. Therefore, understanding the intermediate states that cells pass through during transdifferentiation is crucial given their potential application in regenerative medicine and disease modelling.MethodsIn this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can adopt during neural-like differentiation using live-cell nuclear fluorescence labelling, time-lapse microscopy, and confocal microscopy analysis.ResultsHere, we showed that after neural induction, hBM-MSCs enter an intermediate cellular state in which the nuclei are able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning occur because human cell nuclei somehow sense their environment. In addition, we showed the process of direct interactions between cell nuclei, which opens the possibility of a new level of intercellular interaction.ConclusionsThe present study advances the understanding of the intermediate stage through which hBM-MSCs pass during neural transdifferentiation, which may be crucial to understanding the mechanisms of these cell conversion processes and eventually harness them for use in regenerative medicine. Importantly, our study provides for the first time evidence that the nuclei of hBM-MSC-derived intermediate cells somehow sense their environment, generating cellular protrusions to contact other cells. In summary, human mesenchymal stromal cells could not only help to increase our understanding of the mechanisms underlying cellular plasticity but also facilitate the exact significance of nuclear positioning in cellular function and in tissue physiology.

GFAP serves as a structural element of tunneling nanotubes between glioblastoma cells and could play a role in the intercellular transfer of mitochondria.

Tunneling nanotubes (TNTs) are long F-actin-positive plasma membrane bridges connecting distant cells, allowing the intercellular transfer of cellular cargoes, and are found to be involved in glioblastoma (GBM) intercellular crosstalk. Glial fibrillary acid protein (GFAP) is a key intermediate filament protein of glial cells involved in cytoskeleton remodeling and linked to GBM progression. Whether GFAP plays a role in TNT structure and function in GBM is unknown. Here, analyzing F-actin and GFAP localization by laser-scan confocal microscopy followed by 3D reconstruction (3D-LSCM) and mitochondria dynamic by live-cell time-lapse fluorescence microscopy, we show the presence of GFAP in TNTs containing functional mitochondria connecting distant human GBM cells. Taking advantage of super-resolution 3D-LSCM, we show the presence of GFAP-positive TNT-like structures in resected human GBM as well. Using H2O2 or the pro-apoptotic toxin staurosporine (STS), we show that GFAP-positive TNTs strongly increase during oxidative stress and apoptosis in the GBM cell line. Culturing GBM cells with STS-treated GBM cells, we show that STS triggers the formation of GFAP-positive TNTs between them. Finally, we provide evidence that mitochondria co-localize with GFAP at the tip of close-ended GFAP-positive TNTs and inside receiving STS-GBM cells. Summarizing, here we found that GFAP is a structural component of TNTs generated by GBM cells, that GFAP-positive TNTs are upregulated in response to oxidative stress and pro-apoptotic stress, and that GFAP interacts with mitochondria during the intercellular transfer. These findings contribute to elucidate the molecular structure of TNTs generated by GBM cells, highlighting the structural role of GFAP in TNTs and suggesting a functional role of this intermediate filament component in the intercellular mitochondria transfer between GBM cells in response to pro-apoptotic stimuli.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the hepatocyte growth factor (HGF)-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE) and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (double KO [DKO] cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylationviaimmunoprecipitation, magnitude, and duration of c-Met receptor signaling viaimmunoblot and receptor trafficking by immunofluorescence. Single KO cells displayed a decrease in receptor ubiquitylation and an increase in phosphorylation compared to control. DKO cells had no detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependentinvitrowound healingvialive-cell time-lapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhancesinvitrowound healing. We propose that c-Cbl and Cbl-b are promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Impact of Plastic-Related Compounds on P-Glycoprotein and Breast Cancer Resistance Protein In Vitro.

Plastic in oceans degrades to microplastics and nanoplastics, causing various problems for marine fauna and flora. Recently, microplastic has been detected in blood, breast milk and placenta, underlining their ability to enter the human body with still unknown effects. In addition, plastic contains other compounds such as plasticizers, antioxidants or lubricants, whose impact on human health is also elusive. On the cellular level, two transporters involved in cell protection and detoxification of xenobiotic compounds are the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Despite the great importance of these proteins to maintain the correct cellular balance, their interaction with plastic and related products is evasive. In this study, the possible interaction between different plastic-related compounds and these two transporters was investigated. Applying virtual compound screening and molecular docking of more than 1000 commercially available plastic compounds, we identified candidates most probably interacting with these two transporters. Cytotoxicity and uptake assays confirmed their toxic interaction on P-glycoprotein-overexpressing CEM/ADR5000 and BCRP-overexpressing MDA-MD-231-BCRP cell lines. To specifically visualize the results obtained on the P-glycoprotein inhibitor 2,2'-methylenebis(6-tert-butyl-4-methylphenol), we performed live cell time-lapse microscopy. Confocal fluorescence microscopy was used to understand the behavior of the molecule and the consequences that it has on the uptake of the well-known substrate doxorubicin and, in comparison, with the known inhibitor verapamil. Based on the results, we provide evidence that the compound in question is an inhibitor of the P-glycoprotein. Moreover, it is also possible that 2,2'-methylenebis(6-tert-butyl-4-methylphenol), together with three other compounds, may also inhibit the breast cancer resistance protein. This discovery implies that plastic-related compounds can not only harm the human body but can also inhibit detoxifying efflux pumps, which increases their toxic potential as these transporters lose their physiological functions.

