Biology subjects

Fiolka, R. P.

Publications and source records attributed to Fiolka, R. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Modulation of Oncogenic KRAS Signaling by Branched Actin-driven Cell Membrane Protrusions

For over three decades, we have known that oncogenic RAS alters the actin cytoskeleton organization and cell surface morphology1,2. RAS activates the GTPase RAC1, which triggers the growth of branched actin networks to promote cell membrane protrusions3,4. In melanoma, the hyperactive RAC1 mutant, Rac1P29S, was recently shown to drive extended lamellipodia, which then empower cell proliferation through sequestration and localized inhibition of the merlin tumor suppressor5. This discovery illustrates cell morphological programs not only as outputs but also as regulators of human oncogenic signals. Hence, we wondered whether the pronounced branched actin-driven membrane protrusions (BAMPs) downstream of oncogenic RAS are not mere outputs of RAS signaling but rather an active component in mediating the oncogenic penetrance of RAS mutants. We used volumetric light sheet microscopy and biochemical approaches to investigate the role of BAMPs in regulating the molecular signaling of oncogenic KRAS in pancreatic and lung cancer models. We found that elevated BAMP formation regulated the interaction of oncogenic KRAS with downstream effectors, specifically with the RAC1 GEF TIAM1. This implies that BAMPs amplify their own upstream regulators in a positive feedback. This meritorious cycle upregulates cyclin D1 expression by inactivating the merlin tumor suppressor, independently of the mitogen activated protein kinase pathway (MAPK). In the absence of BAMPs, cells carrying oncogenic KRAS mutations are unable to attain their full penetrance in proliferation. Overall, this work unveils the long-overlooked role of branched actin-driven cell morphology in the functionalization of KRAS mutants as potent oncogenes.

cancer biology↗

bioRxiv2026

Unraveling Subcellular Ultrastructure with Cyclically Multiplexed Expansion Microscopy

Despite advances in fluorescence microscopy, spectral overlap and limited resolution hinder the dense mapping of the cellular ultrastructure. To overcome these challenges, we developed Cy-ExM, a high-plex imaging strategy that integrates optimized cryo-fixation for antigen preservation, expansion microscopy, and iterative immunofluorescence labeling. Using oblique plane microscopy, we perform three-dimensional super-resolution imaging of 20 biological targets encompassing the full cellular volume of individual mammalian cells.

cell biology↗

bioRxiv2025

Multi-immersion Oblique Plane Microscope (miOPM): A reconfigurable platform for high-resolution Light-Sheet Fluorescence Microscopy.

Light-Sheet Fluorescence Microscopy (LSFM) enables gentle, rapid, and efficient volumetric imaging of biological specimens. Despite its potential, LSFM has fragmented into multiple variants, each narrowly optimized for specific samples or imaging regimes, limiting its broad applicability. To overcome this barrier, we introduce the multi-immersion oblique plane microscope (miOPM), an adaptable LSFM platform for high-resolution imaging ranging from subcellular dynamics to whole organisms and cleared tissues. miOPM uniquely supports seamless interchangeability of oil, water, and air objectives, and maintains diffraction limited performance across a refractive index range of 1.33 - 1.51. It is compatible with standard sample mounting, current clearing protocols and high-throughput 3D imaging. We leverage miOPM to image diverse biological specimens at multiple spatial scales. By greatly expanding the adaptability and ease of use of LSFM, miOPM is poised to democratize advanced three-dimensional imaging.

bioengineering↗

bioRxiv2024

Increasing the acquisition speed in oblique plane microscopy via Aliasing

Oblique plane microcopy (OPM), a variant of light-sheet fluorescence microscopy (LSFM), enables rapid volumetric imaging without mechanically scanning the sample or an objective. In an OPM, the sample space is mapped to a distortion free image space via remote focusing, and the oblique light-sheet plane is mapped via a tilted tertiary imaging system onto a camera. As a result, the 3D point-spread function and optical transfer function are tilted to the optical axis of the tertiary imaging system. To satisfy Nyquist sampling, small scanning steps are required to encompass the tilted 3D OTF, slowing down acquisition and increasing sample exposure. Here we show that a judicious amount of under-sampling can lead to a form of aliasing in OPM that can be recovered without a loss of spatial resolution or introducing artifacts. The resulting speed gain depends on the optical parameters of the system and can reach 2-4-fold in our demonstrations. We leverage this speed gain for rapid subcellular 3D imaging of mitochondrial dynamics.

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

Caveolin-1 regulates context-dependent signaling and survival in Ewing Sarcoma.

