scholarly journals Mechanisms regulating targeting of recycling endosomes to the cleavage furrow during cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 391-394 ◽  
Author(s):  
Glenn C. Simon ◽  
Rytis Prekeris
Keyword(s):  
Small Interfering Rna ◽  
Cleavage Furrow ◽  
Gtpase Activating Protein ◽  
Successful Completion ◽  
Interacting Protein ◽  
Recycling Endosomes ◽  
Spatial Regulation ◽  
Dividing Cells ◽  
Temporal And Spatial ◽  
Interfering Rna

Recently, recycling endosomes have emerged as a key components required for the successful completion of cytokinesis. Furthermore, FIP3 (family of Rab11-interacting protein 3), a Rab11 GTPase-binding protein, has been implicated in targeting the recycling endosomes to the midbody of dividing cells. Previously, we have shown that FIP3/Rab11-containing endosomes associate with centrosomes until anaphase, at which time they translocate to the cleavage furrow. At telophase, FIP3/Rab11-containing endosomes move from the furrow into the midbody, and this step is required for abscission. While several other proteins were implicated in regulating FIP3 targeting to the cleavage furrow, the mechanisms regulating the dynamics of FIP3-containing endosomes during mitosis have not been defined. To identify the factors regulating FIP3 targeting to the furrow, we used a combination of siRNA (small interfering RNA) screens and proteomic analysis to identify Cyk-4/MgcRacGAP (GTPase-activating protein) and kinesin I as FIP3-binding proteins. Furthermore, kinesin I mediates the transport of FIP3-containing endosomes to the cleavage furrow. Once in the furrow, FIP3 binds to Cyk-4 as part of centralspindlin complex and accumulates at the midbody. Finally, we demonstrated that ECT2 regulates FIP3 association with the centralspindlin complex. Thus we propose that kinesin I, in concert with centralspindlin complex, plays a role in temporal and spatial regulation of endosome transport to the cleavage furrow during cytokinesis.

scholarly journals Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

2010 ◽  
Vol 299 (3) ◽  
pp. G742-G750 ◽  
Author(s):  
P. Cong ◽  
V. Pricolo ◽  
P. Biancani ◽  
J. Behar
Keyword(s):  
Small Interfering Rna ◽  
Muscle Cells ◽  
High Cholesterol ◽  
Western Blots ◽  
Human Gallbladder ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Δ Opioid Receptor ◽  
Interfering Rna ◽  
Bulk Plasma

The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a δ-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.

scholarly journals Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 401-407
Author(s):  
Daniel Vocelle ◽  
Olivia M Chesniak ◽  
Milton R Smith ◽  
Christina Chan ◽  
S Patrick Walton
Keyword(s):  
Small Interfering Rna ◽  
Temporal Association ◽  
Delivery Vehicle ◽  
Intracellular Location ◽  
Recycling Endosomes ◽  
Intracellular Processing ◽  
Late Endosomes ◽  
Automated Microscopy ◽  
Interfering Rna ◽  
Early Late

Abstract Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.

scholarly journals Menin Missense Mutants Encoded by the MEN1 Gene that Are Targeted to the Proteasome: Restoration of Expression and Activity by CHIP siRNA

2012 ◽  
Vol 97 (2) ◽  
pp. E282-E291 ◽  
Author(s):  
Lucie Canaff ◽  
Jean-François Vanbellinghen ◽  
Ippei Kanazawa ◽  
Hayeon Kwak ◽  
Natasha Garfield ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Target Genes ◽  
Kinase Inhibitors ◽  
Proteasome Inhibitors ◽  
Expression Vectors ◽  
Missense Mutations ◽  
Interacting Protein ◽  
Men1 Gene ◽  
Interfering Rna ◽  
Expression And Activity

Context: In multiple endocrine neoplasia type 1 (MEN1) characterized by tumors of parathyroid, enteropancreas, and anterior pituitary, missense mutations in the MEN1 gene product, menin, occur in a subset of cases. The mutant proteins are degraded by the proteasome. However, whether their expression and activity can be restored is not known. Objective: Our objective was to functionally characterize a panel of 16 menin missense mutants, including W423R and S443Y identified in new MEN1 families, with respect to protein stability, targeting to the proteasome and restoration of expression by proteasome inhibitors and expression and function by small interfering RNA technology. Methods: Flag-tagged wild-type (WT) and missense menin mutant expression vectors were transiently transfected in human embryonic kidney (HEK293) and/or rat insulinoma (Rin-5F) cells. Results: The majority of mutants were short-lived, whereas WT menin was stable. Proteasome inhibitors MG132 and PS-341 and inhibition of the chaperone, heat-shock protein 70 (Hsp70), or the ubiquitin ligase, COOH terminus of Hsp70-interacting protein (CHIP), by specific small interfering RNA, restored the levels of the mutants, whereas that of WT menin was largely unaffected. Inhibition of CHIP restored the ability of mutants to mediate normal functions of menin: TGF-β up-regulation of the promoters of its target genes, the cyclin-dependent kinase inhibitors p15 and p21 as well as TGF-β inhibition of cell numbers. Conclusion: When the levels of missense menin mutants that are targeted to the proteasome are normalized they may function similarly to WT menin. Potentially, targeting specific components of the proteasome chaperone pathway could be beneficial in treating a subset of MEN1 cases.

