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The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores.

Parkinson's disease, an aging-associated neurodegenerative disorder, is characterised by nigrostriatal pathway dysfunction caused by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Human in vitro models are enabling the study of the dopaminergic neurons' loss, but not the dysregulation within the dopaminergic network in the nigrostriatal pathway. Additionally, these models do not incorporate aging characteristics which potentially contribute to the development of Parkinson's disease. Here we present a nigrostriatal pathway model based on midbrain-striatum assembloids with inducible aging. We show that these assembloids can develop characteristics of the nigrostriatal connectivity, with catecholamine release from the midbrain to the striatum and synapse formation between midbrain and striatal neurons. Moreover, Progerin-overexpressing assembloids acquire aging traits that lead to early neurodegenerative phenotypes. This model shall help to reveal the contribution of aging as well as nigrostriatal connectivity to the onset and progression of Parkinson's disease., Competing Interests: Competing interests: J.C.S. is a co-inventor on a patent covering the generation of the here-described midbrain organoids (WO2017060884A1). Furthermore, J.C.S. is a co-founder and shareholder of the company OrganoTherapeutics which makes use of midbrain organoid technology. The other authors declare no competing interests. Ethical approval: Ethics Review Panel (ERP) of the University of Luxembourg and the national Luxembourgish research ethics committee (CNER, Comité National d’Ethique de Recherche) have approved the work with induced pluripotent stem cells (iPSCs). CNER No. 201901/01; ivPD., (© 2024. The Author(s).)

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa., (© 2024. The Author(s).)

Many bacteria produce antimicrobial compounds such as lantibiotics to gain advantage in the competitive natural environments of microbiomes. Epilancins constitute an until now underexplored family of lantibiotics with an unknown ecological role and unresolved mode of action. We discovered production of an epilancin in the nasal isolate Staphylococcus epidermidis A37. Using bioinformatic tools, we found that epilancins are frequently encoded within staphylococcal genomes, highlighting their ecological relevance. We demonstrate that production of epilancin A37 contributes to Staphylococcus epidermidis competition specifically against natural corynebacterial competitors. Combining microbiological approaches with quantitative in vivo and in vitro fluorescence microscopy and cryo-electron tomography, we show that A37 enters the corynebacterial cytoplasm through a partially transmembrane-potential-driven uptake without impairing the cell membrane function. Upon intracellular aggregation, A37 induces the formation of intracellular membrane vesicles, which are heavily loaded with the compound and are essential for the antibacterial activity of the epilancin. Our work sheds light on the ecological role of epilancins for staphylococci mediated by a mode of action previously unknown for lantibiotics., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models., (© 2023. The Author(s).)

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, lipid II, and lipid III WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development., Competing Interests: Declaration of interests The following authors, A.J.P., C.A., A.N., A.L.S., L.L.L., D.H., and K.L., declare competing financial interests because they are employees and consultants of NovoBiotic Pharmaceuticals. A patent US 11,203,616 B2 was issued on 12/21/2021 and describes the use of clovibactin (Novo29) and as an antibiotic, as well as the pharmaceutical composition and antibiotic use of derivatives., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

Bacterial cell wall biosynthesis is the target of many important antibiotics. Its spatiotemporal organization is closely coordinated with cell division. However, the role of peptidoglycan synthesis within cell division is not fully understood. Even less is known about the impact of antibiotics on the coordination of these two essential processes. Visualizing the essential cell division protein FtsZ and other key proteins in Staphylococcus aureus , we show that antibiotics targeting peptidoglycan synthesis arrest cell division within minutes of treatment. The glycopeptides vancomycin and telavancin completely inhibit septum constriction in all phases of cell division. The beta-lactam oxacillin stops division progress by preventing recruitment of the major peptidoglycan synthase PBP2 to the septum, revealing PBP2 as crucial for septum closure. Our work identifies cell division as key cellular target of these antibiotics and provides evidence that peptidoglycan synthesis is the essential driving force of septum constriction throughout cell division of S. aureus .

Common light sheet microscopy comes with a trade-off between light sheet width defining the optical sectioning and the usable field of view arising from the divergence of the illuminating Gaussian beam. To overcome this, low-diverging Airy beams have been introduced. Airy beams, however, exhibit side lobes degrading image contrast. Here, we constructed an Airy beam light sheet microscope, and developed a deep learning image deconvolution to remove the effects of the side lobes without knowledge of the point spread function. Using a generative adversarial network and high-quality training data, we significantly enhanced image contrast and improved the performance of a bicubic upscaling. We evaluated the performance with fluorescently labeled neurons in mouse brain tissue samples. We found that deep learning-based deconvolution was about 20-fold faster than the standard approach. The combination of Airy beam light sheet microscopy and deep learning deconvolution allows imaging large volumes rapidly and with high quality.

