Subcellular Fractionation of Tissue Culture Cells

Aniento, Fernando; Grüenberg, Jean

doi:10.1002/0471140864.ps0403s32

Cited by 14 publications

(17 citation statements)

References 63 publications

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“…In order to isolate endosomes, we developed a modified immunoisolation protocol rather than using a standard subcellular fractionation technique involving high-speed centrifugation that may lead to cross-contamination of the fractions, due to the processing typically associated with postmortem tissue collection (Aniento and Gruenberg, 2003; German and Howe, 2009). In addition, we found that when using magnetic or sepharose beads, there was non-specific binding of some proteins to the beads, including PSD95.…”

Section: Discussionmentioning

confidence: 99%

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

Hammond

McCullumsmith

Funk

et al. 2010

Neuropsychopharmacol

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Several lines of evidence point to alterations of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking in schizophrenia. Multiple proteins, including Synapse Associated Protein 97 (SAP97), Glutamate Receptor Interacting Protein 1 (GRIP1), and N-ethylmaleimide Sensitive Factor (NSF), facilitate the forward trafficking of AMPA receptors toward the synapse. Once localized to the synapse, AMPA receptors are trafficked in a complex endosomal system. We hypothesized that alterations in the expression of these proteins and alterations in the subcellular localization of AMPA receptors in endosomes may contribute to the pathophysiology of schizophrenia. Accordingly, we measured protein expression of SAP97, GRIP1, and NSF in the dorsolateral prefrontal cortex and found an increase in the expression of SAP97 and GRIP1 in schizophrenia. To determine the subcellular localization of AMPA receptor subunits, we developed a technique to isolate early endosomes from postmortem tissue. We found increased GluR1 receptor subunit protein in early endosomes in subjects with schizophrenia. Together, these data suggest that there is an alteration of forward trafficking of AMPA receptors as well as changes in the subcellular localization of an AMPA receptor subunit in schizophrenia.

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Section: Discussionmentioning

confidence: 99%

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

Hammond

McCullumsmith

Funk

et al. 2010

Neuropsychopharmacol

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“…To investigate these possibilities, we infected HeLa cells with AAV2 wt, ⌬VP1, or D9A/D13A/E17A for 16 h and prepared endosomes using floatation centrifugation in a sucrose step gradient (3,18,33). Endosomes were found enriched in fraction 6, as indicated by Western blot analysis of gradient fractions for the endosomal marker protein Rab5 (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…After 24 h, cells were infected with 200 viral genomes (VGs)/cell of double-iodixanol-purified AAV2 wt or AAV2 mutants in 6 ml of DMEM-i. Viruses were attached to the cells for 1 h at 4°C before the cells were shifted to 37°C, followed by incubation for 1 h. The virus was then removed, and the cells were cultivated in 15 ml of culture medium for 15 h. Early endosomal fractions were prepared as described before (3,18,33). Briefly, the cells were harvested and homogenized, and a postnuclear supernatant was prepared, adjusted to 40.6% sucrose-3 mM imidazole (pH 7.4), loaded onto the bottom of an SW60 tube, and overlaid sequentially with 35 and 25% sucrose solutions in 3 mM imidazole (pH 7. brane proteins, where they interact with adaptor proteins (AP) and thereby navigate molecular traffic, either from the plasma membrane or from the trans-Golgi network to endosomal and lysosomal compartments.…”

Section: Methodsmentioning

confidence: 99%

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Popa-Wagner

Porwal

Kann

et al. 2012

J Virol

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Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A 2 domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA 2 activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.

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“…Culture media were collected and cells were then washed in 1x PBS and collected. Subcellular fractionation of BMDM cells was prepared as previously described and either RNA or protein was isolated [ 40 ]. To test the role of caspase-1, we used 250 μ M selective irreversible caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk) (Sigma-Aldrich, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy

DeSantis

Wang

et al. 2015

Mediators of Inflammation

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TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1β secretion. The 17C-6 mice have increased IL-1β in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis.

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Subcellular Fractionation of Tissue Culture Cells

Cited by 14 publications

References 63 publications

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy

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