Biology subjects

Crossman, S.

Publications and source records attributed to Crossman, S..

4 recordsLinked to original sources

bioRxiv2026

Observing concurrent subcellular dynamics in large living tissues

An outstanding question in eukaryotic biology is the mechanistic connection between events occurring at (sub)cellular levels (time scales of milliseconds to minutes) to those at the tissue levels (tens of minutes to months). Deciphering such mechanisms requires imaging approaches capable of simultaneously achieving high spatial and temporal resolutions for large samples over long periods of time. Here, we demonstrate Airy beam-based light sheet microscopy of organelles in tens to hundreds of cells in a few hundred micrometre-wide tissue environments. We achieve a typical resolution of 320 nm over 266 x 266 x 100 m3 volumes at a temporal rate of 0.05 Hz, typically with generally used fluorophores such as Green Fluorescent Protein, over extended periods of time that allow tracking of organelle and protein dynamics. We validated our approach across different length and time scales by imaging mitochondria and endosome dynamics in very large fields of view in zebrafish tissue, molecular assemblies of myosin as gastrulation proceeds in Drosophila embryos, 3D mitochondrial streaming in mouse oocytes, pressure-driven motility and protrusions in amoebae, mitochondrial dynamics in cancer spheroids, 5 -colour fast imaging in iBlastoids, and endosomal dynamics in single cells. Through these model systems, we demonstrate the versatility of Airy beam light sheet microscopy to image large tissues at unprecedented high resolution; to capture dynamics in photosensitive, delicate samples; and to screen 3D samples. We anticipate that our Airy beam-based approach will represent a pivotal advance in cellular biology--especially developmental biology--as it provides, for the first time, true subcellular resolution over large imaging volumes with high temporal resolution.

developmental biology↗

bioRxiv2020

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Optogenetics has been harnessed to shed new mechanistic light on current therapies and to develop future treatment strategies. This has been to date achieved by the correction of electrical signals in neuronal cells and neural circuits that are affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and thereby may modify progression of degenerative disorders, has never been demonstrated in an animal disease model. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinsons disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-[kcy]B pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to overcome limitations of current strategies towards a spatio-temporal regulation of tissue repair. Significance StatementThe death of physiologically important cell populations underlies of a wide range of degenerative disorders, including Parkinsons disease (PD). Two major strategies to counter cell degeneration, soluble growth factor injection and growth factor gene therapy, can lead to the undesired activation of bystander cells and non-natural permanent signaling responses. Here, we employed optogenetics to deliver cell type-specific pro-survival signals in a genetic model of PD. In Drosophila and human cells exhibiting loss of the PINK1 kinase, akin to autosomal recessive PD, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in disease models.

neuroscience↗

bioRxiv2020

Random Forest Regression for Optimizing Variable Planting Rates for Corn and Soybean Using High-Resolution Topographical and Soil Data

In recent years, planting machinery that enables precise control of the planting rates has become available for corn (Zea mays L.) and soybean (Glycine max L.). With increasingly available topographical and soil information, there is a growing interest in developing variable rate planting strategies to exploit variation in the agri-landscape in order to maximize production. A random forest regression-based approach was developed to model the interactions between planting rate, topography, and soil characteristics and their effects on yield based on on-farm variable rate planting trials for corn and soybean conducted at 27 sites in New York between 2014 and 2018 (57 site-years) in collaboration with the New York Corn and Soybean Growers Association. Planting rate ranked highly in terms of random forest regression variable importance while explaining relatively minimal yield variation in the linear context, indicating that yield response to planting rate likely depends on complex interactions with agri-landscape features. Models were moderately predictive of yield within site-years and across years at a particular site, while the ability to predict yield across sites was low. Relatedly, variable importance measures for the topographical and soil features varied considerably across sites. Together, these results suggest that local testing may provide the most accurate optimized planting rate designs due to the unique set of conditions at each site. The proposed method was extended to identify the optimal variable rate planting design for maximizing yield at each site given the topographical and soil data, and empirical validation of the resulting designs is currently underway.

plant biology↗

bioRxiv2019

Lessons in genome engineering: opportunities, tools and pitfalls

CRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome. This technology is now routine for many organisms and cell lines. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. We describe how these tools and protocols can be implemented easily and efficiently. Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. Our tools are designed for use in Drosophila but the lessons we draw are likely to be widely relevant.\n\nAUTHOR SUMMARYThe genome contains all the information that an organism needs to develop and function throughout its life. One of the goal of genetics is to decipher the role of all the genes (typically several thousands for an animal) present in the genome. One approach is to delete each gene and assay the consequences. Deletion of individual genes is now readily achieved with a technique called CRISPR/Cas9. However, simple genetic deletion provides limited information. Here we describe strains and DNA vectors that streamline the generation of more sophisticated genetic tools. We describe general means of creating alleles (genetic variants) that enable gene activity to be measured and experimentally modulated in space and time. Although the tools we describe are universally applicable, each gene requires special consideration. Based on our experience of successes and failures, we suggest measures to maximise the chances that engineered alleles serve their intended purpose. Although our methods are designed for use in Drosophila, they could be adapted to any organism that is amenable to CRISPR/Cas9 genome modification.

genetics↗