NORMIC Oslo imaging seminar
How to build an imaging centre , chemical single molecule movement/interaction and optical tweezers - with coffee avec in the break (bolle or similar)!
Welcome to NORMIC Oslo imaging seminar!
Edna´s talk deals with building up an imaging lab the Singapore way and then mechanical force measurements using high resolution molecular imaging to measure how actin, vinculin tallinn interacts to generate force. She is from the famous lab of Michael Sheetz, focusing on cell motility, motor molecules, integrin-cytoskeleton interactions.
The second talk is by Felix Margadant, a facility manager also from Singapore
Coffee and a snack in the break!
Edna, Xian HU WEDNESDAY Nov 2nd 11.15 -
Mechanobiology Institute, Singapore, National University Medical Institute
National University of Singapore
Title: From Facility Management to Single Molecule Imaging
Mechanobiology Institute (Singapore) has a microscopy core unit that hosts 20 high-end microscope systems that serve the 240 researchers in MBI and their collaborates for cellular and organelle level research. At the time I joined MBI as the microscopy manager, mechanobiology was new territory, selecting and assembling the appropriate instruments was a scientific journey in itself. The early setups and modifications to TIRF and localization microscope systems led to my work on the Talin stretch cycle work.
Talin is a critical focal adhesion adaptor protein that connects actin and integrin directly. The talin dimer has up to 22 cryptic vinculin binding sites (VBS) that are exposed by stretching. A method was developed to dynamically measure both the length of talin and the binding of vinculin at a superresolution level in live cells. Unlike many models of the talin dimer, talin N-termini(integrin-binding domain) are separated by about 180nm, whereas the C-terminal dimerization domains colocalize. The rapid stretching and relaxation cycles of the talin dimers occur in their majority in peripheral cell regions, with a direction that agrees with the actin flow. This is consistent with a stick-slip model for transient binding to flowing actin. By tagging a vinculin-dihydrofaolate reductase(DHFR) chimera with a covalent fluorophore(TMP-atto655), vinculin binding to talin was monitored in parallel with the talin length. A peak of binding was found at the length of 180nm for both full-length vinculin and vinculin head domain, but controls didn’t bind. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. Thus we suggest that talin stretching activates vinculin binding in a cooperative manner, consistent with vinculin dynamics in vivo.
Dr Felix Martin Margadant time 12.15
MBI Microscopy Core Facilities
National University of Singapore
Title: On the viability of high time resolution optical force measurements
Topic: Clear silicone and polymer pillars with hundreds of nanometer to micrometer diameter have been used for more than a decade to assess the forces migrating or moving cells exert on the substrate. Traditionally, the pillars are coated with a thin fluorescent layer so their deflection can be optically observed with ease.
Here I present two advances we introduced to our tracking system. The first one is the lithography assembly of the pillar mold and an isolate stamping step that allows for a nanometer accurate assembly and in consequence a force measurement without ever having to relax the pillars to zero force.
The second one is the transition to far-red high intensity light sources that do not interact with the specimen and allow a speed increase from several seconds per frame to the order of 1000 frames per second. The later approach also allows for the complete isolation of the tracking process from the actual micrography of the cell’s functionality. This introduces very high resolution force maps with millisecond timing, correlated with single molecule activity observation.