Biology subjects

Gustavsson, A.-K.

Publications and source records attributed to Gustavsson, A.-K..

18 recordsLinked to original sources

bioRxiv2026

RhoA effectors ROCK and SLK/LOK oppositely regulate VE-cadherin mobility at endothelial junctions

Cellular contractile forces regulate VE-cadherin dynamics, adhesion, and endothelial junction integrity. RhoA controls these forces through two parallel effector kinases: ROCK, which activates non-muscle myosin II motors, and SLK/LOK, which promote actin-membrane attachment through phosphorylation of ezrin, radixin, moesin proteins (ERM). How these two RhoA effector pathways differentially affect VE-cadherin dynamics at junctions is not known. Using single-molecule tracking of Halo-tagged VE-cadherin, we obtained a detailed characterization of VE-cadherin diffusive behavior inside and outside junctional regions in cultured human umbilical vein endothelial cells (HUVEC). VE-cadherin was substantially less mobile and more confined within junctions than in non-junctional membrane. Inhibiting ROCK with Y-27632 increased VE-cadherin mobility both inside and outside junctions, consistent with RhoA/ROCK-driven contractility restraining cadherin diffusion globally. Unexpectedly, inhibiting SLK/LOK produced the opposite effect, selectively reducing VE-cadherin mobility within junctions with no comparable change outside them. This effect was accompanied by an increase in reticular junctions, suggesting that SLK/LOK inhibition alters VE-cadherin mobility indirectly, through a change in junctional architecture, rather than through a direct effect of actin-membrane coupling on VE-cadherin itself. These findings identify SLK/LOK as a regulator of VE-cadherin dynamics acting in parallel to ROCK downstream of RhoA and point to junctional architecture as a previously unappreciated determinant of VE-cadherin mobility at endothelial cell-cell contacts.

biophysics↗

bioRxiv2026

CaMKII generates actin bundle morphology and mechanics distinct from canonical bivalent cross-linkers

Actin cross-linkers are essential modulators of the actin cytoskeleton, enabling structural diversity and dynamic remodeling. Among them, calcium/calmodulin-dependent protein kinase II (CaMKII) is unique in serving a dual role as a kinase and as a multivalent structural binder of actin in dendritic spines, where it plays a major role in supporting dendritic spine structure via its {beta} subunit. To understand the basis of the structural function of CaMKII, we quantified the morphology and mechanics of actin bundles formed by CaMKII and directly compared them with bundles assembled by the canonical bivalent cross-linkers -actinin and fascin. Using fluorescence microscopy, we measured contour length, straightness ratio, and persistence length across different cross-linker concentrations. Fascin generated progressively shorter, straighter, and stiffer bundles with increasing concentration, whereas -actinin bundles remained largely unchanged. In contrast, increasing CaMKII concentration reduced both bundle straightness and persistence length, revealing a unique concentration-dependent increase in bundle flexibility. To investigate the structural origins of these mechanical behaviors, we complemented the experiments with coarse-grained simulations of cross-linked actin bundles. Simulations revealed that CaMKII-generated bundles retained a substantially larger fraction of their curvature in their time-averaged configuration than bundles formed by -actinin or fascin, indicating that bundle mechanics are strongly influenced by organizational features beyond thermal bending fluctuations alone. CaMKII bundles displayed behavior consistent with additional structural complexity arising from their multivalent architecture and flexible linker domains. Together, our experimental and computational results identify CaMKII as a structurally and mechanically distinct actin cross-linker. We propose that the combination of multivalency and linker flexibility enables CaMKII to function as a molecular structural pivot, promoting flexible, adaptable actin assemblies that are mechanically compatible with the dynamic remodeling required for dendritic spine plasticity.

biophysics↗

bioRxiv2026

Plug-and-Play 3D localization microscopy

Point-spread-function engineering by depth-encoding phase masks enables volumetric super-resolution imaging by 3D single-molecule localization microscopy (SMLM) but usually requires cumbersome relay optics. We demonstrate simple 3D SMLM implementation by phase mask insertion directly into the infinity space of a commercial microscope, along with appropriate computational compensation for field-dependence. The entire inserted component, including the phase mask, is 3D printing-based.

bioengineering↗

bioRxiv2026

High-speed volumetric single-molecule imaging using dual-wavelength light sheets and PSF-engineered enhanced biplane detection

