Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, confocal images showing localization of endogenous α-Rac1 and α-Cdc42 immunoreactivity in chromaffin cells cultured for 3 days. Chromaffin cells were identified by anti-dopamine-β-hydroxylase reactivity as visualized with Alexa488-conjugated secondary antibody. Scale bar = 10 μm. B, time course of Rac1 activation in response to DMPP (20 μM)-mediated nAChR activation (•, n = 5) or elevated K+-induced membrane depolarization (▪, n = 9). Control cells (○, n = 8) from same cell preparations were handled identically, but without stimulation. C, distribution of Rac1 immunoreactivity among cytosol and membrane fractions (particulate) under control conditions and following stimulation with DMPP (20 μM, 2 min, n = 5). D, time course of Cdc42 activation in response to stimulation with DMPP (20 μM; •, n = 7) as compared to control (○, n = 7) cells. Asterisks represent significant differences (P < 0.05) from control.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Cell Culture, Activation Assay

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, chromaffin cells were co-transfected with plasmids carrying HA epitope-tagged Rac1 mutants, and a reporter fusion gene (ANP–EmGFP) that is directed to secretory granules of the regulated exocytotic pathway. Co-expression was evaluated by fluorescence detection of the green fluorescent protein and immunofluorescent detection of the HA tag using an α-HA antibody. B, quantification of Rac1 immunofluorescence in non-transfected (control, n = 27) and Rac1-V12 (n = 10)-transfected chromaffin cells. Scale bars = 10 μm.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Transfection, Expressing, Fluorescence, Immunofluorescence

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, time course of hGH secretion from digitonin-permeabilized chromaffin cells transfected with Rac1-V12 (squares) or an empty parent plasmid (Neo., circles). Secretion was elicited by addition of 30 μM free Ca2+ (filled symbols) and compared to secretion in the absence of Ca2+ (open symbols; n = 3). Co-expression of hGH serves as a reporter of the regulated exocytotic pathway of transfected cells. B, effect of expression of Rac1 mutant proteins on Ca2+-evoked hGH secretion at 15 min (n = 9, Rac1-V12 and Rac1-L61; n = 6, Rac1-N17). Data were normalized to control (Neo.) for comparison between different cell preparations. Asterisks represent statistically significant difference (P < 0.05) from control group.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, co-immunoprecipitation of RhoGDI (RGDI) with Rac1 from chromaffin cell lysate. Cell lysates were immunoprecipitated with Rac1 antibody and aliquots of samples subjected to SDS-PAGE and Western blotting. Blots were probed with a RhoGDI. Lane 1, lysate; 2, immunoprecipitate; 3, supernatant of final wash. B, effect of expression of RhoGDI on Ca2+-evoked (30 μM) hGH secretion, measured at 2 min (open bars) and 15 min (filled bars) from permeabilized cells (n = 6). Asterisks represent statistically significant difference (P < 0.05) from control (Neo.). C, dialysis of RhoGDI from permeabilized chromaffin cells. Immunoblot lanes, 1, cell lysate; 2 and 3, aliquot of medium collected from permeabilized chromaffin cells following 2 min and 15 min of Ca2+ stimulation (30 μM), respectively.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Expressing

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, effect of constitutively active (V12 and L61) or dominant negative (N17) Rac1 mutants on basal (open bars) and evoked (filled bars) hGH secretion. Control (Neo.) cells were transfected with empty parent plasmid. Secretion was evoked by addition of the nACh receptor agonist DMPP (20 μM, 2 min). B, effect of recombinant RhoGDI expression alone and with Rac1-V12 on basal (open bars) and DMPP-induced (20 μM, 2 min; filled bars) hGH secretion. Asterisks represent statistically significant difference (P < 0.05) from control (Neo.). A, Neo, n = 15; Rac1-V12, n = 15; Rac1-L61, n = 3; Rac1-N17, n = 9; B, Neo, n = 21; RhoGDI, n = 17; RhoGDI+Rac1-V12, n = 18.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Dominant Negative Mutation, Transfection, Plasmid Preparation, Recombinant, Expressing

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, whole-cell patch clamp recording from a control ANP–EmGFP transfected cell illustrating evoked ICa and ΔCm responses to a train of repetitive step depolarizations (-90 to +10 mV, 50 ms duration). B, comparison of relationships between cumulative Ca2+ influx and cumulative ΔCm for control (ANP–EmGFP, filled squares, n = 9), Rac1-V12 (filled circles, n = 9) and recombinant RhoGDI (open circles, n = 4) chromaffin cells. Continuous line represents the standard Ca2+-exocytosis relationship previously described for cultured bovine chromaffin cells (Engisch et al. 1997).

