bims-scepro 2024-12-08 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024–12–08
seventeen papers selected by
William Grey, University of York

ERRα is dispensable for hematopoietic stem cell function in the bone marrow.
The dynamics of hematopoiesis over the human lifespan.
Exercise alleviates hematopoietic stem cell injury following radiation via the carnosine/Slc15a2-p53 axis.
Hematopoietic stem cell heterogeneity and age-related platelet bias: implications for bone marrow transplantation and blood disorders.
Versatility of megakaryocytes in homeostasis and disease.
TIFAB Modulates Metabolic Pathways in KMT2A::MLLT3-Induced AML Through HNF4A.
Single-cell transcriptome profiling of m6A regulator-mediated methylation modification patterns in elderly acute myeloid leukemia patients.
Cellular crosstalk in the bone marrow niche.
Targeting Caveolin-1 in Multiple Myeloma Cells Enhances Chemotherapy and Natural Killer Cell-Mediated Immunotherapy.
Interferon gamma-mediated prevention of tumor progression in a mouse model of multiple myeloma.
Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting.
Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.
Evolution of myeloproliferative neoplasms from normal blood stem cells.
The kinetochore protein KNL-1 regulates the actin cytoskeleton to control dendrite branching.
High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myeloid leukemia.
A Pax3 lineage gives rise to transient haematopoietic progenitors.
Systematic Identification of Microtubule Posttranslational Modification "Readers" by Quantitative Proteomics.

Biochem Biophys Res Commun. 2024 Nov 26. pii: S0006-291X(24)01611-5. [Epub ahead of print]741 151075

ERRα is dispensable for hematopoietic stem cell function in the bone marrow.

Hee Eun Bae, Ninib Baryawno, Byoung Soo Kim, Sik Yoon, Sae-Ock Oh, Eun-Kyeong Jo, Chi Dae Kim, Dongjun Lee.

  Hematopoietic stem cells (HSCs) have the ability to self-renew, differentiate into various blood cell types, and reside in the bone marrow (BM) niche. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has a role in mitochondrial biogenesis and metabolic regulation. Previous research has shown that ERRα contributes to the development of acute myeloid leukemia (AML) by acting as a key regulator of mitochondrial processes, though its role in HSC regulation remains mostly unknown. Flow cytometric analysis determined the population of the hematopoietic stem and progenitor cells (HSPC) using Errα knockout (KO) and conditional KO (cKO) mice. Furthermore, we investigated reconstitution with BM transplantation assay. Here, the ablation of Errα in the BM demonstrated that the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the HSPC populations were similar to those of controls. In addition, the ablation of Errα did not perturb HSC function under the stress of transplantation. Collectively, ERRα is not necessary for the control of HSPC populations, as well as for the maintenance of HSC characteristics and functions within the BM.

Keywords:  Bone marrow niche; ERRα; Hematopoietic stem cells

DOI:  https://doi.org/10.1016/j.bbrc.2024.151075
Nat Methods. 2024 Dec 05.

The dynamics of hematopoiesis over the human lifespan.

Hojun Li, Parker Côté, Michael Kuoch, Jideofor Ezike, Katie Frenis, Anton Afanassiev, Laura Greenstreet, Mayuri Tanaka-Yano, Giuseppe Tarantino, Stephen Zhang, Jennifer Whangbo, Vincent L Butty, Enrico Moiso, Marcelo Falchetti, Kate Lu, Guinevere G Connelly, Vivian Morris, Dahai Wang, Antonia F Chen, Giada Bianchi, George Q Daley, Salil Garg, David Liu, Stella T Chou, Aviv Regev, Edroaldo Lummertz da Rocha, Geoffrey Schiebinger, R Grant Rowe.