Plasticity is a hallmark function of cancer cells, yet the mechanisms that enable dynamic switching between survival states remain incompletely understood. Here, we identify Caveolin-1, a membrane-domain scaffolding protein, as a context-dependent regulator of survival signaling in Ewing sarcoma (EwS). Single-cell analyses reveal a distinct subpopulation of EwS cells marked by high CD99 and elevated Caveolin-1 expression. These CD99High cells exhibit unique morphology, transcriptional programs, and markedly enhanced survival both under chemotherapeutic challenge and in vivo. Importantly, CD99High and CD99Low states are reversible, providing EwS cells with a flexible route to survival-oriented plasticity. Mechanistically, we show that Caveolin-1 in CD99High cells orchestrates PI3K/AKT survival signaling by modulating the spatial organization of PI3K activity on the plasma membrane. We propose that the CD99High state establishes a Caveolin-1-driven signaling architecture that supports survival through mechanisms distinct from those used by CD99Low cells. These findings uncover a dynamic state transition in EwS cells and position Caveolin-1 as a key driver of context-specific survival signaling.

cancer biology↗

bioRxiv2024

A general algorithm for consensus 3D cell segmentation from 2D segmented stacks

Cell segmentation is the foundation of a wide range of microscopy-based biological studies. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation, and computation. However, 3D cell segmentation, requiring dense annotation of 2D slices still poses significant challenges. Manual labeling of 3D cells to train broadly applicable segmentation models is prohibitive. Even in high- contrast images annotation is ambiguous and time-consuming. Here we develop a theory and toolbox, u- Segment3D, for 2D-to-3D segmentation, compatible with any 2D method generating pixel-based instance cell masks. u-Segment3D translates and enhances 2D instance segmentations to a 3D consensus instance segmentation without training data, as demonstrated on 11 real-life datasets, >70,000 cells, spanning single cells, cell aggregates, and tissue. Moreover, u-Segment3D is competitive with native 3D segmentation, even exceeding when cells are crowded and have complex morphologies.

bioinformatics↗

bioRxiv2024

Adaptive Optics in an Oblique Plane Microscope

Adaptive optics (AO) can restore diffraction limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both the illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.

cell biology↗

bioRxiv2024

Imaging of cellular dynamics in vitro and in situ: from a whole organism to sub-cellular imaging with self-driving, multi-scale microscopy

Many biological processes span multiple time and length scales, including developmental processes and cancer metastasis. While light-sheet fluorescence microscopy (LSFM) has become a fast and efficient method for imaging of organisms, cells and sub-cellular dynamics, simultaneous observations across these scales have remained challenging. Moreover, continuous high-resolution imaging inside living organisms has mostly been limited to few hours as regions of interest quickly move out of view due to sample movement and growth. Here, we present a self-driving, multi-resolution light-sheet microscope platform controlled by a custom Python-based software, to simultaneous observe and quantify sub-cellular dynamics and entire organisms in vitro and in vivo over hours of imaging. We apply the platform to the study of developmental processes, cancer invasion and metastasis, and we provide quantitative multi-scale analysis of immune-cancer cell interactions in zebrafish xenografts.

bioengineering↗

bioRxiv2024

navigate: an open-source platform for smart light-sheet microscopy.

navigate is a turnkey, open-source software solution designed to enhance light-sheet fluorescence microscopy (LSFM) by integrating smart microscopy techniques into a user-friendly framework. It enables automated, intelligent imaging with a Python-based control system that supports GUI-reconfigurable acquisition routines and the integration of diverse hardware sets. As a comprehensive package, navigate democratizes access to advanced LSFM capabilities, facilitating the development and implementation of smart microscopy workflows without requiring deep programming knowledge or specialized expertise in light-sheet microscopy.

biophysics↗

bioRxiv2023

Mesoscopic Oblique Plane Microscopy via Light-sheet Mirroring

Understanding the intricate interplay and inter-connectivity of biological processes across an entire organism is important in various fields of biology, including cardiovascular research, neuroscience, and developmental biology. Here, we present a mesoscopic oblique plane microscope (OPM) that enables whole organism imaging with high speed and subcellular resolution. A microprism underneath the sample enhances the axial resolution and optical sectioning through total internal reflection of the light-sheet. Through rapid refocusing of the light-sheet, the imaging depth is extended up to threefold while keeping the axial resolution constant. Using low magnification objectives with a large field of view, we realize mesoscopic imaging over a volume of 3.7x1.5x1 mm3 with [~]2.3 microns lateral and [~]9.2 microns axial resolution. Applying the mesoscopic OPM, we demonstrate in vivo and in toto whole organism imaging of the zebrafish vasculature and its endothelial nuclei, and blood flow dynamics at 12 Hz acquisition rate, resulting in a quantitative map of blood flow across the entire organism.

biophysics↗

bioRxiv2023

Projective oblique plane structured illumination microscopy

Structured illumination microscopy (SIM) can double the spatial resolution of a fluorescence microscope and video rate live cell imaging in a two-dimensional format has been demonstrated. However, rapid implementations of 2D SIM typically only cover a narrow slice of the sample immediately at the coverslip, with most of the cellular volume out of reach. Here we implement oblique plane structured illumination microscopy (OPSIM) in a projection format to rapidly image an entire cell in a 2D SIM framework. As no mechanical scanning of the sample or objective is involved, this technique has the potential for rapid projection imaging with doubled resolution. We characterize the spatial resolution with fluorescent nanospheres, compare projection and 3D imaging using OPSIM and image mitochondria and ER dynamics across an entire cell at up to 2.7 Hz. To our knowledge, this represents the fastest whole cell SIM imaging to date.

