Biology subjects

Stepp, W. L.

Publications and source records attributed to Stepp, W. L..

5 recordsLinked to original sources

bioRxiv2025

Smart hybrid microscopy for cell-friendlydetection of rare events

Fluorescence microscopy offers unparalleled access to the spatial organization and dynamics of biological events in living samples, yet capturing rare processes over extended durations remains challenging due to tradeoffs between exposure to excitation light and sample health. Here, we introduce hybrid-EDA, an event-driven acquisition (EDA) framework that combines the gentleness and contextual richness of phase contrast with the functional specificity of fluorescence. We developed surveillance for events of interest in label-free microscopy by novel dynamics-informed neural networks that trigger smart acquisitions in fluorescence upon detection. This allows us to dramatically reduce phototoxic damage while obtaining specific and functional information from fluorescence when beneficial. We demonstrate how hybrid-EDA enables improved imaging acquisitions of organelle contacts and mitochondrial divisions. We envision that hybrid-EDA will enable new insights into a range of dynamic and rare biological processes, providing a powerful and general strategy for cell-friendly imaging.

cell biology↗

bioRxiv2021

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyPs many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterize the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observe an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a more mobile and a less mobile population: small particles in cells unable to make polyP are more likely to be mobile and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP. Our observations suggest that polyP limits cytoplasmic mobility and accessibility during nitrogen starvation, which may help to explain the pleiotropic phenotypes observed in the absence of polyP.

biophysics↗

bioRxiv2021

Event-driven acquisition for content-enriched microscopy

A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition (EDA) framework, in which neural network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope (iSIM). Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biologi-cal events, it acquires data enriched in relevant content.

cell biology↗

bioRxiv2019

Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

The construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors line up in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each others way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

biophysics↗

bioRxiv2019

Resolving kinesin stepping: one head at a time

Kinesins are well-known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin activity have thus been focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the heads. The distal random coil at the C-terminus, termed tail domain, is proposed to mediate this autoinhibition, however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild type conformation selectively impacts one head in the heterodimer but not the other. Our results provide key insights into the regulated kinesin stepping that had escaped experimental scrutiny.

biophysics↗