Over a lifetime, hematopoietic stem cells (HSCs) adjust their lineage output to support age-aligned physiology. In model organisms, stereotypic waves of hematopoiesis have been observed corresponding to defined age-biased HSC hallmarks. However, how the properties of hematopoietic stem and progenitor cells change over the human lifespan remains unclear. To address this gap, we profiled individual transcriptome states of human hematopoietic stem and progenitor cells spanning gestation, maturation and aging. Here we define the gene expression networks dictating age-specific differentiation of HSCs and the dynamics of fate decisions and lineage priming throughout life. We additionally identifiy and functionally validate a fetal-specific HSC state with robust engraftment and multilineage capacity. Furthermore, we observe that classification of acute myeloid leukemia against defined transcriptional age states demonstrates that utilization of early life transcriptional programs associates with poor prognosis. Overall, we provide a disease-relevant framework for heterochronic orientation of stem cell ontogeny along the real time axis of the human lifespan.

DOI: https://doi.org/10.1038/s41592-024-02495-0
Cell Commun Signal. 2024 Dec 03. 22(1): 582

Exercise alleviates hematopoietic stem cell injury following radiation via the carnosine/Slc15a2-p53 axis.

Hao Zeng, Naicheng Chen, Fang Chen, Xiaoyi Zhong, Lijing Yang, Yukai Lu, Mo Chen, Mingqiang Shen, Song Wang, Shilei Chen, Jia Cao, Xi Zhang, Jinghong Zhao, Yang Xu, Junping Wang, Mengjia Hu.

  Ionizing radiation (IR) can cause severe dysfunction of hematopoietic stem cells (HSCs), leading to acute or prolonged myelosuppression. In recent years, physical exercise has been recognized as a healthy lifestyle as it can fight a variety of diseases. However, whether it provides protection against IR is not fully understood. In this study, we revealed that long-term moderate exercise mitigated IR-induced hematopoietic injury by generating carnosine from skeletal muscles. We found that exercised mice displayed reduced loss of HSC number and function after IR, accompanied by alleviated bone marrow damage. Interestingly, these effects were largely abrogated by specific deletion of carnosine synthase Carns1 in skeletal muscles. In contrast, carnosine treatment protected HSCs against IR-induced injury. Mechanistically, we demonstrated that exercise-generated carnosine was specifically transported to HSCs via Slc15a2 and then inhibited p53 transcriptional activity by directly interacting with its core DNA-binding domain, which led to downregulation of the p53 target genes p21 and Puma, thus promoting the proliferation and survival and inhibiting the senescence of irradiated HSCs. More importantly, a similar role of the carnosine/Slc15a2-p53 axis was observed in human cord blood-derived HSCs. Collectively, our data reveal that moderate exercise or carnosine supplementation may be potential antiradiation strategies.

Keywords:  Carnosine; Exercise; Hematopoietic stem cell; Radiation; Senescence; p53 activity

DOI:  https://doi.org/10.1186/s12964-024-01959-2
Stem Cell Res Ther. 2024 Dec 02. 15(1): 459

Hematopoietic stem cell heterogeneity and age-related platelet bias: implications for bone marrow transplantation and blood disorders.

Yalu Chen, Jianqiao Shentu, Hanqi Lou, Yongming Xia, Yinyan Jiang, Shiwei Duan.

  Hematopoietic stem cells (HSCs) are critical for maintaining lifelong blood production and immune function, especially in the context of bone marrow transplantation, where their ability to reconstruct multiple blood lineages is essential. However, recent studies have revealed that certain HSCs exhibit a bias toward platelet differentiation, termed platelet-biased HSCs (P-HSCs). This lineage bias, particularly pronounced with aging, can lead to imbalances in post-transplant blood recovery, negatively affecting patient outcomes. Research by Claus Nerlov's team has provided key insights into the heterogeneity of HSCs, focusing on the age-related expansion of P-HSCs. Using advanced techniques such as single-cell RNA sequencing and molecular barcoding, their work highlights the evolutionary conservation of platelet bias in HSCs across species. This work delves into these findings, discussing their clinical implications for bone marrow transplantation, aging-related blood disorders, and potential therapeutic strategies. Moreover, we address limitations in current methodologies and propose future directions for research to optimize HSC-based therapies and improve clinical outcomes in hematological diseases.

