Decoupling conservative forces and hydrodynamic interactions between optically trapped spheres.

Characterizing the interactions between colloidal particles is important, both from a fundamental perspective as well as due to its technological importance. However, current methods to measure the interaction forces between two colloids have significant limitations. Here we describe a method that exploits the fluctuation spectra of two optically trapped microspheres in order to extract, and decouple, the conservative forces acting between them and their hydrodynamic coupling. We demonstrate the proposed method with two silica microspheres, and find good agreement between our results and previous predictions for the hydrodynamic and electrostatic interactions between the spheres.

Synergy between RecBCD subunits is essential for efficient DNA unwinding.

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity.

Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.