Single-molecule localization microscopy (SMLM) enables nanoscale imaging but remains limited in three-dimensional (3D), high-speed, and high-density applications due to background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints. Here, we present single-objective light-sheet microscopy with PSF-engineering enhanced biplane detection (SoLiD-3D), a versatile imaging platform that integrates dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging for parallel acquisition with PSF engineered detection for high-speed volumetric SMLM. Parallelized single-objective light-sheet excitation combined with PSF engineering overcomes key limitations of conventional wide-field and biplane approaches. Independent control of two excitation wavelengths for optical sectioning enables simultaneous dual-target imaging and single-target dual-color imaging with improved contrast and temporal resolution utilizing dynamically displaced light sheets for volumetric coverage. Using SoLiD-3D, we demonstrate high-speed single- and dual-target dual-color imaging that doubles localization density without sacrificing photon efficiency and continuous volumetric imaging via PSF-engineering enhanced biplane detection for whole-cell 3D imaging with improved axial localization performance over extended depth ranges. We further demonstrate improved speed by utilizing the Hummus PSF, a compact engineered PSF that enables high-precision 3D localization with a substantially reduced spatial footprint, for the first time for super-resolution imaging applications. Taken together, SoLiD-3D mitigates the trade-off between axial range and localization precision and offers improved speed compared to conventional 3D SMLM approaches.

biophysics↗

bioRxiv2026

Increased Telomere Mobility in Progeria is Restored by Isoprenylcysteine Carboxyl Methyltransferase Inhibition

Hutchinson-Gilford Progeria Syndrome (HGPS) is a genetic disease characterized by the accumulation of progerin, a mutant form of lamin A, at the nuclear envelope. Progerin disrupts the stability of the nuclear lamina, leading to genome instability and accelerated aging phenotypes. While structural nuclear defects are well-documented, the impact of progerin on real-time chromatin dynamics and the ability of current therapeutics to rescue these dynamics remains poorly understood. In this work, we employ single-particle tracking to quantify telomere dynamics in HGPS patient fibroblasts. We demonstrate that HGPS cells exhibit significantly increased telomere dynamics, characterized by expanded scan areas, increased diffusion coefficients, and larger jump distances compared to healthy controls. We further evaluated the efficacy of two clinically relevant treatments, the farnesyltransferase inhibitor Lonafarnib and the ICMT inhibitor C75, to determine if emerging treatments can restore chromatin dynamics compared to healthy controls. Our results reveal that Lonafarnib partially rescues telomere dynamics, shifting chromatin motion back towards healthy control levels, and that C75 provides a complete rescue of the dynamics for all parameters quantified. These findings provide a quantitative framework for understanding how nuclear lamina mutations induce aberrant genome dynamics and the efficacy of HGPS therapies on restoring those dynamics.

biophysics↗

bioRxiv2026

Versatile and scalable reflective micromirrors for single-objective light sheet microscopy

We present a tunable microfabrication pipeline for creating robust, reflective inserts that adapt conventional commercial imaging chambers for single-objective light sheet (LS) illumination. This system reduces the complexity associated with dual-objective LS setups and specialized LS chambers while retaining the native functionality and biocompatibility of the original chambers. The fabricated insert features a metalized, 3D nanoprinted micromirror with an angled reflective surface, enabling alignment of a thin LS for sectioning and imaging throughout mammalian cells. Using this pipeline, we demonstrate that single-objective LS illumination achieves an over 4X improvement in the signal-to-background ratio compared with conventional widefield epi-illumination in both fixed and live cell samples. Furthermore, we show substantial resolution enhancement for single-molecule localization microscopy compared to epi-illumination for improved imaging at the nanoscale. The versatile and scalable design offers an easily implemented approach to bring the benefits of single-objective LS microscopy to a wide array of biological studies.

biophysics↗

bioRxiv2026

Lysine Demethylase 4A (KDM4A) Maintains Basal Body Architecture and Protects Against Ciliary Destabilization

Primary cilia are sensory organelles essential for signaling and defects in formation, maintenance or structure underlie diverse ciliopathies. Here, we identify lysine demethylase 4A (KDM4A) as a previously unrecognized mediator of ciliogenesis. Using genetic depletion and pharmacologic inhibition, we show that KDM4A is required for cilia assembly and maintenance. Super-resolution imaging reveals KDM4A localization at the basal body, where it distinctively wraps around the centrioles. We uncover a direct interaction between KDM4A and Rootletin (CROCC), a structural protein mediating centriole cohesion, and demonstrate that KDM4A loss increases inter-centriolar distances, implicating basal body architecture in ciliary failure. Together, these findings define a demethylase-centrosome axis that integrates KDM4A activity with organelle biology, revealing new mechanisms underlying ciliogenesis.