Keywords:  Blood disorders; Bone marrow transplantation; Lineage bias; Platelet-biased HSCs; Therapeutic strategies

DOI:  https://doi.org/10.1186/s13287-024-04084-6
Blood Sci. 2024 Oct;6(4): e00212

Versatility of megakaryocytes in homeostasis and disease.

Daosong Wang, Jiayi Xie, Meng Zhao.

  Megakaryocytes (MKs) constitute a small portion of bone marrow cells and are primarily responsible for producing platelets, which are essential for hemostasis and wound healing. Recent studies have revealed that MKs and platelets perform diverse functions in various physiological and pathological contexts. This comprehensive review highlights the functional diversity of MKs beyond thrombopoiesis, including their roles in regulating hematopoietic stem cells, modulating immune responses, contributing to hematological malignancies, and influencing aging processes.

Keywords:  Aging; Hematopoietic stem cell; Immune responses; Megakaryocyte; Niche; Platelet

DOI:  https://doi.org/10.1097/BS9.0000000000000212
Blood Adv. 2024 Dec 03. pii: bloodadvances.2024013446. [Epub ahead of print]

TIFAB Modulates Metabolic Pathways in KMT2A::MLLT3-Induced AML Through HNF4A.

Yang Wang, Yan Xiu, Qianze Dong, Jinming Zhao, Kelao Neumbo, Masaru Miyagi, Nicholas Borcherding, Lin Fu, Havana Ray De Celis, Nicolas Giuseppe Pintozzi, Daniel T Starczynowski, Chen Zhao.

TRAF-interacting protein with forkhead-associated domain B (TIFAB), an inhibitor of NF-kB signaling, plays critical roles in hematopoiesis, myelodysplastic neoplasms, and leukemia. We previously demonstrated that Tifab enhances KMT2A::MLLT3-driven acute myeloid leukemia (AML) by either upregulating Hoxa9 or through ubiquitin-specific peptidase 15 (USP15)-mediated downregulation of p53 signaling. In this study, we show that Tifab deletion in KMT2A::MLLT3-induced AML impairs leukemia stem/progenitor cell (LSPC) engraftment, glucose uptake, and mitochondrial function. Gene Set Enrichment Analysis reveals that Tifab deletion downregulates MYC, HOXA9/MEIS1, mTORC1 signaling, and genes involved in glycolysis and oxidative phosphorylation (OXPHOS). By comparing genes upregulated in TIFAB-overexpressing LSPCs with those downregulated upon Tifab deletion, we identify hepatocyte nuclear factor 4alpha (Hnf4a) as a key TIFAB target, regulated through the inhibition of NF-kB component RelB, which suppresses Hnf4a in leukemia cells. HNF4A, a nuclear receptor involved in organ development, metabolism, and tumorigenesis, rescues the metabolic defects caused by Tifab deletion and enhances leukemia cell engraftment. Conversely, Hnf4a knockdown attenuates TIFAB-mediated enhancement of LSPC function. These findings highlight the critical role of the TIFAB-HNF4A axis in KMT2A::MLLT3-induced AML and uncover a novel regulator in leukemia biology.

DOI: https://doi.org/10.1182/bloodadvances.2024013446
Mol Biomed. 2024 Dec 06. 5(1): 66

Single-cell transcriptome profiling of m6A regulator-mediated methylation modification patterns in elderly acute myeloid leukemia patients.

Zhe Wang, Xin Du, Peidong Zhang, Meiling Zhao, Tianbo Zhang, Jiang Liu, Xiaolan Wang, Doudou Chang, Xiaxia Liu, Sicheng Bian, Xialin Zhang, Ruijuan Zhang.