cell biology↗

bioRxiv2022

Increasing the Field-of-View in Oblique Plane Microscopy via optical tiling

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial sampling, respectively. In this Letter, we introduce a novel dual-axis scan unit for OPM that enables rapid and high-resolution volumetric imaging throughout a volume of 800 x 500 x 200 microns. This enables imaging of model organisms, such as zebrafish embryos, with subcellular resolution. Furthermore, we combined this microscope with a real-time and multi-perspective projection imaging technique to increase the volumetric interrogation rate to more than 10 Hz.

bioengineering↗

bioRxiv2022

Resolution doubling in light-sheet microscopy via oblique plane structured illumination

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combined with lower photo-toxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1Hz.

biophysics↗

bioRxiv2022

A Microtubule Mechanostat Enables Cells to Navigate Confined Environments

Cells migrating through complex 3D environments experience considerable physical challenges including tensile stress and compression. To move, cells need to resist these forces whilst also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility. Microtubules can both resist compressive forces and sequester key actomyosin regulators to ensure appropriate activation of contractile forces. Yet, how these two roles are integrated to achieve nuclear transmigration in 3D is largely unknown. Here, we demonstrate that compression triggers reinforcement of a dedicated microtubule structure at the rear of the nucleus by the mechanoresponsive recruitment of CLASPs (cytoplasmic linker-associated proteins) which dynamically strengthens and repairs the lattice. These reinforced microtubules form the mechanostat: an adaptive feedback mechanism that allows the cell to both withstand compressive force and spatiotemporally organise contractility signalling pathways. The microtubule mechanostat facilitates nuclear positioning and coordinates force production to enable the cell to pass through constrictions. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture. Our findings reveal a new role for microtubules as cellular sensors which detect and respond to compressive forces, enabling movement and ensuring survival in mechanically demanding environments. One Sentence SummaryMechanically tuned microtubules form a mechanostat to coordinate contractility and nuclear positioning in confined migration.

cell biology↗

bioRxiv2021

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signaling pathways, without which cells undergo anoikis - a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cancer cells often lose firm attachment to surrounding tissue2-5. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs6-13. Bleb function has been thoroughly investigated in the context of amoeboid migration but is far less examined in other scenarios14-19. Here we show by quantitative subcellular 3D imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signaling hubs that confer anoikis resistance. Specifically, we discovered in melanoma cells that blebbing generates plasma membrane contours, which recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signaling hubs activate ERK and PI3K - canonical promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils an unanticipated morphological requirement for mutant NRAS to operate as an effective oncoprotein. Moreover, we find that though some BRAF mutant melanoma do not rely on this survival pathway in a basal state, BRAF/MEK inhibition strongly sensitizes them to both bleb and septin inhibition. Importantly, we demonstrate that fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harboring oncogenic mutations. These data define a role for blebs as potent signaling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

cell biology↗

bioRxiv2021

Heterozygous mutation of Vegfr3 decreases renal lymphatics but is dispensable for renal function

BackgroundLymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. MethodsWe utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy) that abrogates its kinase ability. Vegfr3Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using a combination of tissue clearing, light-sheet microscopy and computational analyses. ResultsIn the kidney, we found Vegfr3 is expressed not only in lymphatic vessels, but also various blood vessels. Vegfr3Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in blood pressure, renal function and histology. Similarly, there was no difference in the degree of renal injury after cisplatin, although Vegfr3Chy/+ mice developed more perivascular inflammation by histology. Control mice treated with cisplatin had a measurable increase in cortical lymphatic density despite no change in cortical lymphatic volume and length. ConclusionsWe demonstrate that Vegfr3 is required for development of renal lymphatics, but a reduction in lymphatic density does not alter renal function and induces only modest histological changes after injury. Our data suggests that an increase in lymphatic density after cisplatin injury may reflect the loss of cortical volume associated with chronic kidney disease rather than growth of lymphatic vessels. SIGNIFICANCE STATEMENTDefects in renal lymphatics occur in various kidney diseases, but their role in maintaining kidney structure and function is unknown. We combine tissue clearing, light-sheet microscopy and computational analysis to characterize lymphatics and find that mice with a heterozygous mutation in Vegfr3 (Vegfr3Chy/+) have severely reduced renal lymphatics. Strikingly, these mice have indistinguishable renal function and histology compared with controls. Even after cisplatin injury, there are no differences in renal function, although Vegfr3Chy/+ mice developed more perivascular inflammation. Our data present a novel method of lymphatic quantification and suggest that a normal complement of renal lymphatics is dispensable for renal structure and function.

physiology↗

bioRxiv2020

u-track 3D: measuring and interrogating intracellular dynamics in three dimensions.

Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes through microscopy. Light-sheet microscopy has opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large and dense sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges. Building on the established framework of particle association in space and time implemented for 2D time-lapse sequences, we first report a complete and versatile pipeline for particle tracking in 3D. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability which defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.

cell biology↗