cell biology↗

bioRxiv2025

Robust fluorescent labeling and tracking of endogenous non-repetitive genomic loci

The spatial organization and dynamics of a genome are central to gene regulation. While a comprehensive understanding of chromatin organization in the human nucleus has been achieved using fixed-cell methods, measuring the dynamics of specific genomic regions over extended periods in individual living cells remains challenging. Here, we present a robust and fully genetically encoded system for fluorescent labeling and long-term tracking of any accessible non-repetitive genomic locus in live human cells using fluorogenic and replenishable nanobody array fusions of the Staphylococcus aureus dCas9, and compact polycistronic single guide (sg)RNAs. First, we characterize the selectivity and photostability of our probes, enabling genome-wide visualization of chromatin dynamics at locally repetitive elements. Next, through multiplexed expression of 8-10 sgRNAs from polycistronic cassettes, we demonstrate efficient and sustained labeling of non-repetitive loci, enabling high-fidelity tracking of gene-proximal regions at exceptional spatial and temporal resolution. Finally, by correlating chromatin mobility with transcriptional activity at multiple genes, we find that local chromatin dynamics at 20 Hz are gene-specific and not necessarily dependent on transcription. Our approach is versatile, minimally invasive, and scalable, enabling multiplexed imaging of regulatory element dynamics involved in gene control, with broad applicability across diverse biological systems and disease contexts.

biophysics↗

bioRxiv2025

Single-objective lattice light sheet microscopy with microfluidics for single-molecule super-resolution imaging of mammalian cells

Single-molecule localization microscopy (SMLM) has redefined optical imaging by enabling imaging beyond the diffraction limit, allowing nanoscale investigation into cellular architecture and molecular dynamics. Light sheet illumination enhances SMLM through optical sectioning of the sample, which drastically improves the signal-to-background ratio and reduces photobleaching and photodamage. Lattice light sheet (LLS) microscopy, in which a 2D optical lattice is implemented for light sheet illumination, can provide exceptional sectioning and extended imaging depth when imaging in scattering samples. However, its conventional dual-objective design poses challenges for certain applications. Here, we present an imaging platform which implements LLS illumination with a reflective single-objective geometry (soLLS) inside a microfluidic chip, enabling the use of a single high numerical aperture objective for both illumination and detection, mitigating constraints of a dual-objective setup. We provide quantitative characterization of the propagation properties of the soLLS and demonstrate that it outperforms conventional Gaussian light sheets in terms of useful field of view and sectioning when propagating through scattering samples. Next, by combining soLLS with point spread function engineering, we demonstrate the platform for improved 3D single-molecule super-resolution imaging of multiple targets across multiple cells. The soLLS imaging platform thus expands investigations of nanoscale cellular and intercellular structures and mechanisms into more challenging samples for a wide range of applications in biology and biomedicine.

biophysics↗

bioRxiv2025

One-click image reconstruction in single-molecule localization microscopy via deep learning

Deep neural networks have led to significant advancements in microscopy image generation and analysis. In single-molecule localization based super-resolution microscopy, neural networks are capable of predicting fluorophore positions from high-density emitter data, thus reducing acquisition time, and increasing imaging throughput. However, neural network-based solutions in localization microscopy require intensive human intervention and computation expertise to address the compromise between model performance and its generalization. For example, researchers manually tune parameters to generate training images that are similar to their experimental data; thus, for every change in the experimental conditions, a new training set should be manually tuned, and a new model should be trained. Here, we introduce AutoDS and AutoDS3D, two software programs for reconstruction of single-molecule super-resolution microscopy data that are based on Deep-STORM and DeepSTORM3D, that significantly reduce human intervention from the analysis process by automatically extracting the experimental parameters from the imaging raw data. In the 2D case, AutoDS selects the optimal model for the analysis out of a set of pre-trained models, hence, completely removing user supervision from the process. In the 3D case, we improve the computation efficiency of DeepSTORM3D and integrate the lengthy workflow into a graphic user interface that enables image reconstruction with a single click. Ultimately, we demonstrate superior performance of both pipelines compared to Deep-STORM and DeepSTORM3D for single-molecule imaging data of complex biological samples, while significantly reducing the manual labor and computation time.

bioengineering↗

bioRxiv2025

Mitochondrial Responses to Conventional and Ultra-high Dose Rate (FLASH) Radiation

