Biology subjects

Cameron, J. C.

Publications and source records attributed to Cameron, J. C..

14 recordsLinked to original sources

bioRxiv2026

A genome-scale ORF screen for resilience factors that protect the RPE and restore vision in aged mice

Aging is a complex phenomenon, yet therapeutic discovery has largely relied on testing individual hypothesis-driven targets. Here, we performed a genome-scale open reading frame (ORF) screen for oxidative-stress resistance in retinal pigment epithelium (RPE) cells, which are vulnerable to oxidative damage during aging. The screen identified nine protective ORFs including antioxidant factors and an epithelial-to-mesenchymal transition regulator. Notably, we also identified three factors that reduce transcriptomic age including the cardiac transcription factor NKX2-5. NKX2-5 conferred robust oxidative resilience in vitro and in vivo. Domain dissection revealed that its homeodomain is dispensable for resilience, enabling development of NKX2-5{Delta}HD, which retained protective activity without detectable toxicity over 14 months. In middle-aged mice, subretinal NKX2-5{Delta}HD restored visual and electrophysiological function, and its systemic delivery improved grip strength and reduced frailty in late-aged mice. These findings highlight the value of unbiased, genome-scale gain-of-function screening, as resilience factors normally expressed in other contexts can be reengineered as potential therapeutics for age-related decline in other tissues.

genetics↗

bioRxiv2026

Interactions of Phototropism and Gravitropism in Cyanobacteria

While gene expression in bacteria has been shown to be affected by near-zero or extremely high gravity, a mechanism has not been established to date. In larger organisms, gravity-sensing mechanisms usually rely on a dense body applying directional pressure which can be detected by the cell. Herein we demonstrate a means of observing the effect of gravity on cyanobacteria by differential expression of native pigments in response to both gravity and light. We observe that in the cyanobacterium Synechococcus sp. PCC 7002, the distribution of pigmentation within the cell, and across cell colonies, is regulated by combined directional sensing of incoming light, adhesion to a surface via extracellular matrix, and applied external force, including the normal force of gravity applied to the cell. Cells grown on a substrate orient their thylakoids on the cell faces proximal and distal to the substrate and locate both chlorophyll and phycobilins in both of these membrane regions; phycobilins are primarily targeted to the membrane region nearest to the light source, while chlorophyll is preferentially expressed in the region opposite the overall external force applied to the cell. The mechanism for distribution of pigments appears to be regulated by presence of polyphosphate bodies within the cell, and removal of polyphosphate negates the cells ability to sense external forces. Furthermore, colonial morphology is affected by application of force, with cells responding to the secretions of other cells along a gradient along the expected response to shading. These results represent a critical step toward understanding basal phototrophic regulatory mechanisms of light use and demonstrate the first known intracellular directional gravity response mechanism in a prokaryote. Statement of SignificanceTo date, no directionally sensitive gravity response mechanism has been observed in any prokaryote. We demonstrate the first evidence of a directional response to external force in a cyanobacterium. This pigment distribution force-directed response interacts with the conventional response to directional light. Furthermore, the cells appear to be able to respond to the presence of other cells above them via intercellular signaling which is not simply due to shading by light.

biophysics↗

bioRxiv2025

Molecular insights into phycobilisome assembly pathway reveal crystalline bodies in cyanobacteria

In oxygenic photosynthetic organisms, light energy is converted to chemical energy to drive CO2 fixation reactions and sustain life on Earth. Cyanobacteria contain phycobilisome (PBS) complexes that play critical roles in light harvesting and directing light energy to the photosystem I and II reaction centers. The proper assembly of PBS components is an intricate process that is required for their activity and association with photosystem I and II. To understand the complex mechanisms regulating the PBS assembly, we knocked out the terminal emitter apcE, which forms the structural scaffold for the PBS core. ApcE knockout led to growth and pigment defects, including elevated levels of photosystem II and abnormal emission spectra. Light microscopy experiments revealed the accumulation of highly fluorescent puncta localized to the pole of apcE knockout cells. Further investigation using electron cryo-tomography identified highly repetitive crystalline arrays of densely packed PBS cylinders. Together, these data indicate that cyanobacteria may accumulate PBS components in the form of highly organized crystalline bodies as intermediates during PBS assembly.