Light sheet fluorescence microscopy (LSFM) is an important tool in developmental biology. In this microscopy technique confocal line detection is often used to improve image contrast. To this end, the image of the illuminating scanned focused laser beam must be mapped onto a line detector. This is not trivial for long-term observations, since the spatial position of the laser beam and therefore its image on the detector may drift. The problem is aggravated in two-photon excitation LSFM, since pulsed laser light sources exhibit a lower laser beam pointing stability than continuous wave lasers. Here, we present a procedure for automatic synchronization between the excitation laser and detector, which does not require any additional hardware components and can therefore easily be integrated into existing systems. Since the recorded images are affected by noise, a specific, noise-tolerant focus metric was developed for calculating the relative displacement, which also allows for autofocusing in the detection direction. Furthermore, we developed an image analysis approach to determine a possible tilt of the excitation laser, which is executed in parallel to the autofocusing and enables the measurement of three solid angles. This allows to automatically correct for the tilting during a measurement. We demonstrated our approach by the observation of the migration of oligodendrocyte precursor cells in two-day-old fluorescent Tg(olig2:eGFP) reporter zebrafish larvae over a time span of more than 20 hours., (© 2022 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Microscopy is a powerful method to evaluate the direct effects of antibiotic action on the single cell level. As with other methodologies, microscopy data is obtained through sufficient biological and technical replicate experiments, where evaluation of the sample is generally followed over time. Even if a single antibiotic is tested for a defined time, the most certain outcome is large amounts of raw data that requires systematic analysis. Although microscopy is a helpful qualitative method, the recorded information is stored as defined quantifiable units, the pixels. When this information is transferred to diverse bioinformatic tools, it is possible to analyze the microscopy data while avoiding the inherent bias associated to manual quantification. Here, we briefly describe methods for the analysis of microscopy images using open-source programs, with a special focus on bacteria exposed to antibiotics., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Expansion microscopy enables super-resolved visualization of specimen without the need of highly sophisticated and expensive optical instruments. Instead, the method is executed with conventional chemicals and lab equipment. Imaging of bacteria is performed using standard fluorescence microscopy. This chapter describes a protocol for the expansion microscopy of Bacillus subtilis expressing DivIVA-GFP. In addition, the cell wall was labeled by wheat germ agglutinin. Here, we place emphasis on the challenges of selecting the protein and organism of interest., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo., (© 2022. The Author(s).)

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s -1 in vivo., (© 2021. The Author(s).)

Background: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS)., Methods: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2 podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants., Results: Crb2 podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O -glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response., Conclusions: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS., (Copyright © 2021 by the American Society of Nephrology.)

Light-sheet fluorescence microscopy (LSFM) helps investigate small structures in developing cells and tissue for three-dimensional localization microscopy and large-field brain imaging in neuroscience. Lattice light-sheet microscopy is a recent development with great potential to improve axial resolution and usable field sizes, thus improving imaging speed. In contrast to the commonly employed Gaussian beams for light-sheet generation in conventional LSFM, in lattice light-sheet microscopy an array of low diverging Bessel beams with a suppressed side lobe structure is used. We developed a facile elementary lattice light-sheet microscope using a micro-fabricated fixed ring mask for lattice light-sheet generation. In our setup, optical hardware elements enable a stable and simple illumination path without the need for spatial light modulators. This setup, in combination with long-working distance objectives and the possibility for simultaneous dual-color imaging, provides optimal conditions for imaging extended optically cleared tissue samples. We here present experimental data of fluorescently stained neurons and neurites from mouse hippocampus following tissue expansion and demonstrate the high homogeneous resolution throughout the entire imaged volume. Utilizing our purpose-built lattice light-sheet microscope, we reached a homogeneous excitation and an axial resolution of 1.2 µm over a field of view of (333 µm) 2 .

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca 2+ -daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