  Millions of people worldwide die of acute myeloid leukaemia (AML) each year. Although N6-methyladenosine (m6A) modification has been reported to regulate the pathogenicity of AML, the mechanisms by which m6A induces dysfunctional hematopoietic differentiation in elderly AML patients remain elusive. This study elucidates the mechanisms of the m6A landscape and the specific roles of m6A regulators in hematopoietic cells of elderly AML patients. Notably, fat mass and obesity-associated protein (FTO) was found to be upregulated in hematopoietic stem cells (HSCs), myeloid cells, and T-cells, where it inhibits their differentiation via the WNT signaling pathway. Additionally, elevated YT521-B homology domain family proteins 2 (YTHDF2) expression in erythrocytes was observed to negatively regulate differentiation through oxidative phosphorylation, resulting in leukocyte activation. Moreover, IGF2BP2 was significantly upregulated in myeloid cells, contributing to an aberrant chromosomal region and disrupted oxidative phosphorylation. m6A regulators were shown to induce abnormal cell-cell communication within hematopoietic cells, mediating ligand-receptor interactions across various cell types through the HMGB1-mediated pathway, thereby promoting AML progression. External validation was conducted using an independent single-cell RNA sequencing (scRNA-Seq) dataset. The THP-1 and MV411 cell lines were utilized to corroborate the m6A regulator profile; in vitro experiments involving short hairpin RNA (shRNA) targeting FTO demonstrated inhibition of cell proliferation, migration, and oxidative phosphorylation, alongside induction of cell cycle arrest and apoptosis. In summary, these findings suggest that the upregulation of m6A regulators in HSCs, erythrocytes, myeloid cells, and T-cells may contribute to the malignant differentiation observed in AML patients. This research provides novel insights into the pathogenesis of AML in elderly patients and identifies potential therapeutic targets.

Keywords:  Acute myeloid leukemia; M6A; Malignant differentiation; Single-cell RNA-seq; WNT signaling

DOI:  https://doi.org/10.1186/s43556-024-00234-7
J Transl Med. 2024 Dec 03. 22(1): 1096

Cellular crosstalk in the bone marrow niche.

Zeqi Huang, Zoya Iqbal, Zhe Zhao, Jianquan Liu, A M Alabsi, Maryam Shabbir, Ayesha Mahmood, Yujie Liang, Wencui Li, Zhiqin Deng.

  The bone marrow niche is a special microenvironment that comprises elements, including hematopoietic stem cells, osteoblasts, and endothelial cells, and helps maintain their characteristic functions. Here, we elaborate on the crosstalk between various cellular components, hematopoietic stem cells, and other cells in the bone marrow niche. We further explain the mechanism of preserving equilibrium in the bone marrow niche, which is crucial for the directional regulation of bone reconstruction and repair. Additionally, we elucidate the intercommunication among osteocytes, the regulation of osteoblast maturation and activation by lymphocytes, the deficiency of megakaryocytes that can markedly impair osteoblast formation, and the mechanism of interaction between macrophages and mesenchymal stem cells in the bone marrow niche. Finally, we discussed the new immunotherapies for bone tumors in the BM niche. In this review, we aimed to provide a candid overview of the crosstalk among bone marrow niche cells and to highlight new concepts underlying the unknown mechanisms of hematopoiesis and bone reconstruction. Thus, this review may provide a more comprehensive understanding of the role of these niche cells in improving hematopoietic function and help identify their therapeutic potential for different diseases in the future.

Keywords:  Bone marrow; Bone reconstruction and repair; Haematopoietic stem cells; Niche

DOI:  https://doi.org/10.1186/s12967-024-05900-6
Adv Sci (Weinh). 2024 Dec 04. e2408373

Targeting Caveolin-1 in Multiple Myeloma Cells Enhances Chemotherapy and Natural Killer Cell-Mediated Immunotherapy.

Dewen Zhan, Zhimin Du, Shang Zhang, Juanru Huang, Jian Zhang, Hui Zhang, Zhongrui Liu, Eline Menu, Jinheng Wang.

