Biology subjects

Bates, M.

Publications and source records attributed to Bates, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Nanoparticle mediated delivery of PD-L1 inhibitor enhances γδ T cell immunotherapy against metastatic ovarian cancer cells

IntroductionImmune checkpoint inhibitors (ICIs) have only shown limited efficacy for patients with ovarian cancer (OC), partly due to the immune suppressive tumour microenvironment (TME) and platelet cloaking of the cancer cells. We tested a nanomedicine strategy to enhance {gamma}{delta} T cell immunotherapy by conjugating the PD-L1 inhibitor (BMS202) to nanodiamonds (NDs). MethodsPatient-derived ascites cells were exposed to activated platelets to model platelet cloaking and the immunosuppressive phenotype in metastatic OC. ND/BMS202 nanocomplexes were then applied to platelet-conditioned OC cells and co-cultured with expanded {gamma}{delta} T cells. Cytotoxicity and immune activation were assessed by quantifying Granzyme B, CD107a, {gamma}-H2AX, cleaved caspase-3, and cleaved caspase-8. ResultsND-mediated delivery of BMS202 significantly enhanced {gamma}{delta} T cell-mediated killing of platelet cloaked OC cells in a dose-dependent manner, with greater efficacy than free BMS202 This enhanced cytotoxicity was supported by increased degranulation (CD107a), Granzyme B release, tumour cell apoptosis (caspase cleavage) and DNA damage ({gamma}-H2AX staining). ConclusionsND-based delivery of the PD-L1 inhibitor (BMS202) enhances {gamma}{delta} T cell-mediated killing of platelet-cloaked metastatic OC cells. While our data support enhanced {gamma}{delta} T cell cytotoxicity following BMS202 delivery, direct evidence of PD-L1 target engagement or PD-1/PD-L1 binding inhibition was not demonstrated in this study. These findings nonetheless justify further validation in patient-derived organoid models to optimize this {gamma}{delta} T cell-based combination immunotherapy and advance its development as a precision therapeutic strategy for metastatic OC.

immunology↗

bioRxiv2026

VLab4Mic: prediction of structural resolvability in super-resolution microscopy

Determining whether a microscopy experiment can resolve a specific feature of a protein assembly remains difficult because researchers must balance imaging modality, labelling strategy, and probe choice. We present VLab4Mic, a simulation platform that predicts structural resolvability before experiments. Starting from atomic models from the PDB or AlphaFold predictions, VLab4Mic places antibodies, nanobodies, chemical linkers, or fluorescent proteins on epitopes, applies stochastic labelling and steric constraints, and generates virtual samples for widefield, confocal, AiryScan, Stimulated Emission Depletion (STED), and Single-Molecule Localisation Microscopy (SMLM). Simulations of nuclear pore complexes agree with experimental data across all five modalities. In case studies, HIV capsid appearance depended strongly on orientation, and STED and SMLM separated domed from flat clathrin lattices where confocal and AiryScan did not. VLab4Mic therefore lets researchers judge which biological questions a given imaging configuration can answer before spending time tuning parameters at the microscope.

bioinformatics↗

bioRxiv2026

Cost-function Optimized Maximal Overlap Drift Estimation for Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) methods enable fluorescence imaging of biological specimens with nanometer-scale resolution. Although fluorophore localization precision is theoretically limited only by photon statistics, in practice the resolution of SMLM images is often degraded by physical drift of the sample and/or the microscope during data acquisition. At present, correcting this effect requires either specialized stabilization systems or computationally intensive post-processing, and established drift correction algorithms based on image cross-correlation suffer from limited temporal resolution. In this study we introduce COMET, a new method for SMLM drift estimation which achieves a substantially higher precision, accuracy, and temporal resolution compared with existing algorithmic approaches. We demonstrate that improved drift estimation translates directly into higher SMLM image resolution, limited by localization precision rather than drift artifacts. COMET is applicable to all types of SMLM data, operating directly on 2D or 3D localization datasets, and is readily integrated into analysis workflows. We benchmark its performance using both simulations and experiments, including STORM, MINFLUX, and Sequential OligoSTORM measurements, where long acquisition times make drift correction particularly challenging. COMET is published as an open-source, Python-based software project and is also available on open cloud-computing platforms.

