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Find video protocols related to scientific articles indexed in Pubmed.
In vivo Screening for Substrates of Protein Kinase A using a combination of proteomic approaches and pharmacological modulation of kinase activity.
Cell Struct. Funct.
PUBLISHED: 11-18-2014
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Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/ 3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ROCK2 was confirmed to be a novel substrate of PKA both in vitro and in vivo. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.
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Pioneering axons regulate neuronal polarization in the developing cerebral cortex.
Neuron
PUBLISHED: 02-25-2014
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The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.
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Neuronal polarization in vivo: Growing in a complex environment.
Curr. Opin. Neurobiol.
PUBLISHED: 01-07-2014
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Neurons are one of the most polarized cell types in the body. During the past three decades, many researchers have attempted to understand the mechanisms of neuronal polarization using cultured neurons. Although these studies have succeeded in discovering the various signal molecules that regulate neuronal polarization, one major question remains unanswered: how do neurons polarize in vivo?
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ERK2-mediated phosphorylation of Par3 regulates neuronal polarization.
J. Neurosci.
PUBLISHED: 08-16-2013
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Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.
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Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein kinases.
Sci Signal
PUBLISHED: 11-17-2011
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Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.
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The role of selective transport in neuronal polarization.
Dev Neurobiol
PUBLISHED: 02-11-2011
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Neurons are functionally and morphologically polarized and possess two distinct types of neurites: axons and dendrites. Key molecules for axon formation are transported along microtubules and accumulated at the distal end of the nascent axons. In this review, we summarize recent advances in the understanding of the mechanisms involved in selective transport in neurons. In addition, we focus on motor proteins, cargo, cargo adaptors, and the loading and unloading of cargo.
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Flexible search for single-axon morphology during neuronal spontaneous polarization.
PLoS ONE
PUBLISHED: 01-24-2011
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Polarization, a disruption of symmetry in cellular morphology, occurs spontaneously, even in symmetrical extracellular conditions. This process is regulated by intracellular chemical reactions and the active transport of proteins and it is accompanied by cellular morphological changes. To elucidate the general principles underlying polarization, we focused on developing neurons. Neuronal polarity is stably established; a neuron initially has several neurites of similar length, but only one elongates and is selected to develop into an axon. Polarization is flexibly controlled; when multiple neurites are selected, the selection is eventually reduced to yield a single axon. What is the system by which morphological information is decoded differently based on the presence of a single or multiple axons? How are stability and flexibility achieved? To answer these questions, we constructed a biophysical model with the active transport of proteins that regulate neurite growth. Our mathematical analysis and computer simulation revealed that, as neurites elongate, transported factors accumulate in the growth cone but are degraded during retrograde diffusion to the soma. Such a system effectively works as local activation-global inhibition mechanism, resulting in both stability and flexibility. Our model shows good accordance with a number of experimental observations.
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CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity.
J. Neurochem.
PUBLISHED: 07-31-2009
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The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2s kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.
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Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27.
Dev. Cell
PUBLISHED: 03-03-2009
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The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.
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Latent process genes for cell differentiation are common decoders of neurite extension length.
J. Cell. Sci.
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A latent process involving signal transduction and gene expression is needed as a preparation step for cellular function. We previously found that nerve growth factor (NGF)-induced cell differentiation has a latent process, which is dependent on ERK activity and gene expression and required for subsequent neurite extension. A latent process can be considered as a preparation step that decodes extracellular stimulus information into cellular functions; however, molecular mechanisms of this process remain unknown. We identified Metrnl, Dclk1 and Serpinb1a as genes that are induced during the latent process (LP) with distinct temporal expression profiles and are required for subsequent neurite extension in PC12 cells. The LP genes showed distinct dependency on the duration of ERK activity, and they were also induced during the latent process of PACAP- and forskolin-induced cell differentiation. Regardless of neurotrophic factors, expression levels of the LP genes during the latent process (0-12 hours), but not phosphorylation levels of ERK, always correlated with subsequent neurite extension length (12-24 hours). Overexpression of all LP genes together, but not of each gene separately, enhanced NGF-induced neurite extension. The LP gene products showed distinct spatial localization. Thus, the LP genes appear to be the common decoders for neurite extension length regardless of neurotrophic factors, and they might function in distinct temporal and spatial manners during the latent process. Our findings provide molecular insight into the physiological meaning of the latent process as the preparation step for decoding information for future phenotypic change.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.