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Patch Clamp, Transfection, Recombinant, Cell Culture

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, chromaffin cells were identified by dopamine-β-hydroxylase immunoreactivity and the cortical cytoskeleton visualized by Alexa568-phalloidin staining. A representative example of a control and DMPP-stimulated (20 μM, 2 min) cell is shown. B, visualization of the cortical actin cytoskeleton by Alexa568-phalloidin in Rac1-V12-transfected chromaffin cells under control conditions and following DMPP stimulation (20 μM, 2 min). Chromaffin cells were identified by cotransfection and expression of ANP–EmGFP directed to secretory granules. Scale bar in A and B = 10 μm.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Staining, Transfection, Cotransfection, Expressing

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, effect of phalloidin on Ca2+-evoked hGH secretion from digitonin-permeabilized chromaffin cells (n = 6). Phalloidin (40 μM) was present during the 2 min permeabilization period and the subsequent 15 min Ca2+ (30 μM) stimulation period. B, representative effect of the membrane-permeant F-actin stabilizing drug jasplakinolide (100 nM) on hGH secretion from intact chromaffin cells stimulated with DMPP (20 μM, 2 min, n = 3). Effect was replicated in 3 separate cell preparations. C, effect of latrunculin-A on Ca2+-evoked hGH secretion from digitonin-permeabilized chromaffin cells (n = 3). Latrunculin-A (10 μM) was present during both the permeabilization period and the 10 min Ca2+ stimulation period. Chromaffin cells were transfected with the constitutively active Rac1 construct (Rac1-V12) or an empty parent plasmid control (Neo.), along with the reporter gene hGH, 3 days prior to initiation of secretion assays.

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Transfection, Construct, Plasmid Preparation

Journal:

Article Title: Facilitation of Ca 2+ -dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

doi: 10.1113/jphysiol.2003.039073

Figure Lengend Snippet: A, effect of Pak1 and ADF overexpression on DMPP-induced (20 μM, 2 min) hGH secretion from intact chromaffin cells (n = 6). B, effect of expression of Rac1-V12 and Rac1-V12 effector domain mutants on Ca2+-induced (30 μM; 15 min) hGH secretion from digitonin-permeabilized chromaffin cells (n = 6).

Article Snippet: As Rac1 is a central regulator of cytoskeletal organization the effects of constitutively active Rac1 on secretion may be associated with localized cytoskeletal reorganization triggered by the rise in intracellular [Ca 2+ ]. compares representative examples of the cortical actin cytoskeleton of control and stimulated (20 μM DMPP, 2 min) chromaffin cells as visualized by Alexa568-phalloidin labelling.

Techniques: Over Expression, Expressing

Journal: Brain

Article Title: Modelling fragile X-associated neuropsychiatric disorders in young inducible 90CGG premutation mice

doi: 10.1093/brain/awaf203

Figure Lengend Snippet: Activation of the 90CGG transgene induces time-dependent alterations in the ventral hippocampal (vH) proteome. ( A ) Mass spectrometry shows that transgene activation in adolescent (DOX+.7w) and young adult (DOX+.12w) mice alters the vH proteome. The volcano plot illustrates the log 2 fold change in protein expression versus the −log 10 of the false discovery rate for upregulated (red) and downregulated (blue) proteins with a false discovery rate > 0.05, comparing DOX−.7w ( n = 4) with DOX+.7w ( n = 3) and DOX−.12w ( n = 4) with DOX+.12w ( n = 3). ( B ) A heat map displays the top 50 enriched proteins in the data set, identified through gene set enrichment analysis (GSEA) for each experimental group. The colour gradient represents expression levels, ranging from lowest (blue) to highest (red). The data were normalized row-wise using z -score normalization. ( C ) Downregulated (blue) and upregulated (red) pathways in the vH proteome of adolescent mice (DOX+.7w) and young adult mice (DOX+.12w). Bars represent the normalized enrichment score with false discovery rate > 25% and a q -value > 0.05, based on the gene ontology (GO) biological process database. In adolescent mice, pathways related to cellular respiration, metabolism and immune response were enriched. In young adult mice, pathways related to cellular/aerobic respiration, oxidative stress and amino acid transport were upregulated, whereas pathways linked to the cytoskeletal organization (e.g. intermediate filament, actin cytoskeleton) and ionotropic glutamatergic signalling were downregulated. ( D ) Although adolescent and young adult mice with premutation show mainly diverging proteomic changes in the vH, several proteins with similar alterations are identified. Only two proteins (GABRA2 and HCN4) show opposite trends in their expression. Venn diagram (blue = decreased expression; red = increased expression) is based on data shown in B . Comparison of this dataset with previously published work revealed several proteins (highlighted in black) that had been identified in the intranuclear inclusions of FXTAS patients (marked with an asterisk), , , differentially expressed proteins in the synaptosome of the CGG KI mouse model (marked with two asterisks) and differentially expressed proteins in the CSF of FXTAS patients (marked with three asterisks). DOX = doxycycline; FDR = false discovery rate; FXTAS = fragile X-associated tremor/ataxia syndrome.

Article Snippet: Conversely, cytoskeletal organization (e.g. actin filament organization, postsynaptic actin cytoskeleton organization, actin filament depolymerization, intermediate filament organization) and glutamate receptor signalling (ionotropic glutamate receptor signalling pathway) were downregulated.

Techniques: Activation Assay, Mass Spectrometry, Expressing, Comparison