Biology subjects

Verboon, J. M.

Publications and source records attributed to Verboon, J. M..

9 recordsLinked to original sources

bioRxiv2026

Spatiotemporal Dynamics of Protein Recruitment During Cell Wound Repair

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

cell biology↗

bioRxiv2022

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

bioRxiv2022

The Centralspindlin proteins Pavarotti and Tumbleweed work with WASH to regulate Nuclear Envelope budding

Nuclear envelope (NE) budding is a nuclear pore independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott-Aldrich Syndrome protein Wash, its regulatory complex (SHRC), and Arp2/3, and requires Washs actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin bundling activities of Wash and Pav are required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development.

cell biology↗

bioRxiv2022

Latent human herpesvirus 6 is reactivated in chimeric antigen receptor T cells

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 super-expressors (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5-7 and has broad implications for the design, production, and monitoring of cell therapies.

immunology↗

bioRxiv2020

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

developmental biology↗

bioRxiv2020

Genetic Analyses of Blood Cell Structure for Biological and Pharmacological Inference

Thousands of genetic associations with phenotypes of blood cells are known, but few are with phenotypes relevant to cell function. We performed GWAS of 63 flow-cytometry phenotypes, including measures of cell granularity, nucleic acid content, and reactivity, in 39,656 participants in the INTERVAL study, identifying 2,172 variant-trait associations. These include associations mediated by functional cellular structures such as secretory granules, implicated in vascular, thrombotic, inflammatory and neoplastic diseases. By integrating our results with epigenetic data and with signals from molecular abundance/disease GWAS, we infer the hematopoietic origins of population phenotypic variation and identify the transcription factor FOG2 as a regulator of platelet -granularity. We show how flow cytometry genetics can suggest cell types mediating complex disease risk and suggest efficacious drug targets, presenting Daclizumab/Vedolizumab in autoimmune disease as positive controls. Finally, we add to existing evidence supporting IL7/IL7-R as drug targets for multiple sclerosis.

genetics↗

bioRxiv2019

Wash and the WASH Regulatory Complex function in Nuclear Envelope budding

Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes can be packaged inside the nucleus and be extruded through the nuclear membranes, completely bypassing nuclear pores. While factors have been identified both as cargos or actively involved in this process, much remains unknown about the molecules that generate the forces and membrane deformations which appear inherent. Using fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model, we identify Wash, its regulatory complex, and Arp2/3 as novel players in NE-budding. Surprisingly, Washs role in this process is bipotent and, independent of SHRC/Arp2/3, its perturbation disrupts the normal homotypic Lamin A/B meshworks that are necessary for NE-budding to occur. In addition to NE-budding emerging as important in additional cellular processes and organisms, its incredible similarity to herpesvirus egress suggests new avenues for exploration in both normal and disease biology.

cell biology↗

bioRxiv2019

The kinesin-like protein Pavarotti functions non-canonically to regulate actin dynamics

Pavarotti, the Drosophila MKLP1 ortholog, is a kinesin-like protein that with Tumbleweed (MgcRacGAP) works together as the centralspindlin complex. This complex is essential for cytokinesis where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. We previously showed that Tumbleweed and Pebble are required for the cell wound repair process. Here, we show that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are utilized during this cellular repair, it is not as the conserved centralspindlin complex. Surprisingly, in vitro and in vivo work show that the classically microtubule-associated Pavarotti binds directly to actin and has a non-canonical role directly regulating actin dynamics. We show that Pavarotti also works independently from Tumbleweed in several actin-related processes during the normal developmental process of oogenesis.

cell biology↗

bioRxiv2018

The Genetic Landscape of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.

genetics↗