Ribosomes are formed of a small and a large subunit (SSU/LSU), both consisting of rRNA and a plethora of accessory proteins. While biochemical and genetic studies identified most of the involved proteins and deciphered the ribosomal synthesis steps, our knowledge of the molecular dynamics of the different ribosomal subunits and also of the kinetics of their intracellular trafficking is still limited. Adopting a labelling strategy initially used to study mRNA export we were able to fluorescently stain the SSU in vivo. We chose DIM2/PNO1 (Defective In DNA Methylation 2/Partner of NOb1) as labelling target and created a stable cell line carrying an inducible SNAP-DIM2 fusion protein. After bulk labelling with a green fluorescent dye combined with very sparse labelling with a red fluorescent dye the nucleoli and single SSU could be visualized simultaneously in the green and red channel, respectively. We used single molecule microscopy to track single SSU in the nucleolus and nucleoplasm. Resulting trajectory data were analyzed by jump-distance analysis and the variational Bayes single-particle tracking approach. Both methods allowed identifying the number of diffusive states and the corresponding diffusion coefficients. For both nucleoli and nucleoplasm we could identify mobile (D = 2.3-2.8 µm 2 /s), retarded (D = 0.18-0.31 µm 2 /s) and immobilized (D = 0.04-0.05 µm 2 /s) SSU fractions and, as expected, the size of the fractions differed in the two compartments. While the fast mobility fraction matches perfectly the expected nuclear mobility of the SSU (D = 2.45 µm 2 /s), we were surprised to find a substantial fraction (33%) of immobile SSU in the nucleoplasm, something not observed for inert control molecules., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The goal of understanding the architecture of neural circuits at the synapse level with a brain-wide perspective has powered the interest in high-speed and large field-of-view volumetric imaging at subcellular resolution. Here, we developed a method combining tissue expansion and light-sheet fluorescence microscopy to allow extended volumetric super resolution high-speed imaging of large mouse brain samples. We demonstrate the capabilities of this method by performing two color fast volumetric super resolution imaging of mouse CA1 and dentate gyrus molecular-, granule cell-, and polymorphic layers. Our method enables an exact evaluation of granule cell and neurite morphology within the context of large cell ensembles spanning several orders of magnitude in resolution. We found that imaging a brain region of 1    mm 3 in super resolution using light-sheet fluorescence expansion microscopy is about 17-fold faster than imaging the same region by a current state-of-the-art high-resolution confocal laser scanning microscope.

Single molecule microscopy techniques allow to visualize the translocation of single transport receptors and cargo molecules or particles through nuclear pore complexes. These data indicate that cargo molecule import into the nucleus takes less than 10ms and nuclear export of messenger RNA (mRNA) particles takes 50-350ms, up to several seconds for extremely bulky particles. This review summarizes and discusses experimental results on transport of nuclear transport factor 2 (NTF2), importin β and mRNA particles. Putative regulatory functions of importin β for the NPC transport mechanism and the RNA helicase Dbp5 for mRNA export kinetics are discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

While transplantation represents a key tool for assessing in vivo functionality of neural stem cells and their suitability for neural repair, little is known about the integration of grafted neurons into the host brain circuitry. Rabies virus-based retrograde tracing has developed into a powerful approach for visualizing synaptically connected neurons. Here, we combine this technique with light sheet fluorescence microscopy (LSFM) to visualize transplanted cells and connected host neurons in whole-mouse brain preparations. Combined with co-registration of high-precision three-dimensional magnetic resonance imaging (3D MRI) reference data sets, this approach enables precise anatomical allocation of the host input neurons. Our data show that the same neural donor cell population grafted into different brain regions receives highly orthotopic input. These findings indicate that transplant connectivity is largely dictated by the circuitry of the target region and depict rabies-based transsynaptic tracing and LSFM as efficient tools for comprehensive assessment of host-donor cell innervation., Competing Interests: O.B. is a co-founder and owns equity of LIFE & BRAIN GmbH. The remaining authors declare no competing financial interests.

The physiological function of epithelia depends on an asymmetric distribution of their membrane domains. Polarity proteins play a crucial role for distribution processes, however, little is known about their mobility in epithelial cells. In this study, we analyzed the intracellular and plasma-membrane-associated mobility of fluorescence-labeled Crb3A and Crb3B. Both variants belong to the Crumbs protein family, which control size and identity of apical membranes in epithelial cells. Fluorescence recovery after photo-bleaching measurements revealed different mobilities for the two Crb3 variants. They also differentially affected mobility and localization of the Pals1/Mpp5 protein, which binds to Crb3A but not to Crb3B. In addition, tracking of intracellular vesicles indicated that Crb3A containing vesicles are slightly more immobile than Crb3B ones. Taken together, our data revealed different intracellular mobility patterns for Crb3A and Crb3B., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Using a combined approach of pulse chase labeling and single-particle tracking of Crb3A or 3B loaded vesicles we collected trajectories of different vesicle population in living podocyte cells and evaluated statistically their different mobility patterns. Differences in their intracellular mobility and in their directed transport correspond well to the role of Crb3A and 3B in renal plasma membrane sorting (Djuric et al., 2016) [1].

RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA., (© 2015. Published by The Company of Biologists Ltd.)

NKCS is an improved mutant of the bioactive peptide NK-2, which shows strong activity against Escherichia coli and low toxicity towards human cells. The different activity demonstrates the relevance of the physico-chemical nature of the target membrane for the biological effect of this peptide. We studied the effect of this potent antimicrobial peptide on model membranes by activity studies, differential scanning calorimetry, single molecule tracking and tracer efflux experiments. We found that NKCS severely distorted, penetrated and perforated model lipid membranes that resembled bacterial membranes, but not those that were similar to human cell membranes. The interactions of NKCS with phosphatidylethanolamine, which is abundant in bacterial membranes, were especially strong and are probably responsible for its antimicrobial activity.