Analysis of Mouse Brain Sections by Live-cell Time-lapse Confocal Microscopy.

The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.

Novel application of live imaging to determine the functional cell biology of endothelial-to-mesenchymal transition (EndMT) within a liver-on-a-chip platform

ObjectiveImaging endothelial cell behaviour under physiological conditions, particularly those associated with chronic fibrotic pathologies, is an incredibly challenging endeavour. While short-term assessments (hours) can be achieved with techniques such as intravital microscopy, vascular changes often occur over days and weeks which is unfeasible with current imaging techniques. These challenges are exemplified within the liver where liver sinusoidal endothelial cells (LSECs) are known to undergo dramatic changes termed endothelial-to-mesenchymal transition (EndMT) during fibrotic liver disease. Despite the established presence of EndMT in liver disease, the inaccessibility of viable liver tissue, and simplicity of 2D culture techniques has meant, the role of EndMT during disease progression remains largely undetermined. This study describes the development of novel fluorescent EndMT reporters to identify, track, and characterise the migratory behaviour of EndMT cells. We show that liver-on-a-chip (LOAC) platforms provide a flexible, optically accessible, and physiologically relevant microenvironment to study the vascular dynamics of EndMT during liver disease.MethodsIdentification, creation, and application of an EndMT-specific fluorescent reporter construct (EndMT-Rep). Transduction of EC using lentiviral packaged CNN1-eGFP construct as an inducible EndMT-Rep (CNN1-Rep) to 2D, 3D, and 4D imaging techniques for fixed and live cell imaging. Combined application of live and fixed imaging technologies to measure EndMT using CNN1-Rep on LOAC platform under physiological conditions. Demonstration of the high-resolution single-cell EndMT tracking by live cell time-lapse microscopy and with post-acquisition processing to perform a comparative study of CNN1-Rep and healthy LSECs within a NASH-like LOAC microenvironment.ConclusionsLOAC enables prolonged, multi-platform imaging of endothelial cell sub-populations such as those undergoing EndMT in 2D and 3D cultures. Our study highlights the application of EndMT reporters, such as CNN1-Rep, to provide high-resolution imaging of EndMT behaviour for the first time under physiologically relevant liver microenvironment. Overall, these methods reveal the adaptability and impact of live-cell imaging on uncovering vascular behaviours, such as EndMT, that are unattainable in viable tissue or conventional 2D in vitro experiments.

Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy

Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform. Here, we developed a fully automated pipeline for camera characterization based solely on thermally generated signal, and implemented it in the popular open-source software Micro-Manager and ImageJ/Fiji. Besides supplying the conventional camera maps of noise, offset and gain, our pipeline also gives access to dark current and thermal noise as functions of the exposure time. This allowed us to avoid structural bias in single-molecule localization microscopy (SMLM), which without correction is substantial even for scientific-grade, cooled cameras. In addition, our approach enables high-quality 3D super-resolution as well as live-cell time-lapse microscopy with cheap, industry-grade cameras. As our approach for camera characterization does not require any user interventions or additional hardware implementations, numerous correction algorithms that rely on camera characterization become easily applicable.

Mining of Single-Cell Signaling Time-Series for Dynamic Phenotypes with Clustering.

Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these experiments and the heterogenous nature of signaling responses due to cell-cell variability hinder the exploration of such datasets. The population averages insufficiently describe the dynamics, yet finding prototypic dynamic patterns that relate to different cell fates is difficult when mining thousands of time-series. Here we demonstrate a protocol where we identify such dynamic phenotypes in a population of PC-12 cells that respond to a range of sustained growth factor perturbations. We use Time-Course Inspector, a free R/Shiny web application to explore and cluster single-cell time-series.

