Biology subjects

Peterman, E. J. G.

Publications and source records attributed to Peterman, E. J. G..

9 recordsLinked to original sources

bioRxiv2026

Intraflagellar transport of tubulin maintains steady-state axoneme integrity in C. elegans cilia

The integrity of the axoneme - the microtubule (MT)-based core of the cilium - and intraflagellar transport (IFT) are interdependent. The mechanisms determining axoneme structure and dynamics have remained largely unknown, especially in primary cilia with a more variable architecture and longer MT singlet parts. Using fluorescence imaging in the phasmid neurons of C. elegans, we here demonstrate that {beta}-tubulin isotype TBB-4 diffuses through the dendrite and employs a combination of anterograde IFT and diffusion to reach the sites of incorporation in the steady-state axoneme. Disrupting tubulins ability to bind to the IFT significantly reduces its share in the axoneme. We suggest that, in phasmid cilia, a constant supply of tubulin by IFT is required for steady-state length maintenance, in order to elevate soluble tubulin concentration near the axonemal tips and to promote MT stability.

cell biology↗

bioRxiv2025

Sensory stimuli and cilium trafficking defects trigger the release of ciliary extracellular vesicles from multiple ciliary locations

The primary cilium is a signaling organelle that extends from many cell types to detect and relay extracellular signals. Beyond its signaling role, the cilium also produces cilia-derived extracellular vesicles (cEVs), although the mechanisms underlying their biogenesis and functions remain poorly understood. We characterized the cEV biogenesis in vivo using ciliated sensory neurons of C. elegans. In response to sensory cues, interruption of the intraflagellar transport (IFT) -a ciliary trafficking machinery carrying cargoes along the cilium-occurs together with ciliary membrane fission, resulting in the release of cEVs. Similarly, mutants disrupting IFT and ciliary receptor trafficking also enhance cEV production. To investigate how IFT influences the rate and location of cEV biogenesis, we selected a membrane marker that spans the entire length of the ciliary membrane independently of IFT. Single-molecule tracking demonstrates that the tetraspanin TSP-6 enters and diffuses within the cilia and does it independently of IFT. Lack of receptor retrieval or receptor entry in the cilium induces membrane budding from ciliary or periciliary membranes, respectively. Prior to fission, these membrane buds get enriched in TSP-6 as well as signaling receptors. Coupling receptor buildup with their export by cEVs provides a mechanism to preserve ciliary function and to modulate ciliary signaling. HIGHLIGHTS- The cone-shaped tetraspanin TSP-6 crosses the transition zone and moves by passive diffusion within the ciliary membrane, independently of IFT. - The production of ciliary EVs loaded with TSP-6 increases upon acute sensory stimulation or when IFT of ciliary membrane proteins is disrupted. - Depending on the nature of the perturbations, ciliary EVs remove excess material from the distal, proximal or periciliary segments of the cilia.

cell biology↗

bioRxiv2025

Condensin I confers Stiffness and Centromeric Cohesion to Mitotic Chromosomes

When cells divide, the newly replicated sister chromatids must be segregated evenly to the daughter cells. During mitosis, mechanical force is applied by spindle microtubules in 2 ways: first by pushing on chromosome arms to promote chromosome congression to the cell equator in metaphase, and then by pulling on kinetochores to promote sister chromatid disjunction during anaphase. For segregation to proceed faithfully, the pliable interphase chromatin must be transformed into stiff mitotic chromosomes able to withstand these forces. However, it is unclear how the cell establishes chromosome stiffness and what the consequences are for dividing cells if this stiffness is disrupted. Many of the structural changes imposed on chromosomes in mitosis are driven by Condensin complexes, in conjunction with Topoisomerase II. Here, we have combined rapid protein depletion and live cell imaging with in-depth mechanical characterization of purified mitotic chromosomes to probe the roles of Condensins I and II in the establishment and maintenance of the mechanical strength of mitotic chromosomes. We show that Condensin I, but not Condensin II, is required to establish chromosome stiffness and chromatin elasticity, and yet ceases to be required for the maintenance of these properties once chromosome formation has been completed. Nevertheless, depletion of Condensin I from already formed chromosomes still impacts centromeric chromatin and leads to a loss of sister centromere cohesion. We propose that the extensive chromatin loop network established by Condensin I is locked in place by Topoisomerase II mediated DNA catenation.

cell biology↗

bioRxiv2023

IFT cargo and motors associate sequentially with IFT trains to enter cilia

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone (TZ). How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors kinesin-II and OSM-3, as well as anterograde cargo proteins IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or TZ function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the TZ.

biophysics↗

bioRxiv2022

Classifying directed and diffusive transport in short, noisy single-molecule trajectories with wMSD

Fluorescence imaging in combination with single-particle tracking analysis has emerged as a powerful tool to study and characterize the motion of proteins moving in biological media. One of the main challenges in this approach is to reliably distinguish between directed and diffusive transport, especially for short and often noisy trajectories showing distinct, time- and place-dependent modes of motility. In this contribution, we present a windowed Mean-Square Displacements classifier (wMSDc) that is able to reliably (i) identify periods of diffusive and directed transport, (ii) extract position-dependent diffusion coefficients and velocities, and (iii) identify the location of switches in direction or motility mode in short (< 50 time points) and noisy single-molecule trajectories. We compare the performance of this approach to a Hidden Markov Model (HMM) method and a Moment Scaling Spectrum based method (DC-MSS) previously published and show that, in most cases, its performance is superior. We present a wide range of applications: from the movement of whole organisms and cells to protein-DNA interactions in vitro and motor-protein dynamics in vivo. Statement of SignificanceExtracting quantitative parameters from single-particle trajectories is a challenging task, especially in biological samples, which often show a high degree of heterogeneity. Trajectories can reveal switches between different types of motion; directed, diffusive and sub-diffusive motion. Usually, the length of these trajectories and their localization precision are limited by the experimental conditions. Here, we present a novel approach to analyse single molecule trajectories, windowed Mean-Square Displacement classifier (wMSDc) to reliably distinguish between directed and diffusive transport in the short trajectories with a finite precision of localization and integration time typically obtained when imaging single fluorescent proteins in living cells or organisms. We show that, using simulated and a wide range of experimental trajectories, wMSDc is a reliable method to extract motility parameters such as diffusion coefficient and velocity.

biophysics↗

bioRxiv2022

Differentiated dynamic response in C. elegans chemosensory cilia

Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function cilia are dependent on intraflagellar transport (IFT). Here we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We found that chemical stimulation resulted in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents resulted in different responses, not only in IFT, ciliary structure and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.

biophysics↗

bioRxiv2020

TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

Sensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

biophysics↗

bioRxiv2019

Supercoiling DNA optically

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures.1,2,3 For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic,4,5,6 angular-optical,7,8,9 micro-pipette,10 and magneto-optical tweezers.11 However, in order to address many open questions, there is a growing need for new techniques that can combine rapid torque control with both spatial manipulation and fluorescence microscopy. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. Supercoiled DNA formed in this way is amenable to fast buffer exchange, and can be interrogated with both force spectroscopy and fluorescence imaging of the whole DNA. We establish this method as a powerful platform to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

biophysics↗

bioRxiv2018

One-dimensional sliding assists σ70-dependent promoter binding by Escherichia coli RNA polymerase

The search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP {sigma}70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP {sigma}70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the \"diffusion facilitation threshold\" - the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding - of 0.2 M RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 M, concentrations of RNAP in cells.\n\nSignificance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site. But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by three-dimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA. Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

biophysics↗