Biology subjects

Ponjavic, A.

Publications and source records attributed to Ponjavic, A..

12 recordsLinked to original sources

bioRxiv2025

HP1β and densely packed chromatin form separate microdomains in mouse ES cells, which are reconfigured upon exit from naive pluripotency

Heterochromatin proteins play a key role in establishing local chromatin structure to control the transcription of target genes. Here we uncover a surprising segregation between regions of high DNA- and high heterochromatin protein 1{beta} (HP1{beta})-density in mouse ES cells. DNA-low/HP1{beta}-high foci retain freely diffusing HP1{beta}, and form via condensation through a multitude of weak interactions on top of HP1{beta} that is bound stably to chromatin. DNA-high/HP1{beta}-low foci exclude freely diffusing HP1{beta} and display reduced chromatin mobility, suggesting a higher degree of chromatin self-interaction and a more repressive environment. Finally, the two types of environments are intertwined in DNA-high/HP1{beta}-high foci, where HP1{beta} maintains heterochromatin in a more compact yet dynamic chromatin state. During the exit from naive pluripotency HP1{beta} is lost from regions of high DNA density as cells transition through the formative state, which might facilitate the reconfiguration of genome structure accompanying a change in cell state that we observed previously. Subsequently, as cells enter primed pluripotency, canonical heterochromatin is established.

molecular biology↗

bioRxiv2025

Single-molecule flow cytometry

Flow cytometry (FC) is a powerful tool for high-throughput characterisation of cell populations, yet its ensemble fluorescence detection and the autofluorescence of cells impose a sensitivity limit of [~]100-1,000 fluorophores per cell. This precludes detection of low-abundance proteins and weak signalling events with biological importance and therapeutic potential. We introduce single-molecule Flow Cytometry (smFC), integrating high-numerical aperture oblique plane microscopy (OPM) with microfluidics to achieve optical sectioning and photon collection efficiencies suitable for single-molecule detection on flowing cells. Using super-bright, large Stokes shift fluorophores, smFC can detect labelled membrane proteins on cells with digital precision down to [~]2 molecules per cell, compared to an unlabelled control. This represents an improvement in the detection limit of 10- to 80-fold depending on the probe. We apply smFC to quantify the distribution of the membrane receptor c-kit in triple-negative breast cancer cells with unprecedented sensitivity. This revealed a previously unidentified distribution of 60% c-kit negative cells and 40% with low-abundance but heterogenous (1-200 mol/cell) surface distribution that is undetectable by conventional FC. These results establish smFC as a robust platform for high-throughput, quantitative single-molecule analysis in cell populations, opening new avenues for detecting rare biomarkers and quantifying the presence of low-abundance membrane proteins and targetable surface molecules with digital precision.

biophysics↗

bioRxiv2024

Active Remote Focus Stabilization in Oblique Plane Microscopy

Light-sheet fluorescence microscopy (LSFM) has demonstrated great potential in the life sciences owing to its efficient volumetric imaging capabilities. For long term imaging, the light-sheet typically needs to be stabilized to the detection focal plane for the best imaging results. Current light-sheet stabilization methods rely on fluorescence emission from the sample, which may interrupt the scientific imaging and add to sample photobleaching. Here we show that for oblique plane microscopes (OPM), a subset of LSFM where a single primary objective is used for illumination and detection, light-sheet stabilization can be achieved without expending sample fluorescence. Our method achieves ~43nm axial precision and maintains the light-sheet well within the depth of focus of the detection system for hour-long acquisition runs in a lab environment that would otherwise detune the system. We demonstrate subcellular imaging of the actin skeleton in melanoma cancer cells with a stabilized OPM.

biophysics↗

bioRxiv2024

Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

Focus stabilisation is vital for long-term fluorescence imaging, particularly in the case of high-resolution imaging techniques. Current stabilisation solutions either rely on fiducial markers that can be perturbative, or on beam reflection monitoring that is limited to high-numerical aperture objective lenses, making multimodal and large-scale imaging challenging. We introduce a beam-based method that relies on astigmatism, which offers advantages in terms of precision and the range over which focus stabilisation is effective. This approach is shown to be compatible with a wide range of objective lenses (10x-100x), typically achieving <10 nm precision with >10 m operating range. Notably, our technique is largely unaffected by pointing stability errors, which in combination with implementation through a standalone Raspberry Pi architecture, offers a versatile focus stabilisation unit that can be added onto most existing microscope setups.

biophysics↗

bioRxiv2022

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

biophysics↗

bioRxiv2020

Single-molecule light-sheet microscopy with local nanopipette delivery

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2{micro}m thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-{beta}, which causes proteotoxicity relevant to Alzheimers disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells. Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instruments capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimers disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

biophysics↗

bioRxiv2020

Single Molecule Light Field Microscopy

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 m depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

biophysics↗

bioRxiv2020

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.

cell biology↗

bioRxiv2020

vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching3. While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced volume) a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.

biophysics↗

bioRxiv2019

The costs of close contact: Visualizing the energy landscape of cell contacts at the nanoscale

Cell-cell contact often underpins signalling between cells. Contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm. Protein binding and accumulation leads to the contact becoming crowded, reducing the rate of protein diffusion, even for unbound proteins. Here we show that, by tracking quantum dots of different dimensions for extended periods of time, it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry and the impact of spatial heterogeneity of adhesion protein density in the contact. By analysing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observe probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous, due to topography- and protein density-related processes. These effects are likely to profoundly influence signalling between cells.\n\nStatement of SignificanceThe spatial distribution and diffusion of proteins has been shown to be important for various signalling machineries. As such size-dependent reorganisation of proteins in the immune cell-contact has been shown to affect activation of immune cells. While these studies relied on bulk measurements to investigate protein exclusion, small scale topographical changes and protein dynamics could not be evaluated. However, recent studies show that T cell activation is mediated by nanoscale structures. In our study the use molecular probes of various sizes to investigate the energy landscape of single molecules in a cell contact. This provides additional information and insights which cannot be determined by performing bulk experiments alone indicates.

biophysics↗

bioRxiv2018

Membrane potential regulates the dynamic localisation of mammalian proteasomes

Proteasomes are abundant molecular machines distributed throughout the eukaryotic cell to facilitate protein degradation. We previously showed that proteasomes can re-organize and assemble into foci bodies in response to proteotoxic stress1, which we termed transient aggregate-associated droplets (TAADs)2. Here, we use single-molecule localization microscopy to quantify re-organization of mammalian proteasomes in different subcellular regions during proteotoxic stress to engage with invading protein aggregates. We determine that [~]60% of 20S proteasomes are capped by at least one 19S particle, and this ratio remains constant during stress. Using single-particle tracking, we show that cells confine global proteasomal movement when experiencing proteotoxic stress via a cytoskeleton-dependent mechanism. Similar limitations on proteasome movement are also observed upon membrane depolarization and repolarization events by patch clamp, which directly induce proteasomes to move away or towards the plasma membrane, respectively. Together, our experiments reveal distinct modes of proteasome motion dependent on cellular requirements, and propose that proteasome transport is restricted upon aggregate invasion to facilitate their subsequent degradation.

cell biology↗

bioRxiv2018

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Of fundamental importance, they are transient structures that generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about the morphological transition and cellular dynamics during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi photon imaging of GFP-transgenic chick embryos. We specifically focussed on the dynamic changes in two principle regions within the somite (the medial and lateral domains) to investigate extensive morphological changes. Furthermore, by using quantitative analysis and cell tracking, we were able to capture for the first time a progenitor cell bulk movement towards the rostral-medial domain of the myotome, where skeletal muscle formation first initiates.

developmental biology↗