A Petri-Dish with Micromolded Pattern as a Coordinate Indicator for Live-Cell Time Lapse Microscopy

A Petri-Dish with Micromolded Pattern as a Coordinate Indicator for Live-Cell Time Lapse Microscopy

Mechanics of microtubule organizing center clustering and spindle positioning in budding yeast Cryptococcus neoformans.

The dynamic process of mitotic spindle assembly depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before forming the mitotic spindle, multiple visible microtubule organizing centers (MTOCs) coalesce into a single focus to serve as an SPB in the pathogenic budding yeast, Cryptococcus neoformans. To explain this unusual phenomenon in the fungal kingdom, we propose a "search and capture" model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and capture each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screen various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning-a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintains spatiotemporal spindle localization.

Harnessing Microfluidic Single Cell (MF-SC) and 3D “Organ-on-Chip” Assay Technologies for the Investigation of Immunotherapy and Study of Tumor Microenvironment (TME) in Antibody-Resistant Non-Hodgkin Lymphoma (NHL)

Abstract Introduction: Interactions between tumor and the TME occur through direct cell contacts and secreted factors such as cytokines and growth factors may influence behavior of tumor cells and response to therapy. Development of clinically applicable tumor models for screening therapeutics is desired to enhance our understanding of these mechanisms. We harnessed a novel MF-SC technology and an innovative 3D biomimetic tumor model to improve our understanding of the role of TME with immunotherapy and NHL tumor biology. Methods: Rituximab (R)- and obinutuzumab (O)-resistant (RR & OR) B-NHL cells (SUDHL4, SUDHL10 & Raji) were developed for evaluation of anti-CD20 resistance. Patient derived cell lines were obtained from newly diagnosed DLBCL patients. Functional response of anti-CD20 resistant & susceptible B-NHL cells were determined at SC levels in a customized MF droplet array platform. Single CD16+ NK cells were co-encapsulated with B-NHL cells at 1:1 effector-target (E:T) ratio in picoliter-volume droplets in presence/absence of R & O. Dynamic cellular interaction between E-T was monitored via live-cell time-lapse microscopy over 4-6 hrs to assess interaction profiles (eg, contact duration, frequency, and single vs serial interaction) and functional cytotoxicity. Target cell kill kinetics was assessed by loss of Calcein AM & ethidium homodimer uptake. Further, we used systems biology analyses (ie, Ingenuity Pathway Analysis (IPA) & Gene Set Enrichment Analysis (GSEA)) to identify associated biologic pathways. Additionally, the MF-SC platform was used to develop microscale 3D multicellular constructs to monitor effect of cancer-stroma interaction in a simulated TME. Heterogeneous tumor spheroids were generated using composite alginate hydrogels and varying combinations of NHL cells, fibroblasts & immune cells. Effect of TME (ie, cell composition and biological activity of immune cells) in the presence/absence of lenalidomide was evaluated based on extent of target cell survival/death (differentially labeled using CellTrace cell labeling & tracking dyes). Results: Development of anti-CD20 resistance in B-NHL cells resulted in decreased expression of CD20, loss of R binding, and decreased cytotoxic activity. Using IPA and GSEA systems biology analyses of RR and OR B-NHL cells, we observed conserved down-regulation of IFN-α/γ, TNF-α, inflammatory response, hypoxia and complement response pathways, and upregulation of MYC targets, which predicted overall immune suppression in all CD20 resistant cells. With MF-SC, we determined that NK cells induced variable duration of contacts with SUDHL10 cells irrespective of anti-CD20 resistance. While the single or multiple contacts made between NK & target cells occurred for an average of 45 minutes, addition of R or O resulted in significantly extended contact time (77 & 79 minutes; P Conclusions: We identified that resistance to R & O occurs through biologically conserved mechanisms. Results in MF-SCs revealed that interactions between NHL tumor and immune cells is a highly dynamic process occurring through multiple cell contacts and that presence of R & O shift these kinetics; this was an important factor in determining cytotoxic immune activity. In addition, experiments based on reconstituted 3D NHL tumor spheroids validated the influence of TME mediating the activity of immunomodulatory drugs. This innovative MF-based 3D NHL spheroid model may be further leveraged for the discovery of immune-based therapeutic applications. Download : Download high-res image (326KB) Download : Download full-size image Disclosures Evens: Celgene: Consultancy; Merck: Consultancy; • Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Pharmacyclics: Consultancy; Kite Pharma: Consultancy; Millennium: Consultancy; Affimed: Consultancy; Amgen: Consultancy.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells.

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

Establishing Regulation of a Dynamic Process by Ypt/Rab GTPases : A Case for Cisternal Progression.