scholarly journals Ablation of PRC1 by Small Interfering RNA Demonstrates that Cytokinetic Abscission Requires a Central Spindle Bundle in Mammalian Cells, whereas Completion of Furrowing Does Not

2005 ◽  
Vol 16 (3) ◽  
pp. 1043-1055 ◽  
Author(s):  
Cristiana Mollinari ◽  
Jean-Philippe Kleman ◽  
Yasmina Saoudi ◽  
Sandra A. Jablonski ◽  
Julien Perard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Cell Cortex ◽  
Central Spindle ◽  
Spatial Regulation ◽  
Daughter Cells ◽  
Cell Cleavage ◽  
Temporal And Spatial ◽  
Interfering Rna ◽  
Passenger Proteins

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

scholarly journals Huntingtin-Interacting Protein 1 Promotes Vpr-Induced G2 Arrest and HIV-1 Infection in Macrophages

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2308
Author(s):  
Tomoyuki Murakami ◽  
Ryosuke Matsuura ◽  
Nopporn Chutiwitoonchai ◽  
Masami Takei ◽  
Yoko Aida
Keyword(s):  
Cell Cycle ◽  
Dna Double Strand Breaks ◽  
G2 Arrest ◽  
Interacting Protein ◽  
Dividing Cells ◽  
Primate Lentiviruses ◽  
Huntingtin Interacting Protein 1 ◽  
Interfering Rna ◽  
Huntingtin Interacting Protein ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) modulates the host cell cycle. The HIV-1 accessory protein Vpr arrests the cell cycle at the G2 phase in dividing cells, and the ability of Vpr to induce G2 arrest is well conserved among primate lentiviruses. Additionally, Vpr-mediated G2 arrest likely correlates with enhanced HIV-1 infection in monocyte-derived macrophages. Here, we screened small-interfering RNA to reveal candidates that suppress Vpr-induced G2 arrest and identified Huntingtin-interacting protein 1 (HIP1) required for efficient G2 arrest. Interestingly, HIP1 was not essential for Vpr-induced DNA double-strand breaks, which are required for activation of the DNA-damage checkpoint and G2 arrest. Furthermore, HIP1 knockdown suppressed HIV-1 infection in monocyte-derived macrophages. This study identifies HIP1 as a factor promoting Vpr-induced G2 arrest and HIV-1 infection in macrophages.

scholarly journals ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment

2015 ◽  
Vol 26 (12) ◽  
pp. 2333-2342 ◽  
Author(s):  
Michitaka Suzuki ◽  
Tatsuro Murakami ◽  
Jinglei Cheng ◽  
Hiroyuki Kano ◽  
Masaki Fukata ◽  
...  
Keyword(s):  
Complex I ◽  
Small Interfering Rna ◽  
Lipid Droplets ◽  
Regulatory Mechanism ◽  
Cellular Lipid ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Triglyceride Lipase ◽  
Interfering Rna ◽  
Hydrolysis Of

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1–coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf–GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.

scholarly journals Delivery of RIPK4 small interfering RNA for bladder cancer therapy using natural halloysite nanotubes

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw6499 ◽  
Author(s):  
Jianye Liu ◽  
Yi Zhang ◽  
Qinghai Zeng ◽  
Hongliang Zeng ◽  
Xiaoming Liu ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Small Interfering Rna ◽  
Bladder Tumor ◽  
Tumor Model ◽  
Halloysite Nanotubes ◽  
Interacting Protein ◽  
Therapeutic Method ◽  
Rnai Technology ◽  
Assisted Delivery ◽  
Interfering Rna

RNA interference (RNAi) technology can specifically silence the expression of a target gene and has emerged as a promising therapeutic method to treat cancer. In the present study, we showed that natural halloysite nanotube (HNT)–assisted delivery of an active small interfering RNA (siRNA) targeting receptor-interacting protein kinase 4 (RIPK4) efficiently silenced its expression to treat bladder cancer. The HNTs/siRNA complex increased the serum stability of the siRNA, increased its circulation lifetime in blood, and promoted the cellular uptake and tumor accumulation of the siRNA. The siRNA markedly down-regulated RIPK4 expression in bladder cancer cells and bladder tumors, thus inhibiting tumorigenesis and progression in three bladder tumor models (a subcutaneous model, an in situ bladder tumor model, and a lung metastasis model), with no adverse effects. Thus, we revealed a simple but effective method to inhibit bladder cancer using RIPK4 silencing, indicating a promising therapeutic method for bladder cancer.

scholarly journals Coordinated transport of phosphorylated amyloid-β precursor protein and c-Jun NH2-terminal kinase–interacting protein-1

2005 ◽  
Vol 171 (4) ◽  
pp. 615-625 ◽  
Author(s):  
Zoia Muresan ◽  
Virgil Muresan
Keyword(s):  
Small Interfering Rna ◽  
Protein C ◽  
Transmembrane Protein ◽  
Amyloid Β ◽  
Precursor Protein ◽  
Down Regulation ◽  
Interacting Protein ◽  
Cellular Context ◽  
Interfering Rna

The transmembrane protein amyloid-β precursor protein (APP) and the vesicle-associated protein c-Jun NH2-terminal kinase–interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr668-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP–JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.

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