Biology subjects

Caplan, J. L.

Publications and source records attributed to Caplan, J. L..

7 recordsLinked to original sources

bioRxiv2025

Toward simple, rapid, and deep plant proteome analysis with an in-cell proteomics strategy

Although modern mass spectrometry (MS) based proteomics has been broadly employed by the plant science community, proteomics analysis of plant tissues remains highly challenging to general plant biology laboratories, mainly due to the lack of effective and easily adaptable strategies that can process plant samples for MS analysis rapidly and reliably. Existing methods often involves lengthy mechanical disruption and protein extraction, which not only require large starting materials but also can cause significant variations, compromising quantitation accuracy. Here, we present an "in-cell proteomics" strategy and benchmark its performance against conventional processing methods using several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Zea mays and Sorghum bicolor. The comprehensive datasets from this study indicate that the on-filter in-cell (OFIC) processing-based "in-cell proteomics" strategy can eliminate cell lysis of plant leaves and pollen grains, and process proteins directly, enabling the identification of 9,000 to 12,000 proteins from leaves, and 7,000 to 9,000 proteins from pollen grains using one-shot LCMS. The OFIC strategy demonstrated broadly equivalent or better performance over conventional approaches in the context of simplicity, speed, digestion effectiveness, and quantitation accuracy. Applying the strategy to investigate plant leaf infection of N. benthamiana leaves revealed interesting signature proteins that respond differentially to host-adapted Colletotrichum destructivum and nonhost-adapted Colletotrichum sublineola fungus. The single-vessel approach bypasses cell disruption, substantially simplifying plant sample preparation while reducing the required sample input and enhancing proteomic sensitivity. It offered a generalizable approach to study plant leaf infection and other significant questions in plant biology and beyond.

biochemistry↗

bioRxiv2023

Characterization of extracellular vesicles isolated from Sorghum bicolor reveals a conservation between monocot and eudicot extracellular vesicle proteomes

Plant extracellular vesicles (EVs) are membrane bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. In this paper, we described the isolation and characterization of extracellular vesicles from Sorghum bicolor. Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, Calcein-acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also being found in the EV proteome of Arabidopsis. These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

plant biology↗

bioRxiv2021

Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

cell biology↗

bioRxiv2020

Pre-meiotic, 21-nucleotide Reproductive PhasiRNAs Emerged in Seed Plants and Diversified in Flowering Plants

Plant small RNAs (sRNAs) are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. They have critical roles in most pathways involved in plant growth and reproductive development. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have not been described in eudicots. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we describe additional variants of 21-nt reproductive phasiRNAs, including those triggered by miR11308 in wild strawberry, a eudicot, and we validate the presence of this pathway in rose. We report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots, with novel biogenesis triggers in the basal eudicot columbine and the rosid flax. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports a singular finding in spruce, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

plant biology↗

bioRxiv2019

Quantitative, super-resolution localization of small RNAs with sRNA-PAINT

Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and around 10 nanometers (nm) in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called "Vetting & Analysis of RNA for in situ Hybridization probes" (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.

cell biology↗

bioRxiv2019

3D Microstructure of Tendon Collagen Fibrils using Serial Block-Face SEM and a Mechanical Model for Load Transfer

Tendons hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer, however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy (SBF-SEM) to investigate the threedimensional microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite element modeling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

bioengineering↗

bioRxiv2018

Sex chromosome evolution via two genes

The origin of sex chromosomes has been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis L.) is an ideal species to test this hypothesis, as the X and Y chromosomes are cytologically homomorphic and recently evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with singlemolecule fluorescence in situ hybridization (smFISH) directly implicate Y-specific genes that respectively suppress female organ development and are necessary for male gametophyte development. Comparison of contiguous X and Y chromosome shows that loss of recombination between the genes suppressing female function (SUPPRESSOR OF FEMALE FUNCTION, SOFF) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION 1, aspTDF1) is due to hemizygosity. We also experimentally demonstrate the function of aspTDF1. These finding provide direct evidence that sex chromosomes can evolve from autosomes via two sex determination genes: a dominant suppressor of femaleness and a promoter of maleness.\n\nOne Sentence SummarySex chromosomes can evolve from autosomes by mutations in two sex determination genes.

genetics↗