Biology subjects

Khariton, M.

Publications and source records attributed to Khariton, M..

7 recordsLinked to original sources

bioRxiv2022

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

bioengineering↗

bioRxiv2022

Comparisons of cell proliferation and cell death across life histories in the hemichordate Schizocardium californicum

BackgroundThere are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when the tissue that will give rise to the adult originates. Here we present patterns of cell proliferation and cell death during larval development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium to answer these questions. ResultsWe identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate prior to the start of any morphological metamorphosis. Additionally, we describe a genetic signature of proliferative cells with an irradiation approach that revealed markers shared between the life history states and others that are unique to larvae or juveniles. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. ConclusionsCell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferate and give rise to the larva and juvenile have a genetic signature that is more suggestive of a heterogeneous pool of proliferative progenitors versus a population of pluripotent set-aside cells. Taken together, we propose that S. californicum has a transformative metamorphosis that may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

developmental biology↗

bioRxiv2021

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system. MotivationThe study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

bioengineering↗

bioRxiv2020

Mapping single-cell atlases throughout Metazoa unravels cell type evolution

Comparing single-cell transcriptomic atlases from diverse organisms can elucidate the origins of cellular diversity and assist the annotation of new cell atlases. Yet, comparison between distant relatives is hindered by complex gene histories and diversifications in expression programs. Previously, we introduced the self-assembling manifold (SAM) algorithm to robustly reconstruct manifolds from single-cell data (Tarashansky et al., 2019). Here, we build on SAM to map cell atlas manifolds across species. This new method, SAMap, identifies homologous cell types with shared expression programs across distant species within phyla, even in complex examples where homologous tissues emerge from distinct germ layers. SAMap also finds many genes with more similar expression to their paralogs than their orthologs, suggesting paralog substitution may be more common in evolution than previously appreciated. Lastly, comparing species across animal phyla, spanning mouse to sponge, reveals ancient contractile and stem cell families, which may have arisen early in animal evolution.

bioinformatics↗

bioRxiv2020

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput phenotyping via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying size and morphology as well as a heterogeneous cell mixture of a whole dissociated flatworm (5 - 25 m in diameter) within highly monodisperse double emulsions (35 m in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS phenotyping of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional screening. ABSTRACT FIGURE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/139311v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9d2028org.highwire.dtl.DTLVardef@1aa55b3org.highwire.dtl.DTLVardef@1e05eb3org.highwire.dtl.DTLVardef@1695a88_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Mechanically Resolved Imaging of Bacteria using Expansion Microscopy

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (ExM) in which cells are physically expanded prior imaging and their expansion patterns depend on the structural and mechanical properties of their cell walls, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation in order to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of ExM to distinguish species within a dense community through in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

biophysics↗

bioRxiv2018

‘Chromatic’ Neuronal Jamming in a Primitive Brain

Jamming models developed in inanimate matter have been widely used to describe cell packing in tissues1-7, but predominantly neglect cell diversity, despite its prevalence in biology. Most tissues, animal brains in particular, comprise a mix of many cell types, with mounting evidence suggesting that neurons can recognize their respective types as they organize in space8-11. How cell diversity revises the current jamming paradigm is unknown. Here, in the brain of the flatworm planarian Schmidtea mediterranea, which exhibits remarkable tissue plasticity within a simple, quantifiable nervous system12-16, we identify a distinct packing state, chromatic jamming. Combining experiments with computational modeling, we show that neurons of distinct types form independent percolating networks barring any physical contact. This jammed state emerges as cell packing configurations become constrained by cell type-specific interactions and therefore may extend to describe cell packing in similarly complex tissues composed of multiple cell types.

biophysics↗