scholarly journals Cadherin activity is required for activity-induced spine remodeling

2004 ◽  
Vol 167 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Ko Okamura ◽  
Hidekazu Tanaka ◽  
Yoshiki Yagita ◽  
Yoshinaga Saeki ◽  
Akihiko Taguchi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Cytochalasin D ◽  
Dominant Negative ◽  
Spine Head ◽  
Green Fluorescent ◽  
Activity Dependent

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging

2006 ◽  
Vol 66 (6) ◽  
pp. 564-577 ◽  
Author(s):  
Janis E. Lochner ◽  
Leah S. Honigman ◽  
Wilmon F. Grant ◽  
Sarah K. Gessford ◽  
Alexis B. Hansen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Tissue Plasminogen ◽  
Activity Dependent

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

NRSF Causes cAMP-Sensitive Suppression of Sodium Current in Cultured Hippocampal Neurons

2002 ◽  
Vol 88 (1) ◽  
pp. 409-421 ◽  
Author(s):  
H. Nadeau ◽  
H. A. Lester
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Sodium Current ◽  
Striking Similarity ◽  
Secondary Effects ◽  
Functional Effect ◽  
Green Fluorescent ◽  
Altered Sensitivity ◽  
Cultured Hippocampal Neurons

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na+channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1–2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na+channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K+currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic “silencer” and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons

2000 ◽  
Vol 84 (2) ◽  
pp. 1062-1075 ◽  
Author(s):  
H. Nadeau ◽  
S. McKinney ◽  
D. J. Anderson ◽  
H. A. Lester
Keyword(s):  
Cell Death ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Input Resistance ◽  
Firing Frequency ◽  
Compensatory Mechanisms ◽  
Spike Shape ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Potential Threshold

Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2–4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at −144 mV in 5.4 mM external K+blockable by 500 μM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

scholarly journals Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

2000 ◽  
Vol 11 (4) ◽  
pp. 1213-1224 ◽  
Author(s):  
Christoph Kaether ◽  
Paul Skehel ◽  
Carlos G. Dotti
Keyword(s):  
Membrane Proteins ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Microscopy Study ◽  
Video Microscopy ◽  
Axonal Membrane ◽  
Color Video ◽  
Green Fluorescent ◽  
Living Neurons

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis

2005 ◽  
Vol 168 (2) ◽  
pp. 329-338 ◽  
Author(s):  
Emily Osterweil ◽  
David G. Wells ◽  
Mark S. Mooseker
Keyword(s):  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Dominant Negative ◽  
Myosin Vi ◽  
Receptor Endocytosis ◽  
Synapse Loss ◽  
Synaptic Structure ◽  
Acid Type ◽  
Significant Deficit ◽  
The Brain

Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid–type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.

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