  The cell membrane transport capacity and surface targets of multiple myeloma (MM) cells heavily influence chemotherapy and immunotherapy. Here, it is found that caveolin-1 (CAV1), a primary component of membrane lipid rafts and caveolae, is highly expressed in MM cells and is associated with MM progression and drug resistance. CAV1 knockdown decreases MM cell adhesion to stromal cells and attenuates cell adhesion-mediated drug resistance to bortezomib. CAV1 inhibition in MM cells enhances natural killer cell-mediated cytotoxicity through increasing CXCL10, SLAMF7, and CD112. CAV1 suppression reduces mitochondrial membrane potential, increases reactive oxygen species, and inhibits autophagosome-lysosome fusion, resulting in the disruption of redox homeostasis. Additionally, CAV1 knockdown enhances glutamine addiction by increasing ASCT2 and LAT1 and dysregulates glutathione metabolism. As a result of CAV1 inhibition, MM cells are more sensitive to starvation, glutamine depletion, and glutamine transporter inhibition, and grow more slowly in vivo in a mouse model treated with bortezomib. The observation that CAV1 inhibition modulated by 6-mercaptopurine, daidzin, and statins enhances the efficacy of bortezomib in vitro and in vivo highlights the translational significance of these FDA-approved drugs in improving MM outcomes. These data demonstrate that CAV1 serves as a potent therapeutic target for enhancing chemotherapy and immunotherapy for MM.

Keywords:  caveolin‐1; glutathione metabolism; immunotherapy; multiple myeloma; natural killer cell; redox homeostasis

DOI:  https://doi.org/10.1002/advs.202408373
Hemasphere. 2024 Dec;8(12): e70047

Interferon gamma-mediated prevention of tumor progression in a mouse model of multiple myeloma.

Zoltán Kellermayer, Sabrin Tahri, Madelon M E de Jong, Natalie Papazian, Cathelijne Fokkema, Elodie C G Stoetman, Remco Hoogenboezem, Gregory van Beek, Mathijs A Sanders, Louis Boon, Chelsea Den Hollander, Annemiek Broijl, Pieter Sonneveld, Tom Cupedo.

Malignant plasma cells in multiple myeloma patients reside in the bone marrow and continuously interact with local immune cells. Progression and therapy response are influenced by this immune environment, highlighting the need for a detailed understanding of endogenous immune responses to malignant plasma cells. Here we used the 5TGM1 murine transfer model of multiple myeloma to dissect early immune responses to myeloma cells. We modeled stable and progressive disease by transferring 5TGM1 murine myeloma cells into C57Bl/6 mice and KaLwRij mice, respectively. We used flow cytometry and single-cell and bulk transcriptomic analyses to characterize differential immune responses in stable and progressive disease. Transfer of 5TGM1 cells in C57Bl/6 mice led to stable disease with low tumor burden in a subset of animals. Stable disease was associated with sustained activation and expansion of NK cells, ILC1, and CD8+ T cells, a response that was lost upon disease progression. Single-cell RNA-sequencing of immune cells and bulk RNA sequencing of immune and mesenchymal stromal cells implicated the activation of interferon responses as a central immune pathway during stable disease. Experimentally, neutralization of IFNγ significantly increased myeloma development and progression in C57Bl/6 mice, testifying to the importance of this pathway in early disease control. In conclusion, we provide a framework for studying immune responses to multiple myeloma progression in immunocompetent and genetically modifiable mice and highlight the importance of bone marrow immunity in tumor control.

DOI: https://doi.org/10.1002/hem3.70047
Nat Commun. 2024 Dec 05. 15(1): 10614

Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting.

James M Fulcher, Lye Meng Markillie, Hugh D Mitchell, Sarah M Williams, Kristin M Engbrecht, David J Degnan, Lisa M Bramer, Ronald J Moore, William B Chrisler, Joshua Cantlon-Bruce, Johannes W Bagnoli, Wei-Jun Qian, Anjali Seth, Ljiljana Paša-Tolić, Ying Zhu.

Single-cell multiomics provides comprehensive insights into gene regulatory networks, cellular diversity, and temporal dynamics. Here, we introduce nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), an integrated platform that enables global profiling of the transcriptome and proteome from same single cells via RNA sequencing and mass spectrometry-based proteomics, respectively. Benchmarking of nanoSPLITS demonstrates high measurement precision with deep proteomic and transcriptomic profiling of single-cells. We apply nanoSPLITS to cyclin-dependent kinase 1 inhibited cells and found phospho-signaling events could be quantified alongside global protein and mRNA measurements, providing insights into cell cycle regulation. We extend nanoSPLITS to primary cells isolated from human pancreatic islets, introducing an efficient approach for facile identification of unknown cell types and their protein markers by mapping transcriptomic data to existing large-scale single-cell RNA sequencing reference databases. Accordingly, we establish nanoSPLITS as a multiomic technology incorporating global proteomics and anticipate the approach will be critical to furthering our understanding of biological systems.