biophysics↗

bioRxiv2026

Profibrotic Changes Following Tension Application in a Fetal Lamb Model of Long Gap Esophageal Atresia

IntroductionEsophageal atresia is a common congenital anomaly, occurring in 1 in 3,500 live births. The Foker process has revolutionized the treatment of long gap esophageal atresia (LGEA). It is well established that the Foker process causes tension accelerated growth of the esophagus, but what occurs at the molecular level during tension accelerated growth is still unknown. We aimed to create tension accelerated growth in a fetal lamb model of LGEA in order to answer this question. MethodsFollowing IACUC approval, time-dated fetal lambs (108 to 120 days of gestation) underwent thoracic esophagectomy. Both esophageal ends were ligated and sutured together to create an internal pexy under high tension. Lambs were delivered on postoperative day 2 (POD2) (n=7), POD6 (n=9) or term (n=5). The native esophagus collected at model creation served as control tissue. Specimens were bluntly separated into two layers: inner layer (IL) (epithelium, lamina propria, muscularis mucosa, submucosa) and outer layer (OL) (submucosa, muscle layer, adventitia). RNA sequencing (RNAseq), proteomics, immunohistochemistry, western blotting and real-time qRT-PCR were performed on the specimens. Mann-Whitneys or unpaired t-test were used for statistical analyses. Esophageal fibroblast cell lines established from human biopsy specimens were cultured and stimulated with TGF-beta for in vitro studies on collagen expression. Results23 lambs underwent esophagectomy with tension suture placement at 108 to 120 days gestation. Histologic analysis of tension conditioned compared to control esophagus by trichrome staining demonstrated an increase in collagen deposition in tension conditioned esophagus compared to controls. High throughput bulk RNA sequencing and proteomic analysis were performed with a focus on pathways implicated in fibrosis. GSEA analysis of the inner layer demonstrates upregulation of TGFB signaling, extracellular matrix organization, and collagen deposition at all timepoints. Further analysis was performed to evaluate specific collagen subtypes contributing to this profibrotic phenotype, and COL8A1 and COL12A1 were both significantly upregulated in both RNA and proteomic analysis at all timepoints, with Western blotting confirming up regulation in stretched tissue. In order to evaluate the relationship between TGFB signaling and collagen deposition in the esophagus, we stimulated esophageal fibroblasts with TGFB, qRT-PCR was performed to evaluate the expression of COL8A1, COL12A1, and COL6A3. Expression of all three of these collagen subtypes was noted to be significantly upregulated at all timepoints following TGFB stimulation when compared to non-stimulated controls. ConclusionsTension accelerated growth can safely be achieved in a fetal ovine model of long gap esophageal atresia. Additionally, esophageal atresia can be modeled in the ovine fetus as early as 92 days gestation. Our results demonstrate that esophageal tissue subjected to sustained tension undergoes significant profibrotic changes, as evidenced by upregulation of TGFB signaling, alterations in extracellular matrix organization, and increased collagen deposition. While it is well documented that patients with LGEA have an increased risk of post operative esophageal strictures, these findings provide the first in vivo proof of the role of tension in conferring a profibrotic phenotype in the tension-lengthened esophagus.

developmental biology↗

bioRxiv2025

Spontaneous peripheral oxygen desaturation and apnea events in mice vary by strain and inspired oxygen level

