Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Label-free measurement of amyloid elongation by suspended microchannel resonators.

Protein aggregation is a widely studied phenomenon that is associated with many human diseases and with the degradation of biotechnological products. Here, we establish a new label-free method for characterizing the aggregation kinetics of proteins into amyloid fibrils by suspended microchannel resonators (SMR). SMR devices are unique in their ability to provide mass-based measurements under reaction-limited conditions in a 10 pL volume. To demonstrate the method, insulin seed fibrils of defined length, characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM), were covalently immobilized inside microchannels embedded within a micromechanical resonator, and the elongation of these fibrils under a continuous flow of monomer solution (rate ∼1 nL/s) was measured by monitoring the resonance frequency shift. The kinetics for concentrations below ∼0.6 mg/mL fits well with an irreversible bimolecular binding model with the rate constant kon = (1.2 ± 0.1) × 10(3) M(-1) s(-1). Rate saturation occurred at higher concentrations. The nonlinear on-rate for monomer concentrations from 0 to 6 mg/mL and for temperatures from 20 to 42 °C fit well globally with an energy landscape model characterized by a single activation barrier. Finally, elongation rates were studied under different solution conditions and in the presence of a small molecule inhibitor of amyloid growth. Due to the low volume requirements, high precision, and speed of SMR measurements, the method may become a valuable new tool in the screening for inhibitors and the study of fundamental biophysical mechanisms of protein aggregation processes.

Poly(2-oxazoline)-Based Microgel Particles for Neuronal Cell Culture.

An increasing number of in vivo and in vitro neuro-engineering applications are making use of colloidal particles as neuronal cell carriers. Recent studies highlight the shortcomings of commercial glass particles and stress the benefit of using soft microgel particles (MGPs) instead. This study describes first the fabrication of MGPs from telechelic poly(2-methyl-2-oxazoline)s (PMeOx) cross-linkers and hydrophilic neutral (hydroxyethyl)methacrylate (HEMA) or charged 2-methacryloxyethyltrimethylammonium (METAC) monomers by emulsion polymerization, and it discusses their ability to support cell growth. It establishes that uncharged copolymers lead to MGPs with nonfouling properties inappropriate for cell culture, and it provides a protocol to amend their surface properties to enable cell adhesion. Finally, it demonstrates that the introduction of positive charges by METAC is necessary to obtain surface properties suitable for neuronal cell development. Through the optimization of the PMeOx30 MGP properties, this work provides general guidelines to evaluate and tune MGP chemistry to obtain microcarriers for neuro-engineering applications.

Hypoxia induces EMT in low and highly aggressive pancreatic tumor cells but only cells with cancer stem cell characteristics acquire pronounced migratory potential.

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC(low)) and highly aggressive CSC-like cells (CSC(high)) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC(high) cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC(low) cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC(high) cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC(high) cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.