A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning.

Sensory neurons (SNs) detect a wide range of information from the body and the environment that is critical for homeostasis. There are three main subtypes of SNs: nociceptors, mechanoreceptors, and proprioceptors, which express different membrane proteins, such as TRKA, TRKB, or TRKC, respectively. Human pluripotent stem cell technology provides an ideal platform to study development and diseases of SNs, however there is not a viable method to isolate individual SN subtype for downstream analysis available. Here, we employ the method immunopanning to isolate each SN subtype. This method is very gentle and allows proper survival after the isolation. We use antibodies against TRKA, TRKB, and TRKC to isolate nociceptors, mechanoreceptors, and proprioceptors, respectively. We show that our cultures are enriched for each subtype and express their respective subtype markers. Furthermore, we show that the immunopanned SNs are electrically active and respond to specific stimuli. Thus, our method can be used to purify viable neuronal subtypes using respective membrane proteins for downstream studies.

RAPIDS, a method for sub-compartmental identification of protein interactomes.

Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H2O2 oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS.

Sequestosome 1 Is Part of the Interaction Network of VAPB.

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments.

The Interactome of the VAP Family of Proteins: An Overview.

Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.

Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane.

Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.

MINDY1 Is a Downstream Target of the Polyamines and Promotes Embryonic Stem Cell Self-Renewal.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170-1178.