Mouse models of chronic intermittent hypoxia are widely used in research to understand the role of sleep apnea in disease pathogenesis. Mice exposed to periodic reductions in FIO2 model arterial desaturations observed in humans and recapitulate many comorbidities of sleep apnea. Here, we perform a detailed characterization and confirm reports that mice in room air experience spontaneous, periodic desaturation events. We measured peripheral oxygen saturation in the four mouse strains most commonly used in intermittent hypoxia research (C57BL/6J, CD1, BALB/c, and 129S1) and subjected them to conscious barometric plethysmography to measure oxygen desaturations and apneas simultaneously and took measurements across a range of fractional inspired oxygen (FIO2). As expected, all strains experienced periodic apneas that were followed by desaturations and decreasing FIO2 resulted in a reduction of spontaneous apneic events (p = 0.001). Surprisingly, most oxygen desaturations were not preceded by apneas or hypopneas, and mice experienced more desaturations at lower FIO2 (p < 0.001), despite less frequent apneas. Furthermore, we found strain differences in ventilatory response consistent with prior findings and a novel strain difference in 129S1 mice. These data suggest that spontaneous desaturations are caused not only by apneas and hypopneas but also by other mechanisms, independent of respiration. Our findings provide important context for mouse models of sleep apnea and associated diseases, and future work should explore the extent to which these findings are relevant in humans.

physiology↗

bioRxiv2025

Extracting biological structure and heterogeneity from the nano to the macro scale

Fluorescence microscopy is an essential tool in biology. It has revealed great variability at multiple scales, in macromolecular complexes, cells, and organisms. Understanding this variability will reveal the mechanisms by which genetically or biochemically identical systems adopt different biological states. Achieving this requires the ability to extract both the underlying biological structure and how it varies across the population. Currently the field lacks general techniques to deal with arbitrary structures and different types of variability. Here we present SQUASSH, a new convolutional neural network-based approach to freely fit structural models to fluorescence microscopy data that simultaneously quantifies variability to reveal correlations, dynamics, and systematic distortions. SQUASSH is highly versatile: it accommodates diverse imaging modalities at length scales from nm to mm. This approach opens up applications such as imaging nanoscale macromolecular structures, revealing patterns in shape changes from organelle to tissue scale, and characterizing systems biology of dynamical processes.

cell biology↗

bioRxiv2025

Novel Laser Technology Enables 10x Faster SRS Imaging and Rapid Tuning in Biological Samples

Stimulated Raman Scattering (SRS) microscopy was developed for the label-free detection of molecular groups, addressing the speed limitations of spontaneous Raman microscopy. Standard SRS microscopy typically operates with laser sources at an 80 MHz repetition rate and a color-tuning speed of approximately 0.1 Hz to target different molecular groups. Here, we present a novel laser system that overcomes these speed limitations, achieving an order-of-magnitude improvement in both color-tuning and imaging speed. Our system features a reduced repetition rate of 40 MHz, enabling SRS imaging that is ten times faster than standard systems while maintaining the same average power at the sample. This is achieved through increased pulse energy and laser modulation at half the repetition rate. Furthermore, the system provides nearly ten times faster color-tuning across an extended range (660-1010 nm) by employing angle-tuning of nonlinear crystals instead of temperature-tuning. The improved performance is demonstrated in direct comparison with a standard SRS laser system, showcasing the potential for significantly enhanced imaging capabilities.

biophysics↗

bioRxiv2024

Experimental Phage Evolution Results in Expanded Host Ranges Against MDR and XDR Klebsiella pneumoniae Isolates