DOI: https://doi.org/10.1038/s41467-024-54099-z
J Biol Chem. 2024 Nov 29. pii: S0021-9258(24)02547-X. [Epub ahead of print] 108045

Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.

Dina Franić, Mihaela Pravica, Klara Zubčić, Shawna Miles, Antonio Bedalov, Mirta Boban.

  Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

Keywords:  autophagy; inclusions; nucleus-vacuole junction; proteasome; protein aggregation; protein degradation; protein quality control; quiescence; spatial sequestration; ubiquitin

DOI:  https://doi.org/10.1016/j.jbc.2024.108045
Haematologica. 2024 Dec 05.

Evolution of myeloproliferative neoplasms from normal blood stem cells.

Sahand Hormoz, Vijay G Sankaran, Ann Mullally.

Over the course of the last decade, genomic studies in the context of normal human hematopoiesis have provided new insights into the early pathogenesis of myeloproliferative neoplasms (MPN). A preclinical phase of MPN, termed clonal hematopoiesis (CH) was identified and subsequent lineage tracing studies revealed a multi-decade long time interval from acquisition of an MPN phenotypic driver mutation in a hematopoietic stem cell (HSC) to the development of overt MPN. Multiple germline variants associated with MPN risk have been identified through genome-wide association studies (GWAS) and in some cases functional interrogation of the impact of the variant has uncovered new insights into HSC biology and MPN development. Increasingly sophisticated methods to study clonal contributions to human hematopoiesis and measure HSC fitness have helped discern the biology underlying the tremendous clinical heterogeneity observed in MPN. Despite these advances, significant knowledge gaps remain particularly with respect to germline genetic contributors to both MPN pathogenesis and phenotypic diversity, as well as limitations in the ability to prospectively quantify rates of clonal expansion in individual MPN patients. Ultimately, we envisage a personalized approach to MPN care in the future, where an individualized genetic assessment can predict MPN trajectory and this information will be used to inform and guide therapy. MPN is particularly amenable to precision medicine strategies and our increased understanding of the evolution of MPN from normal blood stem cells provides a unique opportunity for early therapeutic intervention approaches and potentially MPN prevention strategies.

DOI: https://doi.org/10.3324/haematol.2023.283951
J Cell Biol. 2025 Feb 03. pii: e202311147. [Epub ahead of print]224(2):

The kinetochore protein KNL-1 regulates the actin cytoskeleton to control dendrite branching.

Henrique Alves Domingos, Mattie Green, Vasileios R Ouzounidis, Cameron Finlayson, Bram Prevo, Dhanya K Cheerambathur.

The function of the nervous system is intimately tied to its complex and highly interconnected architecture. Precise control of dendritic branching in individual neurons is central to building the complex structure of the nervous system. Here, we show that the kinetochore protein KNL-1 and its associated KMN (Knl1/Mis12/Ndc80 complex) network partners, typically known for their role in chromosome-microtubule coupling during mitosis, control dendrite branching in the Caenorhabditis elegans mechanosensory PVD neuron. KNL-1 restrains excess dendritic branching and promotes contact-dependent repulsion events, ensuring robust sensory behavior and preventing premature neurodegeneration. Unexpectedly, KNL-1 loss resulted in significant alterations of the actin cytoskeleton alongside changes in microtubule dynamics within dendrites. We show that KNL-1 modulates F-actin dynamics to generate proper dendrite architecture and that its N-terminus can initiate F-actin assembly. These findings reveal that the postmitotic neuronal KMN network acts to shape the developing nervous system by regulating the actin cytoskeleton and provide new insight into the mechanisms controlling dendrite architecture.