microbiology↗

bioRxiv2025

Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites

Lysosomal dysfunction is a well-recognized feature of aging, yet its systematic molecular investigation remains limited. Here, we employ a suite of tools for rapid lysosomal isolation to construct a multi-tissue atlas of the metabolite changes that murine lysosomes undergo during aging. Aged lysosomes in brain, heart, muscle and adipose accumulate glycerophosphodiesters and cystine, metabolites that are causally linked to juvenile lysosomal storage disorders like Batten disease. Levels of these metabolites increase linearly with age, preceding organismal decline. Caloric restriction, a lifespan-extending intervention, mitigates these changes in the heart but not the brain. Our findings link lysosomal storage disorders to aging-related dysfunction, uncover a metabolic lysosomal "aging clock," and open avenues for the mechanistic investigation of how lysosomal functions deteriorate during aging and in age-associated diseases. One-Sentence SummaryAging in mice is tracked by a lysosomal "clock", where glycerophosphodiesters and cystine - metabolites causally linked to juvenile lysosomal storage disorders - gradually accumulate in lysosomes of the brain, heart, skeletal muscle and adipose tissue.

cell biology↗

bioRxiv2025

Reprogramming Factors Activate a Non-Canonical Oxidative Resilience Pathway That Can Rejuvenate RPEs and Restore Vision

Oct4, Sox2, and Klf4 (OSK) Yamanaka factors induce pluripotency and reverse age-related epigenetic changes, yet the mechanisms by which they promote rejuvenation remain poorly explored. Oxidative stress contributes to CNS aging and retinal pigmented epithelium (RPE) degeneration in age-related macular degeneration. We find that OSK expression in RPE restores retinal structure and visual function in aged mice and promotes oxidative resilience through a non-canonical, Tet2-independent pathway. Integrative functional genomics identifies GSTA4, a detoxifying enzyme that clears the lipid peroxidation byproduct 4-HNE, as a necessary and sufficient OSK effector. Dynamic GSTA4 regulation by OSK recapitulates a stem cell derived stress resilience program. GSTA4 overexpression alone enhances mitochondrial resilience, rejuvenates the aged RPE transcriptome, and reverses visual decline. GSTA4 is consistently upregulated across diverse lifespan-extending interventions suggesting a broader pro-longevity role. These findings uncover a previously unrecognized protective axis driven by Yamanaka factors that circumvents reprogramming, providing therapeutic insights for age-related diseases. HIGHLIGHTSO_LIOSK-GSTA4 provides a dynamic, Tet2-independent stress-resilience axis. C_LIO_LIFunctional genomics pinpoints GSTA4 as a direct downstream effector activated by OSK. C_LIO_LIRPE aging involves progressive accumulation of 4-HNE that can be detoxified by GSTA4. C_LIO_LIEnhancing GSTA4 rejuvenates RPE cells, restores vision and is associated with lifespan-extending interventions. C_LI

genetics↗

bioRxiv2025

Calcite Precipitation by a Nitrogen-Fixing Cyanobacterium

Microbiologically induced calcium carbonate precipitation (MICP) is the process through which the metabolic activity of microorganisms causes the precipitation of calcium carbonate, which can result in solidification of sediment. In cyanobacteria, MICP is thought to occur primarily because cells sequester bicarbonate for the photosynthetic process, thereby lowering the pH of the surrounding media. However, these mechanisms are still poorly understood. Here we show direct evidence of MICP caused by the filamentous cyanobacterium Anabaena. Anabaena differentiates into photosynthetic vegetative cells and nitrogen-fixing heterocysts. Using quantitative microscopy, we show that MICP occurs due to two distinct mechanisms: Firstly, mechanical stress on vegetative cells can cause leakage and/or lysis, releasing sequestered bicarbonate into the environment, resulting in formation of new crystals. Secondly, contact between a heterocyst and a calcite crystal seed appears to cause rapid crystal growth. Our results suggest an evolutionary benefit of contact-mediated precipitation to anchor cyanobacteria growing in tidal regions. By providing greater insight into MICP caused by Anabaena, these results could be used to optimize bio-cement production, thereby enabling a green construction material that could assist with carbon sequestration and reducing the impact of climate change.

microbiology↗

bioRxiv2024

Machine Learning Models for Segmentation and Classification of Cyanobacterial Cells