The prevailing model for transport within the Golgi is cisternal maturation. This process can be viewed as switching of cisternal markers using live-cell microscopy in yeast cells since the Golgi cisternae are separated, as opposed to the stacked Golgi seen in other organisms. It is also possible to determine which trafficking machinery components are required for this process by studying mutants depleted for these components. However, determining how cisternal maturation is regulated has been more challenging. The key for demonstrating regulation is not solely to stop the maturation when depleting a vesicular trafficking component, but also to illustrate a change in the speed. The obvious candidates for such regulation are the Ypt/Rab GTPases because of their established mode of action as regulators. Since the precise localization of the Golgi Ypts, Ypt1 and Ypt31, to specific Golgi cisternae has been controversial, we started by carefully colocalizing these Ypts with the Golgi cisternal markers using two independent approaches: immunofluorescence and live-cell microscopy. Next, the opposite effects of depletion versus constitutively activating Ypt mutations on Golgi morphology were determined. Finally, the ability of constitutively activating Ypt mutations to accelerate a specific cisternal-maturation step was established by live-cell time-lapse microscopy. Using these approaches, we defined three dynamic Golgi cisternae, early, intermediate, and late, separated two independent steps of cisternal maturation and showed their regulation by two different Ypts. Here, we discuss the major principles and precautions needed for each phase of this research, the main point being definition of a new criterion for regulation of a dynamic process versus requirement of a machinery structural component: acceleration of the dynamics.

Development of a reliable bovine neuronal cell culture system and labeled recombinant bovine herpesvirus type-1 for studying virus-host cell interactions

Bovine herpesvirus type 1 (BoHV-1) is the viral causative agent of infectious bovine rhinotracheitis and a component of the bovine respiratory complex commonly referred to as shipping fever in calves. BoHV-1 is also responsible for losses of aborted calves and reductions in dairy productivity. BoHV-1 belongs to the neurotropic alphaherpesviruses which have a predilection to infect and establish latency in sensory neurons. Neuronal cell cultures provide a useful platform for experiments investigating neuronal entry, retrograde and anterograde transport, and the establishment of latency. Rodent neuronal cell lines and primary rabbit neuronal cells have been utilized for BoHV-1, though a reliable host-specific neuronal cell culture system has not been developed. In this study, BoHV-1 readily infected bovine-derived immortalized neuronal progenitor cells (FBBC-1) differentiated in cell culture producing neurite-like projections and exhibiting neuronal cell markers NeuN and L1CAM. FBBC-1 cells expressed both nectin-1 and nectin-2 alphaherpesvirus receptors on their cell surfaces, however, nectin-2 was detected in much greater abundance than nectin-1. To facilitate investigations of BoHV-1 infection, a recombinant BoHV-1 virus expressing the green fluorescent protein (GFP) cloned into a bacterial artificial chromosome (BAC) was used to generate an mCherry-VP26 fusion protein. The BoHV-1 GFP expressing VP26mCherry labeled virus infected differentiated FBBC-1 cells as evidenced by the production of infectious virions and the expression of both GFP and mCherry fluorophores. Time-lapse live cell microscopy revealed the presence of mCherry fluorescent capsids in neuronal projections immediately after virus entry moving retrograde in a saltatory manner. Proximity ligation assays (PLA) using MDBK cells demonstrated that BoHV-1 glycoprotein D (gD) interacted more efficiently with nectin-1 than nectin-2. However, the gD interaction with nectin-2 predominated in differentiated FBBC-1 cells in comparison to the gD nectin-1 interaction. The efficiently differentiated FBBC-1 neuronal cell line and fluorescently labeled BoHV-1 virions will assist experimentation aiming to elucidate specific mechanisms of virus entry and transport in a homologous bovine neuronal cell culture system.

Plasmodium falciparum parasites exit the infected erythrocyte after haemolysis with saponin and streptolysin O.

Malaria is caused by unicellular parasites of the genus Plasmodium, which reside in erythrocytes during the clinically relevant stage of infection. To separate parasite from host cell material, haemolytic agents such as saponin are widely used. Previous electron microscopy studies on saponin-treated parasites reported both, parasites enclosed by the erythrocyte membrane and liberated from the host cell. These ambiguous reports prompted us to investigate haemolysis by live-cell time-lapse microscopy. Using either saponin or streptolysin O to lyse Plasmodium falciparum-infected erythrocytes, we found that ring-stage parasites efficiently exit the erythrocyte upon haemolysis. For late-stage parasites, we found that only approximately half were freed, supporting the previous electron microscopy studies. Immunofluorescence imaging indicated that freed parasites were surrounded by the parasitophorous vacuolar membrane. These results may be of interest for future work using haemolytic agents to enrich for parasite material.