RESUMO
Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.
Assuntos
Cálcio , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Cálcio/metabolismo , Humanos , Animais , Vesículas Sinápticas/metabolismo , Multimerização Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Transição de Fase , Mutação/genética , Células HEK293 , Fusão de Membrana , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Separação de FasesRESUMO
Exposure to carbon blacks (CBs) has been associated with the progression of pulmonary fibrosis, whereas the mechanism is still not clear. We therefore aimed to investigate the effect of RhoA/ROCK pathway on pulmonary fibrosis caused by CBs exposure. Western blot analysis indicated that CBs could promote the activation of RhoA/ROCK pathway and phosphorylation of p65 and IκBα in mice lung. However, ROCK inhibitor Y-27632 could attenuate phosphorylation levels of p65 and IκBα and restore histopathological changes of the lung tissue. Then, we evaluated the effect of RhoA/ROCK pathway on pulmonary fibrosis by detecting the expression levels of α-SMA, vimentin, and Collagen type-I (Col-I), which could be partly inhibited by Y-27632. It was assumed that inhibition of ROCK could be a promising therapeutic candidate for CBs-induced pulmonary fibrosis, which possibly through the blockage of RhoA/ROCK/NF-κB pathway.