The premetazoan ancestry of the synaptic toolkit and appearance of first neurons

Neurons, especially when coupled with muscles, allow animals to interact with and navigate through their environment in ways unique to life on earth. Found in all major animal lineages except sponges and placozoans, nervous systems range widely in organization and complexity, with neurons possibly representing the most diverse cell-type. This diversity has led to much debate over the evolutionary origin of neurons as well as synapses, which allow for the directed transmission of information. The broad phylogenetic distribution of neurons and presence of many of the defining components outside of animals suggests an early origin of this cell type, potentially in the time between the first animal and the last common ancestor of extant animals. Here, we highlight the occurrence and function of key aspects of neurons outside of animals as well as recent findings from non-bilaterian animals in order to make predictions about when and how the first neuron(s) arose during animal evolution and their relationship to those found in extant lineages. With advancing technologies in single cell transcriptomics and proteomics as well as expanding functional techniques in non-bilaterian animals and the close relatives of animals, it is an exciting time to begin unraveling the complex evolutionary history of this fascinating animal cell type.publishedVersio

Evolutionary insights into premetazoan functions of the neuronal protein homer.

Reconstructing the evolution and ancestral functions of synaptic proteins promises to shed light on how neurons first evolved. The postsynaptic density (PSD) protein Homer scaffolds membrane receptors and regulates Ca(2+) signaling in diverse metazoan cell types (including neurons and muscle cells), yet its ancestry and core functions are poorly understood. We find that the protein domain organization and essential biochemical properties of metazoan Homer proteins, including their ability to tetramerize, are conserved in the choanoflagellate Salpingoeca rosetta, one of the closest living relatives of metazoans. Unlike in neurons, Homer localizes to the nucleoplasm in S. rosetta and interacts directly with Flotillin, a protein more commonly associated with cell membranes. Surprisingly, we found that the Homer/Flotillin interaction and its localization to the nucleus are conserved in metazoan astrocytes. These findings suggest that Homer originally interacted with Flotillin in the nucleus of the last common ancestor of metazoans and choanoflagellates and was later co-opted to function as a membrane receptor scaffold in the PSD

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery

Histone demethylase Lsd1 is required for the differentiation of neural cells in Nematostella vectensis

Chromatin regulation is a key process in development but its contribution to the evolution of animals is largely unexplored. Chromatin is regulated by a diverse set of proteins, which themselves are tightly regulated in a cell/tissue-specific manner. Using the cnidarian Nematostella vectensis as a basal metazoan model, we explore the function of one such chromatin regulator, Lysine specific demethylase 1 (Lsd1). We generated an endogenously tagged allele and show that NvLsd1 expression is developmentally regulated and higher in differentiated neural cells than their progenitors. We further show, using a CRISPR/Cas9 generated mutant that loss of NvLsd1 leads to developmental abnormalities. This includes the almost complete loss of differentiated cnidocytes, cnidarian-specific neural cells, as a result of a cell-autonomous requirement for NvLsd1. Together this suggests that the integration of chromatin modifying proteins into developmental regulation predates the split of the cnidarian and bilaterian lineages and constitutes an ancient feature of animal development.publishedVersio

Evolution of the ribbon-like organization of the Golgi apparatus in animal cells

The ‘‘ribbon,’’ a structural arrangement in which Golgi stacks connect to each other, is considered to be restricted to vertebrate cells. Although ribbon disruption is linked to various human pathologies, its functional role in cellular processes remains unclear. In this study, we investigate the evolutionary origin of the Golgi ribbon. We observe a ribbon-like architecture in the cells of several metazoan taxa suggesting its early emergence in animal evolution predating the appearance of vertebrates. Supported by AlphaFold2 modeling, we propose that the evolution of Golgi reassembly and stacking protein (GRASP) binding by golgin tethers may have driven the joining of Golgi stacks resulting in the ribbon-like configuration. Additionally, we find that Golgi ribbon assembly is a shared developmental feature of deuterostomes, implying a role in embryogenesis. Overall, our study points to the functional significance of the Golgi ribbon beyond vertebrates and underscores the need for further investigations to unravel its elusive biological roles

Nitrated α–Synuclein Immunity Accelerates Degeneration of Nigral Dopaminergic Neurons

