Biology subjects

Scheiderer, L.

Publications and source records attributed to Scheiderer, L..

4 recordsLinked to original sources

bioRxiv2023

Uncovering kinesin dynamics in neurites with MINFLUX

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on microtubules polymerized on glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.

biophysics↗

bioRxiv2023

MINSTED tracking of single biomolecules

We show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatio-temporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within < 250 microseconds using about 13 photons. The power of MINSTED tracking is demonstrated by quantifying side-steps of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

biophysics↗

bioRxiv2022

Spontaneously blinking fluorophores for accelerated MINFLUX nanoscopy

Spontaneously blinking fluorophores, a class of molecules switching rapidly between a dark and a brightly emitting state, have emerged as a popular core to build fluorescent markers for super-resolution microscopy. With typical on-times in the order of tens of milliseconds, they are most suitable for STORM and related nanoscopy methods. Recent MINFLUX nanoscopy, however, can localize molecules even within a millisecond and achieve an up to ten times higher localization precision. Here, we present a series of spontaneous blinkers with short on-times (1-3 ms) matching MINFLUX recording time-scales. Our design builds upon a silicon rhodamine fluorescent core with a modified thiophene- or a benzothiophene-fused spirolactam fragment, which shifts the spirocyclization equilibrium toward the dark closed form at physiological conditions, imparting cell permeability. Concurrently, we obtain a highly photostable, short-lived open form with bright red emission. Characterizing the blinking behavior of single fluorophores bound to three different protein tags (antibodies, nanobodies, and HaloTag self-labeling enzyme) allowed us to select the best candidate for MINFLUX microscopy. The short on-times speed up MINFLUX localization by up to 30-fold.

biophysics↗

bioRxiv2022

MINFLUX dissects the unimpeded walking of kinesin-1

We report on an interferometric MINFLUX microscope that records protein movements with down to 1.7 nm precision within less than 1 ms. While such spatio-temporal resolution has so far required linking a strongly scattering 30-500 nm diameter bead to the much smaller protein, MINFLUX localization requires the detection of only down to 20 photons from an ~1-nm sized fluorophore. Harnessing this resolution, we dissect the unhindered stepping of the motor protein kinesin-1 on microtubules at up to physiological ATP concentrations. By attaching the fluorophore to different kinesin-1 sites and resolving steps and substeps of these protein constructs, we uncover a three-dimensional orientation change of the unbound kinesin head. We also find that kinesin-1 takes up ATP while only one head is bound, whereas hydrolysis of ATP occurs with both heads bound to the microtubule, resolving a long-standing conundrum of its mechanochemical cycle. Our results establish MINFLUX as a non-invasive tool for tracking protein movements and probing submillisecond structural rearrangements with nanometer resolution.

biophysics↗