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All Posts in Category: Recent Publications

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Ablation of arginyl‐tRNA‐protein transferase in oligodendrocytes impairs central nervous system myelination

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Anabela Palandri, Laura Vanesa Bonnet, Maria Gimena Farias, Marta Elena Hallak, Mauricio Raul Galiano

Addition of arginine (Arg) from tRNA can cause major alterations of structure and function of protein substrates. This post-translational modification, termed protein arginylation, is mediated by the enzyme arginyl-tRNA-protein transferase 1 (Ate1). Arginylation plays essential roles in a variety of cellular processes, including cell migration, apoptosis, and cytoskeletal organization. Ate1 is associated with neuronal functions such as neurogenesis and neurite growth. However, the role of Ate1 in glial development, including oligodendrocyte (OL) differentiation and myelination processes in the central nervous system, is poorly understood. The present study revealed a peak in Ate1 protein expression during myelination process in primary cultured OLs. Post-transcriptional downregulation of Ate1 reduced the number of OL processes, and branching complexity, in vitro. We conditionally ablated Ate1 from OLs in mice using 2′,3′-cyclic nucleotide 3′-phosphodiesterase-Cre promoter (“Ate1-KO” mice), to assess the role of Ate1 in OL function and axonal myelination in vivo. Immunostaining for OL differentiation markers revealed a notable reduction of mature OLs in corpus callosum of 14-day-old Ate1-KO, but no changes in spinal cord, in comparison with wild-type controls. Local proliferation of OL precursor cells was elevated in corpus callosum of 21-day-old Ate1-KO, but was unchanged in spinal cord. Five-month-old Ate1-KO displayed reductions of mature OL number and myelin thickness, with alterations of motor behaviors. Our findings, taken together, demonstrate that Ate1 helps maintain proper OL differentiation and myelination in corpus callosum in vivo, and that protein arginylation plays an essential role in developmental myelination.

Authors: Anabela Palandri, Laura Vanesa Bonnet, Maria Gimena Farias, Marta Elena Hallak, Mauricio Raul Galiano

Article:

Ablation of arginyl-tRNA-protein transferase in oligodendrocytes impairs central nervous system myelination

Palandri A., Bonnet L.V., Farias M.G., Hallak M.E:, Galiano M.R.. Glia. October 2021. https://doi.org/10.1002/glia.24107

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The antimicrobial peptide Polybia-MP1 differentiates membranes with the hopanoid, diplopterol from those with cholesterol

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Dayane S. Alvares, Mariela R. Monti, Joao Neto Ruggiero, Natalia Wilke

Polybia-MP1 is an antimicrobial peptide that shows a decreased activity in membranes with cholesterol (CHO). Since it is now accepted that hopanoids act as sterol-surrogates in some sterol-lacking bacteria, we here inquire http://ciquibic.fcq.unc.edu.ar/integrantes/wilke-natalia/about the impact of Polybia-MP1 on membranes containing the hopanoid diplopterol (DP) in comparison to membranes with CHO. We found that, despite the properties induced on lipid membranes by DP are similar to those induced by CHO, the effect of Polybia-MP1 on membranes with CHO or DP was significantly different. DP did not prevent dye release from LUVs, nor the insertion of Polybia-MP1 into monolayers, and peptide-membrane affinity was higher for those with DP than with CHO. Zeta potentials (ζ ) for DP-containing LUVs showed a complex behavior at increasing peptide concentration. The effect of the peptide on membrane elasticity, investigated by nanotube retraction experiments, showed that peptide addition softened all membrane compositions, but membranes with DP got stiffer at long times. Considering this, and the ζ results, we propose that peptides accumulate at the interface adopting different arrangements, leading to a non-monotonic behavior. Possible correlations with cell membranes were inquired testing the antimicrobial activity of Polybia-MP1 against hopanoid-lacking bacteria pre-incubated with DP or CHO. The fraction of surviving cells was lower in cultures incubated with DP compared to those incubated with CHO. We propose that the higher activity of Polybia-MP1 against some bacteria compared to mammalian cells is not only related to membrane electrostatics, but also the composition of neutral lipids, particularly the hopanoids, could be important.

Keywords: Targeting of AMPs, Bacterial resistance, Membrane dynamic elasticity, Optical tweezers, Lytic activity, Zeta potential

Authors: Dayane S. Alvares, Mariela R. Monti, Joao Neto Ruggiero, Natalia Wilke

Article:

The antimicrobial peptide Polybia-MP1 differentiates membranes with the hopanoid, diplopterol from those with cholesterol

Alvares D.S, Monti M.R., Ruggiero Neto J., N Wilke. BBA Advances Volume 1, 2021, 100002. https://doi.org/10.1016/j.bbadva.2021.100002

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Unraveling the etiology of myelin disorders: the P2 case in Charcot-Marie-Tooth disease

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Julius pusterla, Guillermo G. Montich, Rafael G. Oliveira. 

There are several examples of single mutations that lead to a well-defined disease through a well-known mechanism. In other cases, a collection of mutations of the same protein produces a pathology with different degrees of severity. The accompanying work by Uusitalo et al. studies several mutants of the fatty acid binding protein P2 of the peripheral nervous system myelin. They conserve the native tertiary structure but a remarkable difference in the capacity to interact with lipids. This could be a clue to unravel the complex way in which these mutations affect myelin structure and function in a variant of Charcot-Marie-Tooth disease. Comment on: https://doi.org/10.1111/febs.16079.

Keywords: FABP; P2; emergence; myelin disorders; protein-lipid interaction.

Authors: Julius pusterla, Guillermo G. Montich, Rafael G. Oliveira. 

Article:

Pusterla J, Montich GG, Oliveira RG. Unraveling the etiology of myelin disorders: the P2 case in Charcot-Marie-Tooth disease. FEBS J. 2021 Jul 29. doi: 10.1111/febs.16131. Epub ahead of print. PMID: 34327848.

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Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M

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Cardozo Gizzi et al. Nat Protoc. 2020

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4–5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1–2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

 

Authors: Cardozo Gizzi AM, Espinola SM, Gurgo J, Houbron C, JB plug, DI Cats, Nollmann M.

 

Article: Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M. Cardozo Gizzi AM, Espinola SM, Gurgo J, Houbron C, JB plug, DI Cats, Nollmann M. Nat Protoc. 2020 Jan 22. doi: 10.1038/s41596-019-0269-9

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