SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains

2006 ◽  
Vol 26 (6) ◽  
pp. 399-412 ◽  
Author(s):  
Ilaria Decimo ◽  
Renza Roncarati ◽  
Silvia Grasso ◽  
Marcel Clemens ◽  
Christian Chiamulera ◽  
...  
Keyword(s):  
Cell Body ◽  
Fluorescent Protein ◽  
Full Length ◽  
Differential Distribution ◽  
Calmodulin Binding ◽  
Quantitative Expression ◽  
Terminal Domain ◽  
Control Fusion ◽  
Green Fluorescent

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (49) ◽  
pp. 16211-16220 ◽  
Author(s):  
Timothy I. Wood ◽  
David P. Barondeau ◽  
Chiharu Hitomi ◽  
Carey J. Kassmann ◽  
John A. Tainer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

scholarly journals Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium

2009 ◽  
Vol 192 (2) ◽  
pp. 456-466 ◽  
Author(s):  
Nina Grantcharova ◽  
Verena Peters ◽  
Claudia Monteiro ◽  
Katherina Zakikhany ◽  
Ute Römling
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Population Based ◽  
Quantitative Expression ◽  
Serovar Typhimurium ◽  
Cellular Aggregation ◽  
Green Fluorescent ◽  
Biofilm Development ◽  
Biofilm Structure

ABSTRACT Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

scholarly journals Inactivation of a Heterocyst-Specific Invertase Indicates a Principal Role of Sucrose Catabolism in Heterocysts of Anabaena sp.

2010 ◽  
Vol 192 (20) ◽  
pp. 5526-5533 ◽  
Author(s):  
Rocío López-Igual ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Fluorescent Protein ◽  
Northern Analysis ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Vegetative Cells ◽  
Anabaena Sp ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N2 fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO2. The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

1999 ◽  
Vol 112 (1) ◽  
pp. 111-125 ◽  
Author(s):  
M.R. Amieva ◽  
P. Litman ◽  
L. Huang ◽  
E. Ichimaru ◽  
H. Furthmayr
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Full Length ◽  
Cell Behavior ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, &gt;90% of N-moesin and &gt;60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

2000 ◽  
Vol 278 (5) ◽  
pp. E825-E831 ◽  
Author(s):  
Aimee W. Kao ◽  
Chunmei Yang ◽  
Jeffrey E. Pessin
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Wild Type ◽  
Cell Cytoplasm ◽  
Functional Specificity ◽  
Glut 4 ◽  
Green Fluorescent ◽  
Dynamin 2

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.

Sign in / Sign up

Export Citation Format

Share Document