Resistance to antibiotics is approaching crisis levels for organisms such as the ESKAPEE pathogens (includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) that often are acquired in hospitals. These organisms sometimes have acquired plasmids that confer resistance to most if not all beta-lactam antibiotics such as those produced by Carbapenem Resistant Enterobacterales (CREs). We have been developing alternative means for dealing with antibiotic resistant microbes that cause infections in humans by developing viruses (bacteriophages) that attack and kill them. We have been working with one of the ESKAPEE pathogens, K. pneumoniae, that has one of the highest propensities for antimicrobial resistance, to develop phages that target and kill it. We identified a number of phages that have lytic capacity against only a few clinical isolates, and through experimental evolution over the course of 30 days, were able to vastly expand the host ranges of these phages to kill a broader range of clinical K. pneumoniae isolates including MDR (multi-drug resistant) and XDR (extensively-drug resistant) isolates. Most interestingly, they were capable of inhibiting growth of clinical isolates both on solid and in liquid medium over extended periods. That we were able to extend the host ranges of multiple naive MDR and XDR K. pneumoniae through experimental phage evolution suggests that such a technique may be applicable to other antibiotic-resistant organisms to help stem the tide of antibiotic resistance and offer further options for medical treatments. ImportanceBacterial pathogens are becoming greater threats given the rise in antibiotic resistance, where traditional therapies may no longer work to cure some infections. Chief amongst these multidrug resistant infections (MDR) and extensively drug-resistant infections (XDR) is Klebsiella pneumoniae, which is known to sometimes harbor genetic elements that render it incredibly difficult to treat with conventional antibiotics. Treatments like bacteriophages have not had much success against such pathogens because resistance to the phages used often develops rapidly. We adapted a co-evolutionary technique to develop K. pneumoniae phages to be highly active longitudinally against K. pneumoniae clinical isolates. In as few as 30 days, we were able to vastly expand the host ranges of K. pneumoniae phages against MDR and XDR clinical isolates and that maintain their infectivity over clinically relevant time periods. By adapting these established techniques to clinical MDR and XDR K. pneumoniae isolates, we believe we can establish similar techniques for expanding phage host ranges against most antibiotic-resistant bacteria. As such, phages can be viable alternatives to antibiotics when antibiotic resistance exists in hospitals and communities.

microbiology↗

bioRxiv2024

Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking

MINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with more relaxed fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, and over typical time scales ({micro}s-10ms), sample and excitation conditions used in MINFLUX imaging. By subsequent simulations of representative MINFLUX localization procedures, we found that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. However, these localization errors could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.

biophysics↗

bioRxiv2024

Dynamical forces drive organ morphology changes during embryonic development

Cells, tissues, and organs must change shape in precise ways during embryonic development to execute their functions. Multiple mechanisms including biochemical signaling pathways and biophysical forces help drive these morphology changes, but it has been difficult to tease apart their contributions, especially from tissue-scale dynamic forces that are typically ignored. We use a combination of mathematical models and in vivo experiments to study a simple organ in the zebrafish embryo called Kupffers vesicle. Modeling indicates that dynamic forces generated by tissue movements in the embryo produce shape changes in Kupffers vesicle that are observed during development. Laser ablations in the zebrafish embryo that alter these forces result in altered organ shapes matching model predictions. These results demonstrate that dynamic forces sculpt cell and organ shape during embryo development. Significance StatementWe aim to understand the mechanisms that control precise cell and tissue shape changes required for organ function. Many studies focused on cell shapes have ignored the role of dynamic forces self-generated by slow tissue flows, but recent work showing tissues are near a jamming transition with diverging relaxation timescales suggests slow motion could give rise to large forces. Through a combination of mathematical modeling, imaging, and mechanical perturbations to in vivo experiments, our work demonstrates that tissue-scale dynamic forces are sculpting the shape of an epithelial organ in the zebrafish embryo called Kupffers vesicle (KV). Because there are many processes during development that occur at similarly slow rates, this suggests that self-generated dynamic forces should be investigated more broadly.

developmental biology↗

bioRxiv2024

Super-resolution going viral: T4 virus particles as perfect nature-designed 3D-Bio-NanoRulers

In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the three-dimensional domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of three-dimensional subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub-diffraction-limit distances. In this context, we introduce the use of the non-human-infecting virus, bacteriophage T4, as an effective and straightforward bio-ruler for three-dimensional super-resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) along with the technique of astigmatic imaging, we uncover the icosahedral capsid of the bacteriophage T4, measuring 120 nm in length and 86 nm in width, and its hollow viral tail. This level of detail in light microscopy represents a significant advancement. We further outline a simple protocol for the production and preparation of samples. Moreover, we explore the extensive potential of bacteriophage T4 as a multi-faceted 3D bio-ruler, proposing its application as a novel benchmark for three-dimensional super-resolution imaging in biological studies.

biophysics↗

bioRxiv2023

Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same temporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wildtype cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK-single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings corroborates the toroidal apoptotic pore model.

cell biology↗

bioRxiv2023

The Drosophila MIC10 orthologue has a propensity to polymerize into cristae-shaping filaments