PurposeUltra-high dose rate (>40 Gy/s, FLASH) radiation therapy (RT) provides equivalent tumor control while reducing normal tissue toxicity relative to conventional dose rate (CONV) RT. However, the mechanisms underlying the observed FLASH effect are unknown. We hypothesized that the preservation of mitochondrial integrity in nontumorigenic cells by FLASH RT could be a key factor in reducing normal tissue toxicity and improving overall treatment outcomes. MethodsWe examined mitochondrial health and function after CONV and FLASH in vitro, ex vivo, and in vivo through assays of metabolic flux, mitochondrial membrane potential, mitochondrial reactive oxygen species (ROS), mitochondrial DNA damage and copy number, mitochondrial morphology, and tumor growth and survival. ResultsIn in vitro assays, murine pancreatic cancer (PDAC) cells showed evidence of equal mitochondrial damage in response to CONV and FLASH, but nontumorigenic pancreatic cells were spared by FLASH. These results were recapitulated ex vivo, and mice treated with FLASH showed higher response rates and longer survival time than mice treated with CONV in an in vivo tumor model. ConclusionsCollectively, these results suggest that FLASH spares mitochondrial function in nontumorigenic cells, but not in PDAC cells, relative to CONV. The preservation of mitochondrial integrity in nontumorigenic cells may be a key mechanism underlying the reduced normal tissue toxicity observed with FLASH RT.

cancer biology↗

bioRxiv2024

Long axial-range double-helix point spread functions for 3D volumetric super-resolution imaging

Single-molecule localization microscopy (SMLM) is a powerful tool for observing structures beyond the diffraction limit of light. Combining SMLM with engineered point spread functions (PSFs) enables 3D imaging over an extended axial range, as has been demonstrated for super-resolution imaging of various cellular structures. However, super-resolving structures in 3D in thick samples, such as whole mammalian cells, remains challenging as it typically requires acquisition and post-processing stitching of multiple slices to cover the entire sample volume or more complex analysis of the data. Here, we demonstrate how the imaging and analysis workflows can be simplified by 3D single-molecule super-resolution imaging with long axial-range double-helix (DH)-PSFs. First, we experimentally benchmark the localization precisions of short- and long axial-range DH-PSFs at different signal-to-background ratios by imaging of fluorescent beads. The performance of the DH-PSFs in terms of achievable resolution and imaging speed was then quantified for 3D single-molecule super-resolution imaging of mammalian cells by DNA-PAINT imaging of the nuclear lamina protein lamin B1 in U-2 OS cells. Furthermore, we demonstrate how the use of a deep learning-based algorithm allows the localization of dense emitters, drastically improving the achievable imaging speed and resolution. Our data demonstrate that using long axial-range DH-PSFs offers stitching-free, 3D super-resolution imaging of whole mammalian cells, simplifying the experimental and analysis procedures for obtaining volumetric nanoscale structural information.

biophysics↗

bioRxiv2024

Lysine Demethylase 4A is a Centrosome Associated Protein Required for Centrosome Integrity and Genomic Stability

Centrosomes play a fundamental role in nucleating and organizing microtubules in the cell and are vital for faithful chromosome segregation and maintenance of genomic stability. Loss of structural or functional integrity of centrosomes causes genomic instability and is a driver of oncogenesis. The lysine demethylase 4A (KDM4A) is an epigenetic eraser of chromatin methyl marks, which we show also localizes to the centrosome with single molecule resolution. We additionally discovered KDM4A demethylase enzymatic activity is required to maintain centrosome homeostasis, and is required for centrosome integrity, a new functionality unlinked to altered expression of genes regulating centrosome number. We find rather, that KDM4A interacts with both mother and daughter centriolar proteins to localize to the centrosome in all stages of mitosis. Loss of KDM4A results in supernumerary centrosomes and accrual of chromosome segregation errors including chromatin bridges and micronuclei, markers of genomic instability. In summary, these data highlight a novel role for an epigenetic eraser regulating centrosome integrity, mitotic fidelity, and genomic stability at the centrosome.

cell biology↗

bioRxiv2024

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise, 3D nanoscale studies of a wide range of cellular structures. Here we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi-and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D singlemolecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

biophysics↗

bioRxiv2023

Whole-cell multi-target single-molecule super resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable novel microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. By combining these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we demonstrate whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.

biophysics↗

bioRxiv2023

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, C3ORF14) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assay revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.

cell biology↗

bioRxiv2021

Fast and parallel nanoscale 3D tracking of heterogeneous mammalian chromatin dynamics

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent , and a transition to more isotropic dynamics in 3D after such treatment. We think that our methodology in the future will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.

biophysics↗

bioRxiv2017

3D Single-Molecule Super-Resolution Microscopy With A Tilted Light Sheet

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSFs for fiducial bead tracking and live axial drift correction.

biophysics↗