Protein–Protein Interaction Network and Subcellular Localization of the Arabidopsis Thaliana ESCRT Machinery

The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein–protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants

The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells

The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)-localized omega-3 fatty acid desaturases (Fad3s) are regulated extensively at the post-transcriptional level by both temperature-dependent changes in translational efficiency, as well as modulation of protein half-life. While the N-terminal sequences of Fad3 proteins were shown to contain information that mediates their rapid, proteasome-dependent protein turnover in both plant and yeast cells, it is currently unknown whether these sequences alone are sufficient to direct protein degradation. In this report, we fused the N-terminal sequences of two different Fad3 proteins to an ER-localized fluorescent protein reporter, consisting of the green fluorescent protein and the ER integral membrane protein cytochrome b5, and then measured (via microscopy) the degradation of the resulting fusion proteins in plant suspension-cultured cells relative to a second, co-expressed fluorescent reporter protein. Overall, the results demonstrate that the N-termini of both Fad3 proteins are sufficient for conferring rapid, proteasome-dependent degradation to an ER-bound marker protein

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.12 month embargo; published: 26 September 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms

Single‐Cell, Single‐Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single-cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation-related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver

Clinical Phenomapping and Outcomes after Heart Transplantation

BackgroundSurvival after heart transplantation (HTx) is limited by complications related to alloreactivity, immune suppression, and side effects of pharmacological therapies. We hypothesize that time-dependent phenomapping of clinical and molecular datasets is a valuable approach to clinical assessments and guiding medical management to improve outcomes.MethodsWe analyzed clinical, therapeutic, biomarker, and outcome data from 94 adult HTx patients and 1557 clinical encounters performed between January 2010 and April 2013. Multivariate analyses were employed to evaluate the association between immunosuppression therapy, biomarkers, and the combined clinical endpoint of death, allograft loss, retransplantation, and rejection. Data were analyzed by K-means clustering (k=2) to identify patterns of similar combined immunosuppression management, and percentile slopes were computed to examine the changes in dosages over time. Findings were correlated with clinical parameters, HLA antibody titers, peripheral blood mononuclear cell gene expression of the AlloMap test genes, and an intragraft, heart tissue gene co-expression network analysis was performed.ResultsUnsupervised cluster analysis of immunosuppressive therapies identified two groups, one characterized by a steeper immunosuppression minimization, associated with a higher likelihood for the combined endpoint, and the other by a less pronounced change. A time-dependent phenomap suggested that patients in the higher event rate group had increased HLA class I and II antibody titers, higher expression of the FLT3 AlloMap gene, and lower expression of the March8 and WDNR40A AlloMap genes. Intramyocardial biomarker-related co-expression network analysis of the FLT3 showed an immune system-related network underlying this biomarker.ConclusionTime-dependent precision phenotyping is a mechanistically insightful, data-driven approach to characterize patterns of clinical care and identify ways to improve clinical management and outcomes

Clinical Phenomapping and Outcomes after Heart Transplantation

BackgroundSurvival after heart transplantation (HTx) is limited by complications related to alloreactivity, immune suppression, and side effects of pharmacological therapies. We hypothesize that time-dependent phenomapping of clinical and molecular datasets is a valuable approach to clinical assessments and guiding medical management to improve outcomes.MethodsWe analyzed clinical, therapeutic, biomarker, and outcome data from 94 adult HTx patients and 1557 clinical encounters performed between January 2010 and April 2013. Multivariate analyses were employed to evaluate the association between immunosuppression therapy, biomarkers, and the combined clinical endpoint of death, allograft loss, retransplantation, and rejection. Data were analyzed by K-means clustering (k=2) to identify patterns of similar combined immunosuppression management, and percentile slopes were computed to examine the changes in dosages over time. Findings were correlated with clinical parameters, HLA antibody titers, peripheral blood mononuclear cell gene expression of the AlloMap test genes, and an intragraft, heart tissue gene co-expression network analysis was performed.ResultsUnsupervised cluster analysis of immunosuppressive therapies identified two groups, one characterized by a steeper immunosuppression minimization, associated with a higher likelihood for the combined endpoint, and the other by a less pronounced change. A time-dependent phenomap suggested that patients in the higher event rate group had increased HLA class I and II antibody titers, higher expression of the FLT3 AlloMap gene, and lower expression of the March8 and WDNR40A AlloMap genes. Intramyocardial biomarker-related co-expression network analysis of the FLT3 showed an immune system-related network underlying this biomarker.ConclusionTime-dependent precision phenotyping is a mechanistically insightful, data-driven approach to characterize patterns of clinical care and identify ways to improve clinical management and outcomes

Clinical phenomapping and outcomes after heart transplantation

BackgroundSurvival after heart transplantation (HTx) is limited by complications related to alloreactivity, immune suppression, and adverse effects of pharmacologic therapies. We hypothesize that time-dependent phenomapping of clinical and molecular data sets is a valuable approach to clinical assessments and guiding medical management to improve outcomes.MethodsWe analyzed clinical, therapeutic, biomarker, and outcome data from 94 adult HTx patients and 1,557 clinical encounters performed between January 2010 and April 2013. Multivariate analyses were used to evaluate the association between immunosuppression therapy, biomarkers, and the combined clinical end point of death, allograft loss, retransplantation, and rejection. Data were analyzed by K-means clustering (K = 2) to identify patterns of similar combined immunosuppression management, and percentile slopes were computed to examine the changes in dosages over time. Findings were correlated with clinical parameters, human leucocyte antigen antibody titers, and peripheral blood mononuclear cell gene expression of the AlloMap (CareDx, Inc., Brisbane, CA) test genes. An intragraft, heart tissue gene coexpression network analysis was performed.ResultsUnsupervised cluster analysis of immunosuppressive therapies identified 2 groups, 1 characterized by a steeper immunosuppression minimization, associated with a higher likelihood for the combined end point, and the other by a less pronounced change. A time-dependent phenomap suggested that patients in the group with higher event rates had increased human leukocyte antigen class I and II antibody titers, higher expression of the FLT3 AlloMap gene, and lower expression of the MARCH8 and WDR40A AlloMap genes. Intramyocardial biomarker-related coexpression network analysis of the FLT3 gene showed an immune system-related network underlying this biomarker.ConclusionsTime-dependent precision phenotyping is a mechanistically insightful, data-driven approach to characterize patterns of clinical care and identify ways to improve clinical management and outcomes