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1.
Anal Chem ; 91(14): 8973-8978, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31190528

RESUMO

It has recently been reported that polystyrene microbeads may be modified to realize plasticizer-free ion-selective optical sensors (optodes) on the basis of solvatochromic dye transducers. We show here that the functionalized microbeads, individually isolated by flow cytometry, exhibit unexpectedly poor fluorescent properties and that the sensor response is instead attributed to the supernatant. A more thorough study reveals that such optical microemulsion sensors can be made operationally functional and chemically selective, seemingly in the absence of any solvent matrix or added surfactant. Instead, it is shown that residual THF used in the fabrication of the emulsified sensors may solubilize the sensing components and give a functional optode response. To evaluate this further, the number of sensing components was stepwise simplified to assess their need. Variation of residual THF levels has no effect on the ion optode response when plasticizer is present, in support of established results. Lipophilic solvatochromic dye transducers are also shown not to require an added surfactant as their nature already endows the emulsified sensors with a stabilizing ionic surface charge. The ionophores are shown to exhibit much larger stability constants in the surfactant-free formulations than surfactant-based ones (valionomycin, log ß > 9.2 compared to 6.1; Na+-ionophore X, 6.7 vs 4.7), which is attributed to a less polar solvent environment for the ionophore. Potassium-, sodium-, and calcium-selective sensors were used as model systems in this study.

2.
Nat Cell Biol ; 19(7): 787-798, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28604678

RESUMO

The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.


Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Membranas Intracelulares/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Microscopia de Força Atômica , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , ATPases Vacuolares Próton-Translocadoras/genética
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