Membrane nanotubes are ancient machinery for cell-to-cell communication and transport. Their interference with the immune system.

Nanotubular connections between mammalian cell types came into the focus only two decades ago, when "live cell super-resolution imaging" was introduced. Observations of these long-time overlooked structures led to understanding mechanisms of their growth/withdrawal and exploring some key genetic and signaling factors behind their formation. Unbelievable level of multiple supportive collaboration between tumor cells undergoing cytotoxic chemotherapy, cross-feeding" between independent bacterial strains or "cross-dressing" collaboration of immune cells promoting cellular immune response, all via nanotubes, have been explored recently. Key factors and "calling signals" determining the spatial directionality of their growth and their overall in vivo significance, however, still remained debated. Interestingly, prokaryotes, including even ancient archaebacteria, also seem to use such NT connections for intercellular communication. Herein, we will give a brief overview of current knowledge of membrane nanotubes and depict a simple model about their possible "historical role".

Live cell superresolution-structured illumination microscopy imaging analysis of the intercellular transport of microvesicles and costimulatory proteins via nanotubes between immune cells.

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication. These tethers can form spontaneously between many cell types, including cells of the immune and nervous systems. Traffic of viral proteins, vesicles, calcium ions, mRNA, miRNA, mitochondria, lysosomes and membrane proteins/raft domains have all been reported so far via the open ended tunneling nanotubes (TNTs). Recently we reported on existence of plasma membrane derived GM1/GM3 ganglioside enriched microvesicles and costimulatory proteins in nanotubes connecting B lymphocytes, the way they are formed and transported across TNTs, however, still remained unclear. Here, using live cell confocal and Structured Illumination (SR-SIM) superresolution imaging, we show that B cells respond to bacterial (Cholera) toxin challenge by their subsequent internalization followed by rapid formation of intracellular microvesicles (MVs). These MVs are then transported between adjacent B cells via nanotubes. Selective transport-inhibition analysis of two abundant motor proteins in these cell types demonstrated that actin-based non-muscle myosin 2A dominantly mediates intercellular MV-transport via TNTs, in contrast to the microtubule-based dynein, as shown by the unchanged transport after inhibition of the latter. As suggested by SR-SIM images of GFP-CD86 transfected macrophages, these costimulatory molecules may be transferred by unusually shaped MVs through thick TNTs connecting macrophages. In contrast, in B cell cultures the same GFP-CD86 is dominantly transported along the membrane wall of TNTs. Such intercellular molecule-exchange can consequently improve the efficiency of antigen-dependent T cell activation, especially in macrophages with weak costimulator expression and T cell activation capacity. Such improved T cell activating potential of these two cell types may result in a more efficient cellular immune response and formation of immunological memory. The results also highlight the power of superresolution microscopy to uncover so far hidden structural details of biological processes, such as microvesicle formation and transport.

Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.

The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes.

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca2+-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca2+ in regulation of TNT formation. Finally, we observed transport of various GM1/GM3+ vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

Synaptic mitochondria: a brain mitochondria cluster with a specific proteome.

The synapse is a particularly important compartment of neurons. To reveal its molecular characteristics we isolated whole brain synaptic (sMito) and non-synaptic mitochondria (nsMito) from the mouse brain with purity validated by electron microscopy and fluorescence activated cell analysis and sorting. Two-dimensional differential gel electrophoresis and mass spectrometry based proteomics revealed 22 proteins with significantly higher and 34 proteins with significantly lower levels in sMito compared to nsMito. Expression differences in some oxidative stress related proteins, such as superoxide dismutase [Mn] (Sod2) and complement component 1Q subcomponent-binding protein (C1qbp), as well as some tricarboxylic acid cycle proteins, including isocitrate dehydrogenase subunit alpha (Idh3a) and ATP-forming ß subunit of succinyl-CoA ligase (SuclA2), were verified by Western blot, the latter two also by immunohistochemistry. The data suggest altered tricarboxylic acid metabolism in energy supply of synapse while the marked differences in Sod2 and C1qbp support high sensitivity of synapses to oxidative stress. Further functional clustering demonstrated that proteins with higher synaptic levels are involved in synaptic transmission, lactate and glutathione metabolism. In contrast, mitochondrial proteins associated with glucose, lipid, ketone metabolism, signal transduction, morphogenesis, protein synthesis and transcription were enriched in nsMito. Altogether, the results suggest a specifically tuned composition of synaptic mitochondria. BIOLOGICAL SIGNIFICANCE: Neurons communicate with each other through synapse, a compartment metabolically isolated from the cell body. Mitochondria are concentrated in presynaptic terminals by active transport to provide energy supply for information transfer. Mitochondrial composition in the synapse may be different than in the cell body as some examples have demonstrated altered mitochondrial composition with cell type and cellular function in the muscle, heart and liver. Therefore, we posed the question whether protein composition of synaptic mitochondria reflects its specific functions. The determined protein difference pattern was in accordance with known functional specialties of high demand synaptic mitochondria. The data also suggest specifically tuned metabolic fluxes for energy production by means of interaction with glial cells surrounding the synapse. These findings provide possible mechanisms for dynamically adapting synaptic mitochondrial output to actual demand. In turn, an increased vulnerability of synaptic mitochondria to oxidative stress is implied by the data. This is important from theoretical but potentially also from therapeutic aspects. Mitochondria are known to be affected in some neurodegenerative and psychiatric disorders, and proteins with elevated level in synaptic mitochondria, e.g. C1qbp represent targets for future drug development, by which synaptic and non-synaptic mitochondria can be differentially affected.

TGFß activated kinase 1 (TAK1) at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR), receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R) and the innate receptor, Toll-like receptor 9 (TLR9). However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF) and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs), ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1) is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation.

Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans ­ in contrast to mice ­ the C3a peptide is most probably not involved directly in the T cell activation process.

The AC8 IgG3 monoclonal anti-cholesterol antibody modulates uptake and presentation of antigens for T cell activation.

Natural anti-cholesterol antibodies (ACHAs) exist in mammalian species, moreover their level sensitively changes in pathological situations, such as atherosclerosis or HIV infection. The conditions of their production and functional role, however, still remained ill defined. Recently we developed IgG3 type monoclonal ACHAs that selectively react with 'clustered cholesterol' of live immune cells, such as membrane microdomains (lipid rafts and caveolas). These antibodies inhibited HIV-1 infection of Th cells and macrophages by remodeling the HIV-1 receptor/coreceptor distribution in the plasma membrane of target cells. As a novel modulatory effect, here we show that the AC8 IgG3 monoclonal anti-cholesterol antibody (mACHA), but not the AC9 IgM mACHA, spontaneously bind to all professional APCs, such as murine macrophages (Mfs) or bone marrow derived dendritic cells (DCs) and B lymphocytes. Upon binding, AC8 mAb remarkably enhanced the efficiency of yeast uptake by macrophages, but not the uptake of OVA-Ig immune complexes by DCs. Binding to B lymphoma APCs, AC8 mAb remodeled their surface membrane by microclustering rafts and recruiting MHC-II and the CD80 costimulators to common microdomains. The modulated APCs induced an enhanced activation signaling (higher Ca(2+)-signals and NFAT1 activation) in Th cells conjugated with them, relative to untreated APCs. The results presented herein highlight the modulatory potential of the IgG3 type AC8 mAb on both innate and adaptive effector cell functions.