Timelapse microscopy has recently been employed to study the metabolism and physiology of cyanobacteria at the single-cell level. However, the identification of individual cells in brightfield images remains a significant challenge. Traditional intensity-based segmentation algorithms perform poorly when identifying individual cells in dense colonies due to a lack of contrast between neighboring cells. Here, we describe a newly developed software package called Cypose which uses machine learning (ML) models to solve two specific tasks: segmentation of individual cyanobacterial cells, and classification of cellular phenotypes. The segmentation models are based on the Cellpose framework, while classification is performed using a convolutional neural network named Cyclass. To our knowledge, these are the first developed ML-based models for cyanobacteria segmentation and classification. When compared to other methods, our segmentation models showed improved performance and were able to segment cells with varied morphological phenotypes, as well as differentiate between live and lysed cells. We also found that our models were robust to imaging artifacts, such as dust and cell debris. Additionally, the classification model was able to identify different cellular phenotypes using only images as input. Together, these models improve cell segmentation accuracy and enable high-throughput analysis of dense cyanobacterial colonies and filamentous cyanobacteria.

microbiology↗

bioRxiv2024

Cyanobacteria form a procarboxysome-like structure in response to high CO2

Fixing 25% of CO2 globally, cyanobacteria are integral to climate change efforts. The cyanobacterial CO2 concentrating mechanism (CCM) features the carboxysome, a bacterial microcompartment which houses their CO2 fixing machinery. The proteinaceous shell of the carboxysome restricts diffusion of CO2, both inward and outward. While necessary for CCM function in air (0.04% CO2), when grown in high CO2 levels (3% CO2) representative of early earth, the shell would harmfully limit CO2 fixation. To understand how carboxysomes change form and function in response to increased CO2 conditions, we used a Grx1-roGFP2 redox sensor and single cell timelapse fluorescence microscopy to track subcellular redox states of Synechococcus sp. PCC 7002 grown in air or 3% CO2. Comparing different levels of compartmentalization, we targeted the cytosol, a shell-less carboxysomal assembly intermediate called the procarboxysome, and the carboxysome. The carboxysome redox state was dynamic and, under 3% CO2, procarboxysome-like structures formed and mirrored cytosolic redox states, indicating that a more permeable shell architecture may be favorable when [CO2] is high. This work represents a step in understanding how cyanobacteria respond to changing CO2 concentrations and the selective forces driving carboxysome evolution.

biochemistry↗

bioRxiv2024

High-density CRISPRi screens reveal adaptive transcriptional gradients in cyanobacteria

Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the biochemical basis of environmental adaptation in these microbes, it remains poorly understood and challenging to re-wire. This study uses a high-density, genome-wide CRISPR interference (CRISPRi) screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extrema. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Notably, transcriptional repression of a gene for a core subunit of the NDH-1 complex, which is important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. In general, most genes with fitness-improving knockdowns were distinct to each light color, evidencing unique stress responses and alleviation mechanisms. Multi-target transcriptional repression produced nonadditive effects. Findings reveal diverse mechanisms of environmental adaptation in cyanobacteria and provide a new approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells. SIGNIFICANCE STATEMENTCyanobacteria are the most abundant photosynthetic organisms on Earth, where they endure a striking variety of environmental fluctuations. This study examines the biochemical basis of environmental adaptation in Synechococcus sp. PCC 7002, an important model strain, by modulating the expression of every gene in its genome. Results show that partial, but not complete, reduction in the expression of a subset of influential genes can improve growth under cold monochromatic conditions. Optimal expression levels differ between red and blue light and shift with multi-gene adjustments. Findings show how minor transcriptional adjustments can yield major improvements in growth under environmental extrema and provide a powerful systems-level approach for studying--and fine-tuning--the adaptive capacity of microbes.

microbiology↗

bioRxiv2022

Kinetics and targeting of Vipp1 aggregation in cyanobacteria

The kinetics of regulation of vesicle-inducing protein in plastids (Vipp1), an essential protein in all oxygenic phototrophs, are not completely understood. Vipp1 is responsible for both membrane maintenance and damage repair processes, aggregating at sites of damage (proton leakage) or physical strain (responding to presence of anionic lipids). Herein we spatiotemporally resolve exchange between inactive cytoplasmic Vipp1 and these two functionalities. We demonstrate that Vipp1 can be aggregated to a specific site in the thylakoid membrane by laser damage, but this behavior appears to be mediated by a rapidly resolved secondary process, as cytoplasmic Vipp1 is rendered unresponsive to nearby damage on a time scale three orders of magnitude faster than the aggregation process itself. One Sentence SummaryVipp1 aggregation to points on the thylakoid is induced in response to specific damage and repair kinetics are observed.