DOI: https://doi.org/10.1083/jcb.202311147
Biofabrication. 2024 Dec 02.

High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myeloid leukemia.

Magdalena Rudzinska-Radecka, Laura Turos-Korgul, Debjita Mukherjee, Paulina Podszywalow-Bartnicka, Katarzyna Piwocka, Jan Guzowski.

  Leukemic microenvironment has been recognized as a factor that strongly supports the mechanisms of resistance. Therefore, targeting the microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available leukemia 3D culture models, the reproducible generation of miniaturized leukemic microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use droplet microfluidics to generate tens of thousands of highly monodisperse leukemic-bone marrow microenvironments within minutes. We employ gelatin methacryloyl (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, check the impact of other components of the ECM (hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads to establish the optimal conditions for microtissue formation. We administer model kinase inhibitor, imatinib, at various concentrations to the encapsulated leukemic microtissues, and, via comparing mono- and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity. With that we demonstrate that our system mimics the physiological stroma-dependent protection. We discuss applicability of our model to (i) studying the role of direct- or close-contact interactions between the leukemia and bone marrow cells embedded in microscale 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized leukemia therapies.

Keywords:  3D cell models; cancer microenvironment; droplet microfluidics; drug resistance; extracellular matrix; myeloid leukemia; stroma-mediated protection

DOI:  https://doi.org/10.1088/1758-5090/ad998d
Development. 2024 Dec 01. pii: dev202924. [Epub ahead of print]151(23):

A Pax3 lineage gives rise to transient haematopoietic progenitors.

Giovanni Canu, Rosamaria Correra, Guillermo Diez-Pinel, Raphaël F P Castellan, Laura Denti, Alessandro Fantin, Christiana Ruhrberg.

  During embryonic development, muscle tissues, skin, and a subset of vascular endothelial cells arise from Pax3-expressing embryonic progenitors defined as paraxial mesoderm. By contrast, haemogenic potential is well established for extra-embryonic mesoderm and intra-embryonic lateral plate mesoderm, which do not express Pax3. To date, it is not known whether the haematopoietic system also contains Pax3 lineage cells. Here, we show that the mouse foetal liver and foetal circulation contain a transient population of Pax3 lineage cells with hallmarks of haematopoietic progenitors and the potential to generate both myeloid and erythroid cells. We propose that Pax3 lineage haematopoietic cells should be investigated to better understand normal haematopoietic development from different mesodermal derivatives. Further, genetic alterations of Pax3 lineage haematopoietic cells should be investigated for their potential to cause haematopoietic malignancies.

Keywords:  Foetal liver; Hematopoietic development; Macrophage origin; Mouse; Paraxial mesoderm; Pax3; Vascular endothelial cell

DOI:  https://doi.org/10.1242/dev.202924
Methods Mol Biol. 2025 ;2872 189-203

Systematic Identification of Microtubule Posttranslational Modification "Readers" by Quantitative Proteomics.

Takashi Hotta, Ryoma Ohi.

  Microtubules, dynamic polymers assembled from α, β-tubulin dimers, contribute to myriad cellular processes. This is largely attributed to microtubule-associated proteins (MAPs). How MAPs selectively bind microtubules to carry out various functions is not known. The "Tubulin Code" theory proposes that posttranslational modifications (PTMs) of microtubules serve as signs that can be read by specific MAPs, thereby conferring specific functional properties to the microtubules. In support of this hypothesis, "reader" MAPs have been identified for various tubulin PTMs, but, until recently, no systematic screening had been performed to identify readers in an unbiased manner. We addressed this by developing a reader identification pipeline that uses quantitative mass spectrometry to interrogate the microtubule proteome of cells programmed to express specific PTMs. This pipeline can be used to identify readers for any tubulin PTM from various cell types as long as the writer enzymes are known. We also provide an alternative, complementary approach to obtain modified microtubules using a generic writer enzyme in vitro.

Keywords:  Microtubule; Microtubule-associated proteins; Posttranslational modifications; Quantitative proteomics

DOI:  https://doi.org/10.1007/978-1-0716-4224-5_13