Mitochondria are essential eukaryotic double-membrane organelles. The convoluted mitochondrial inner membrane forms highly organized invaginations, termed cristae, which are crucial for energy metabolism. Cristae formation requires MICOS, a conserved hetero-oligomeric inner membrane complex. The MICOS core subunit MIC10 is a small transmembrane protein that oligomerizes through highly conserved glycine-rich motifs to control cristae formation. Sequence alignments show that D. melanogaster exhibits three MIC10-like proteins with different tissue-specific expression patterns. Here, we show that the ubiquitously expressed Dmel_CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Loss of DmMIC10b disturbs cristae architecture of mitochondria and reduces the life-span and fertility of flies. Moreover, using fluorescence nanoscopy and electron tomography, we demonstrate that despite its high similarity to the MIC10 proteins from yeast and humans, DmMIC10b exhibits the unique ability to polymerize into elongated filaments upon overexpression. DmMIC10b filaments form bundles which accumulate in the intermembrane space and alter the shape of mitochondrial cristae membranes. We show that the formation of the filaments relies on conserved glycine and cysteine residues and is suppressed by co-expression of other MICOS proteins. Thereby, our findings provide new insights into the regulation of MICOS in flies.

cell biology↗

bioRxiv2022

Temporal Application of Lysyl Oxidase during Hierarchical Collagen Fiber Formation Differentially Effects Mechanics in Engineered Tissues

The primary source of strength in musculoskeletal menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native alignment and fiber diameters by 6 weeks. However, tissue mechanics plateau at 1 MPa, suggesting crosslinking is lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but its not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX treatment at different stages of hierarchical fiber formation in our culture system to produce functional engineered replacements and to better understand factors effecting collagen crosslink maturation. We found LOXL2 treatment did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. Statement of SignificanceCollagen fibers are the primarily source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body.

bioengineering↗

bioRxiv2022

Heparins enhance C1 esterase inhibitor activity: a promising remedy for acute hereditary angioedema

RationaleHereditary angioedema (HAE) is a potentially life-threatening illness most commonly due to deficiency or dysfunction of C1-esterase inhibitor (C1-INH). While specific treatments are available to thwart acute exacerbations, they are extremely costly and some can be associated with rare but serious side effects. The heparins are long known to augment C1-INH activity and case reports / series have documented their efficacy in treating HAE. Objectiveto determine if unfractionated heparin and two low-molecular weight heparins (enoxaparin and nadroparin) can augment C1-INH activity ex vivo in the sera of patients with HAE and in an in vitro biochemical assay. MethodsC1-INH activity in the absence or presence of the heparin formulations were analyzed by two different methods. To measure C1-INH activity ex vivo, a commercially available assay was utilized with patient sera, excess amounts of C1s, and a substrate of C1s which, upon cleavage by C1s, produces a chromogenic product. To determine biochemically the C1-INH activity in vitro, a pharmacologic grade C1-INH, recombinant C1s (C1s-CCP12SP), and a peptide substrate of C1s were employed. Microscale thermophoresis was used to determine whether C1-INH binds to heparin. Main resultsin patient sera, nadroparin was superior to enoxaparin and unfractionated heparin in augmenting C1-INH activity, followed by enoxaparin and then unfractionated heparin. In the in vitro biochemical assay, all three heparins augmented C1-INH-C1s binding linearly in a dose-dependent fashion. Microscale thermophoresis assay demonstrated that nadroparin binds to C1-INH, providing a mechanism by which heparin facilitates the interaction between C1-INH and the proteases known to produce bradykinin, the mediator of HAE. Conclusionlow-molecular weight heparin augments C1-INH activity and should be studied as a potential treatment for acute HAE.

pharmacology and toxicology↗

bioRxiv2022

Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosomes pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans and zebrafish is needed for PCM maintenance through mediating PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexins C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

cell biology↗

bioRxiv2021

Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

biophysics↗

bioRxiv2021

Rab11 endosomes coordinate centrosome number and movement following mitotic exit

The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

cell biology↗