biochemistry↗

bioRxiv2022

Asymmetric survival in single-cell lineages of cyanobacteria in response to photodamage

Summary paragraphOxygenic photosynthesis is driven by the coupled action of the light-dependent pigment protein complexes, photosystem I and II, located within the internal thylakoid membrane system. However, photosystem II is known to be prone to photooxidative damage. Thus, photosynthetic organisms have evolved a repair cycle to continuously replace the damaged proteins in photosystem II. However, it has remained difficult to deconvolute the damage and repair processes using traditional ensemble approaches. Here we demonstrate an automated approach using time-lapse fluorescence microscopy and computational image analysis to study the dynamics and effects of photodamage in single cells at sub-cellular resolution in cyanobacteria. By growing cells in a two-dimensional layer, we avoid shading effects, thereby generating uniform and reproducible growth conditions. Using this platform, we analyzed the growth and physiology of multiple strains simultaneously under defined photoinhibitory conditions stimulated by UV-A light. Our results reveal an asymmetric cellular response to photodamage between sibling cells and the generation of an elusive subcellular structure, here named a photoendosome, derived from the thylakoid which could indicate the presence of a previously unknown photoprotective mechanism. We anticipate these results to be a starting point for further studies to better understand photodamage and repair at the single-cell level.

biochemistry↗

bioRxiv2020

Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index: a Rapid and Accessible Tool that Exploits Genomic Data in Public Health and Clinical Microbiology Applications

As microbial genomics makes increasingly important contributions to clinical and public health microbiology, the interpretation of whole genome sequence data by non-specialists becomes essential. In the absence of capsule-based vaccines, two protein-based vaccines have been used for the prevention of invasive serogroup B meningococcal disease (IMD), since their licensure in 2013/14. These vaccines have different components and different coverage of meningococcal variants. Hence, decisions regarding which vaccine to use in managing serogroup B IMD outbreaks require information about the index case isolate including: (i) the presence of particular vaccine antigen variants; (ii) the expression of vaccine antigens; and (iii) the likely susceptibility of its antigen variants to antibody-dependent bactericidal killing. To obtain this information requires a multitude of laboratory assays, impractical in real-time clinical settings, where the information is most urgently needed. To facilitate assessment for public health and clinical purposes, we synthesised genomic and experimental data from published sources to develop and implement the Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index, which is publicly-available on PubMLST (https://pubmlst.org). Using whole genome sequences or individual gene sequences obtained from IMD isolates or clinical specimens, MenDeVAR provides rapid evidence-based information on the presence and possible immunological cross-reactivity of different meningococcal vaccine antigen variants. The MenDeVAR Index enables practitioners who are not genomics specialists to assess the likely reactivity of vaccines for individual cases, outbreak management, or the assessment of public health vaccine programmes. MenDeVAR has been developed in consultation with, but independently of, both vaccine manufacturers.

genomics↗

bioRxiv2020

CyAn: A MATLAB toolbox for image and data analysis of cyanobacteria

Studies of photosynthetic cyanobacteria are often carried out by growing the bacteria in large volume cultures. However, this results in an uneven amount of light received by the cells due to cell-cell shading. Fluorescence microscopy solves this issue as cells can be grown in a single layer, eliminating shading issues. However, microscopy brings its own challenge as hundreds of cells are imaged at the same time, making it difficult to analyze the resulting data by hand. Here we present CyAn, a toolbox to process and analyze microscope images of cyanobacteria. The toolbox automates cyanobacteria identification in microscope images, tracks moving and dividing cells, and computes relevant biological data such as growth rates. As proof-of-principle, we demonstrate that wild type and mutants can be grown on the same slide and identified using the software, allowing a much more accurate probe into photosynthetic processes that are often masked due to cellular heterogeneity or those requiring precise light control to prevent cell-cell shading.

microbiology↗

bioRxiv2020

Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002

Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the structure-function relationships between the intracellular structures of cyanobacteria and their roles in cells. While these studies have made great progress in understanding cyanobacterial physiology, the previous fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We have performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a PsbU-APEX2 gene fusion. This study demonstrates the power of APEX2 as a tool to study the cell biology of intracellular features of cyanobacteria with enhanced spatiotemporal resolution.

biochemistry↗