11 research outputs found
Multiphoton-excited fluorescence imaging and correlation of single quantum dots using a combined approach of laser scanning microscopy and FCS
Single-molecule imaging to characterise the transport mechanism of the Nuclear Pore Complex
In the eukaryotic cell, a large macromolecular channel, known as the Nuclear Pore
Complex (NPC), mediates all molecular transport between the nucleus and cytoplasm.
In recent years, single-molecule fluorescence (SMF) imaging has emerged as a
powerful tool to study the molecular mechanism of transport through the NPC. More
recently, techniques such as Single-Molecule Localisation Microscopy (SMLM) have
enabled the spatial and temporal distribution of cargos, transport receptors and even
structural components of the NPC to be determined with nanometre accuracy. In this
protocol, we describe a method to study the position and/or motion of individual
molecules transiting through the NPC with high spatial and temporal precision
Graduierte Analyse der Ras-vermittelten Signaltransduktion in Systemen unterschiedlicher Komplexität
Specialities of Fluorescing Semiconductor Nanocrystals (Quantumdots)in Conjunction with Nonlinear Laser Microscopic Techniques (LSM, FCS, MCS)
Specialities of Fluorescing Semiconductor Nanocrystals (Quantumdots)in Conjunction with Nonlinear Laser Microscopic Techniques (LSM, FCS, MCS)
Blocking endocytosis enhances short-term synaptic depression under conditions of normal availability of vesicles
It is commonly thought that clathrin-mediated endocytosis is the rate-limiting step of synaptic transmission in small CNS boutons with limited capacity for synaptic vesicles, causing short-term depression during high rates of synaptic transmission. Here, we show by analyzing synaptopHluorin fluorescence that 200 action potentials evoke the same cumulative amount of vesicle fusion, irrespective of the frequency of stimulation (5-40 Hz), implying the absence of vesicle reuse, since the method used (alkaline-trapping) measures only first-round exocytosis. After blocking all slow or specifically clathrin-mediated endocytosis, however, the same stimulation patterns cause a rapid stimulation-frequency-dependent release depression. This form of depression does not reflect insufficient vesicle supply, but appears to be the result of slow clearance of vesicular components from the release site. Our findings uncover an important yet overlooked role of endocytic proteins for release site clearance in addition to their well-characterized role in endocytosis itself
An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms
We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors