During clathrin-mediated endocytosis, dynamin promotes the formation of clathrin-coated vesicles, but its mode of action is unresolved. In a recent study, Macia and colleagues made use of dynasore, a dynamin-specific inhibitor, to show that dynamin plays a dual role in endocytosis: they confirmed that dynamin is involved in detaching fully formed coated pits from the membrane, and also propose a new role for dynamin earlier in the process at the point of invagination.
"After our pharmacologic study (see earlier) that indicated activation of signaling pathways involved in both autophagy and phagocytosis, and because giant vacuoles containing MSU appeared comparatively late versus the rapid generation of autophagosomes, was the primum movens to destroy these solid particles autophagy or phagocytosis? Dynasore, a dynamin inhibitor, was used to abrogate the phagocytic pathways by blocking vesicle formation [75,76]. Interestingly, pretreatment of OBs with dynasore totally abolished the MSU-induced cleavage of LC3-I into LC3-II (Figure 5E), suggesting that phagocytosis precedes autophagy and that MSU-activated autophagy directly depends on crystal phagocytosis by OBs. "
[Show abstract][Hide abstract] ABSTRACT: Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.
Normal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.
Most of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.
MSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.
"Dynasore affects a number of dynamin isoforms, disabling not only dynamin 1, dynamin 2 and Drp1, the mitochondrial dynamin (Macia et al., 2006), but also potentially dynamin 3 (Lou et al., 2012). It is also possible that Dynasore is targeting another unidentified GTPase required at the early stages of invagination (Nankoe and Sever, 2006). This broad range block of dynamin activity is sufficient to impair internalization of ATP7A from the PM, while blocking only either dynamin 1 or dynamin 2 is not. "
[Show abstract][Hide abstract] ABSTRACT: The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking remain poorly understood. We assessed the role of clathrin, adaptor complexes, lipid rafts and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNAi-mediated depletion of caveolin 1, 2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant negative mutant not only disrupted clathrin-regulated pathways but also blocked PM to TGN internalization of ATP7A. Depletion of the μ subunits of either AP-2 or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Importantly, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and wash-out, suggesting forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane associated factors including clathrin, AP-2, AP-1 and Rab22 are regulators of ATP7A trafficking.
Molecular biology of the cell 04/2013; 24(11). DOI:10.1091/mbc.E12-08-0625 · 4.47 Impact Factor
"It has been reported that non-viral gene carriers including magnetic nanoparticles, are energy dependent and exploit the endocytic pathway to gain entry into their target cells , , . Hence we used varying doses of endocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride (EIPA, a macropinocytosis blocker) , , Chlorpromazine (a drug that disrupts clathrin-mediated endocytosis) , , Dynasore (a dynamin inhibitor – dynamin promotes the formation of clathrin-coated vesicles, a crucial stage in clathrin-mediated endocytosis) , , , Genistein and Filipin (a tyrosine kinase inhibitor that inhibits the caveolae-mediated endocytosis) ,  to determine the pathway by which the nucleic acid-nanoparticle complexes enter into HeLa cells. In order to assay the effect of these blockers on transfection, we complexed nTMag with pEGFP-N1 plasmid (which over expresses Green Fluorescent Protein) to report on the entry of the magnetic nanoparticles. "
[Show abstract][Hide abstract] ABSTRACT: Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.
PLoS ONE 12/2012; 7(12):e51350. DOI:10.1371/journal.pone.0051350 · 3.23 Impact Factor
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