The neuropathology of Parkinson's disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated alpha-synuclein (N-alpha-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.Nitrotyrosine (NT)-modified alpha-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated alpha-Syn. Mice immunized with the NT-modified C-terminal tail fragment of alpha-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-alpha-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss.These data show that NT modifications within alpha-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in alpha-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease

Comparative investigations on the regulation of SNARE complex assembly by Sec1/Munc18-like proteins

Membranfusionsereignisse zwischen den verschiedenen Kompartimenten eukaryotischer Zellen werden durch zwei konservierte Proteinfamilien vermittelt: SNARE- und Sec1/Munc18 (SM)-Proteine. Die SNARE-Proteine Syntaxin1a, SNAP-25 und Synaptobrevin2 sind in Nervenzellen an der Fusion synaptischer Vesikel mit der Plasmamembran beteiligt. Syntaxin1a und SNAP-25 sind auf der Plasmamembran lokalisiert, Synaptobrevin2 auf der Vesikelmembran. Die Komplexbildung der drei Membranproteine zwischen Plasma- und Vesikelmembran leitet die Fusion ein. SM-Proteine interagieren mit SNARE-Proteinen, allerdings ist der genaue molekulare Mechanismus dieser Interaktion noch nicht wirklich aufgeklärt. So bindet das SM-Protein Munc18a fast die gesamte zytosolische Domäne von Syntaxin1a in der so genannten geschlossenen Konformation. Mehrere andere SM-Proteine (i.e. Munc18c, Sly1, Vps45) scheinen dagegen nur ein kurzes N-terminales Peptid für die Bindung an ihren Syntaxinpartner zu verwenden. Im ersten Teil dieser Arbeit konnte ich mittels ITC zeigen, dass für eine hochaffine Bindung von Munc18a und Syntaxin1a nicht nur Kontakte von der geschlossenen Konformation, sondern zusätzlich Kontakte vom N-terminalen Peptid von Syntaxin1a nötig sind. In der Tat konnte das N-terminale Peptid in eine zuvor übersehene Elektronendichte in der Kristallstruktur eingefügt werden. Diese befindet sich an der äußeren Oberfläche der Domäne 1 von Munc18a. Die Elektronendichte konnte verfeinert werden und die Aminosäuren 2-9 vom N-terminalen Syntaxin1a-Peptid in die Elektronendichte eingebaut werden. Des Weiteren wurde ein zweites SM-Protein/Syntaxin-Paar untersucht. Auch für eine hochaffine Interaktion von Vps45 und Syntaxin16 ist das N-terminale Peptid und der gesamte Rest des Syntaxin16-Moleküls, sehr wahrscheinlich in einer geschlossenen Konformation, an der Bindung beteiligt. Meine Arbeit zeigt, dass SM-Protein/Syntaxin-Paare über einen konservierten Bindungsmechanismus interagieren. An der kooperativen Bindung scheinen jeweils zwei Bereiche von Syntaxin1a beteiligt. Im zweiten Abschnitt der Arbeit wurde der Einfluss von Munc18a auf die SNARE-Komplexbildung untersucht. Es ist seit einiger Zeit bekannt, dass Munc18a Syntaxin1a bindet und die SNARE-Komplexbildung in vitro blockiert. Bisher konnte allerdings nicht geklärt werden, wie Syntaxin1a sich aus der festen Umklammerung von Munc18a befreien kann. In der hier vorliegenden Arbeit konnte gezeigt werden, dass die Interaktion von Munc18a mit dem N-Peptid nötig ist, um die SNARE-Komplexbildung unterbinden zu können. Wurde das N-terminale Peptid von Syntaxin1a entfernt oder die Bindung durch Punktmutationen im N-terminalen Peptid bzw. in der Munc18a-Bindestelle gestört, war die SNARE-Komplexbildung in Anwesenheit von Munc18a nicht mehr blockiert. Dieser Befund weist auf einen möglichen Konformationswechsel im Munc18a/Syntaxin1a-Komplex hin. Abschließend wurde die Interaktion von Munc18 und Syntaxin1 und die Rolle von Munc18 bei der SNARE-Komplexbildung in dem Choanoflagellaten Monosiga brevicollis, einem den Tieren nahe verwandter Einzeller, untersucht. Es stellte sich heraus, dass nicht nur die Bindung von Munc18 an das N-terminale Peptid und die geschlossene Konformation von Syntaxin1 in Choanoflagellaten hoch konserviert ist, sondern auch die regulative Funktion von Munc18 bei der SNARE-Komplexbildung. Des Weiteren legen meine licht- und elektronenmikroskopischen Untersuchungen nahe, dass Monosiga brevicollis einen Sekretionsapparat besitzt, welcher sich in der apikalen Region der Zelle befindet.Membrane fusion events between various intracellular