RESUMO
Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.
Assuntos
Neurônios , Protocaderinas , Neuritos , Corpo Celular , Comunicação CelularRESUMO
Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.
Assuntos
Anexinas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Anexinas/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Transporte Biológico Ativo/fisiologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Estabilidade Proteica , Proteínas de Transporte Vesicular/genéticaRESUMO
cIPAD is a fluorescent indicator that allows the visualization of trans-interactions of clustered protocadherin (Pcdh), a cell adhesion molecule that mediates neuronal self-recognition. We describe steps for using HEK293T cells to visualize Pcdh trans-interactions across cells as a preliminary experiment before using dissociated mouse neurons. We then detail procedures for visualizing Pcdh trans-interactions between processes originating from the same neurons, which are considered as Pcdh-mediated neuronal self-recognition. For complete details on the use and execution of this protocol, please refer to Kanadome et al.1.
Assuntos
Caderinas , Protocaderinas , Humanos , Animais , Camundongos , Caderinas/genética , Caderinas/metabolismo , Células HEK293 , Neurônios/metabolismo , Adesão CelularRESUMO
Clustered protocadherin (Pcdh), a cell adhesion protein, is involved in the self-recognition and non-self-discrimination of neurons by conferring diversity on the cell surface. Although the roles of Pcdh in neurons have been elucidated, it has been challenging to visualize its adhesion activity in neurons, which is a molecular function of Pcdh. Here, we present fluorescent indicators, named IPADs, which visualize the interaction of protocadherin-α4 isoform (α4). IPADs successfully visualize not only homophilic α4 trans-interactions, but also combinatorial homophilic interactions between cells. The reversible nature of IPADs overcomes a drawback of the split-GFP technique and allows for monitoring the dissociation of α4 trans-interactions. Specially designed IPADs for self-recognition are able to monitor the formation and disruption of α4 trans-interactions between processes originating from the same neurons. We expect that IPADs will be useful tools for obtaining spatiotemporal information on Pcdh interactions in neuronal self-recognition and non-self-discrimination processes.
RESUMO
N-cadherin (NCad) is a classical cadherin that mediates cell-cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.
Assuntos
Caderinas , Neurônios , Caderinas/metabolismo , Adesão Celular , Neurônios/metabolismoRESUMO
Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Protocaderinas/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte ProteicoRESUMO
Palmitoylation is a reversible posttranslational lipid modification of proteins involved in a wide range of cellular functions. More than a thousand proteins are estimated to be palmitoylated. In neurons, PSD-95, a major postsynaptic scaffold protein, requires palmitoylation for its specific accumulation at the synapse and dynamically cycles between palmitoylated and depalmitoylated states. Although palmitoylating enzymes of PSD-95 have been well characterized, little is known about the depalmitoylating enzymes (e.g., thioesterases for palmitoylated PSD-95). An elegant pharmacological analysis has suggested that subsets of α/ß hydrolase domain (ABHD)-containing proteins of the metabolic serine hydrolase superfamily involve thioesterases for palmitoylated proteins. Here, we describe a systematic method to screen the ABHD serine hydrolase genes, which unveiled ABHD17 as the depalmitoylating enzyme for PSD-95. Furthermore, we introduce the acyl-PEGyl exchange gel-shift (APEGS) method that enables quantification of palmitoylation levels/stoichiometries on proteins in various biological samples and can be used to monitor the dynamic depalmitoylation process of proteins.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Hidrolases/metabolismo , Lipoilação , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células COS , Chlorocebus aethiops , Proteína 4 Homóloga a Disks-Large , Células HEK293 , HumanosRESUMO
Apoptosis-linked gene 2 (ALG-2), which is a gene product of PDCD6, is a 22-kDa Ca2+ -binding protein. Accumulating evidence points to a role for ALG-2 as a Ca2+ -responsive adaptor protein. On binding to Ca2+ , ALG-2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG-2. However, the differences in cellular roles for the ALG-2 homodimer and ALG-2/peflin heterodimer are unclear. In the present study, we found that Trk-fused gene (TFG) protein interacted with the ALG-2 homodimer. Immunostaining analysis revealed that TFG and ALG-2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII-mediated protein transport from the endoplasmic reticulum. Time-lapse live-cell imaging demonstrated that both green fluorescent protein-fused TFG and mCherry-fused ALG-2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+ levels. Furthermore, overexpression of ALG-2 induced the accumulation of TFG at ERES. TFG has an ALG-2-binding motif and deletion of the motif decreased TFG binding to ALG-2 and shortened its half-life at ERES, suggesting a critical role for ALG-2 in retaining TFG at ERES. We also demonstrated, by in vitro cross-linking assays, that ALG-2 promoted the polymerization of TFG in a Ca2+ -dependent manner. Collectively, the results suggest that ALG-2 acts as a Ca2+ -sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG-2 in COPII-dependent trafficking from the endoplasmic reticulum.