compartments in eukaryotic cells are mediated by two conserved protein families: SNARE- and Sec1/Munc18 (SM)-proteins. The SNARE proteins syntaxin1, SNAP-25, and synaptobrevin2 play a central role during fusion of synaptic vesicles with the plasma membrane. Syntaxin1 and SNAP-25 are located in the plasma membrane, whereas synaptobrevin2 resides on synaptic vesicles. Their assembly into a membrane-bridging ternary SNARE complex is believed to drive fusion. SM-proteins interact with SNARE-proteins but the molecular basis for this interaction is not entirely understood. Munc18a binds the cytosolic domain of syntaxin1a in a so called closed conformation. A binding mode distinct from that of Munc18a/syntaxin1a appears to govern the interaction of several other SM-proteins (i.e. Munc18c, Sly1, Vps45) with their cognate syntaxins. In these complexes the far N-terminal region of syntaxin binds to the SM-protein. In the first part of this thesis it is shown that Munc18a binds simultaneously to the closed conformation of syntaxin1a and to the N-terminal peptide of syntaxin1a by ITC. In addition, residual electron density on the outer surface of domain 1 of Munc18a was uncovered, a finding that had been overlooked until now. Re-refinement of the Munc18a/syntaxin1a-structure improved the electron density within this region, and residues 2 9 of syntaxin1a could be modelled. A second SM-protein/syntaxin-pair was also investigated. It was observed that the N-terminal peptide of syntaxin 16 binds to the SM-protein Vps45, while the remainder of syntaxin 16, probably in a closed conformation strongly enhances the affinity of the interaction. Collectively these data indicate that SM-proteins interact with their cognate syntaxins via a conserved binding mechanism. Two regions of syntaxin appear to cooperatively bind Munc18a. Next, the role of Munc18a in controlling SNARE complex assembly was investigated. It has previously been established, that Munc18a binds syntaxin1a and thereby blocks SNARE-complex assembly in vitro. However, how syntaxin1a can escape the tight grip of Munc18a, thus enabling its participation in SNARE complex formation remains unclear. Here it is demonstrated, that the interaction of Munc18a with the N-terminal peptide of syntaxin1a is essential for the inhibition of SNARE-complex formation. Removal of the N-terminal peptide of syntaxin1a, and either point mutations in the peptide, or in the Munc18a binding site, allowed for SNARE complex formation of Munc18a-bound syntaxin1a. These results suggest a conformational change in the Munc18a/syntaxin1a-complex. In the third section, the interaction of Munc18 and syntaxin1 and the role of Munc18 in SNARE-complex assembly in the choanoflagellate Monosiga brevicollis, a unicellular organism believed to be the closest known relative of animals, was investigated. The results indicate that not only is the binding of Munc18 to the N-terminal peptide and the closed conformation of syntaxin1 conserved, but also the regulative function of Munc18 in controlling SNARE-complex assembly in chaonoflagellates. Furthermore using light- and electron microscopy it was shown that Monosiga brevicollis appears to possess a secretion apparatus localized to the apical region of the cell

Exciting times to study the identity and evolution of cell types

The EMBO/EMBL Symposium on ‘The Identity and Evolution of Cell Types’ took place in Heidelberg, Germany, on 15-19 May 2019. The symposium, which brought together a diverse group of speakers addressing a wide range of questions in multiple model systems, provided a platform to discuss how the concept of a cell type should be considered in the era of single cell omics techniques and how cell type evolution can be studied

Insights into the origin of metazoan filopodia and microvilli

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery. © 2013 The Author.This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, a European Research Council Starting Grant (ERC-2007-StG- 206883), a grant (BFU2011-23434) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T., funding from NIH NIGMS GM089977 to N.K., pregraduate Formacion Profesorado Universitario grant from MICINN to A.S-P., a Deutsche Forschungsgemeinschaft (DFG) postdoctoral fellowship to P.B., and the Canadian Research Chair program grant to B.F.L.Peer Reviewe