Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent ...morphology, and even differentiation 1–8. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant 9. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.
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•MSCs on soft matrix exhibit a less spread nucleus with low lamin-A,C levels•Lamin-A,C is rapidly phosphorylated in response to reduced cytoskeletal tension•Phosphorylation leads to nuclear softening and lamin-A,C turnover•Levels of lamin-A,C and myosin-IIA are coregulated in response to matrix elasticity
Buxboim et al. show that the nucleoskeletal protein lamin-A,C is phosphorylated upon relaxation of actomyosin tension on the nucleus. Mobility of lamin-A,C increases, as does protein turnover and nuclear flexibility. Downstream of lamin-A,C, the serum response factor (SRF) pathway feeds back into actomyosin expression and a contractile phenotype.
The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells
. This process requires major molecular adaptations to allow HSCs to meet ...the regulatory and metabolic requirements for cell division
. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)
, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
The cost of competency? Kao, Yun-Ruei; Will, Britta
Cell,
02/2023, Letnik:
186, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Curtailed protein translation ensures stemness and multipotency in embryonic and adult tissue-specific stem cells. In this issue of Cell, a study led by Zhao and colleagues uncovered increased ...susceptibility of hematopoietic stem cells (HSC) to iron-dependent programmed necrotic cell death (ferroptosis) as a consequence of low protein synthesis.
Curtailed protein translation ensures stemness and multipotency in embryonic and adult tissue-specific stem cells. In this issue of Cell, a study led by Zhao and colleagues uncovered increased susceptibility of hematopoietic stem cells (HSC) to iron-dependent programmed necrotic cell death (ferroptosis) as a consequence of low protein synthesis.
Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to ...AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell-focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation.
Eltrombopag (EP), a small-molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in ...patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. We investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO-R-deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Our mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Together, our study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation-mediated molecular reprogramming and indicates that labile iron pool-regulated pathways can modulate HSC function.
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant ...IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term ...maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron—particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.
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•Quiescent HSCs strongly restrict their pool of cytoplasmic labile iron•Regenerating HSCs activate iron homeostasis pathways•Iron homeostasis is functionalized for stem cell fate determination and relies on Tip60•Reversible modest iron loading drives the functional decline of aging HSCs
The preservation of adult stem cell identity following cell division is essential for sustained tissue maintenance and repair. Will, Kao, and colleagues identify iron as a crucial regulator allowing hematopoietic stem cells to orchestrate metabolic and gene regulatory control during regenerative fate determination.
•Ciclopirox Ethanolamine (CPX) is a regulator of HSCs and preserves the stem cell state in culture.•CPX-induced HSC maintenance is mediated by intracellular iron chelation, which restricts ...respiratory capacity and proliferation.
Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood (CB) cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC-specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.
Iron is an important bioelement essential for catalyzing various electron transfer reactions in cells. It occurs as ferric and ferrous forms mostly incorporated in iron-sulfur clusters, or bound to ...iron-binding proteins. Only a small fraction of transitory free iron is present at all times limiting the generation of hydroxyl radicals; the amount of this labile iron pool (LIP) depends on the cells’ energetic needs. Our resent study of iron homeostasis-regulatory pathways in hematopoietic stem cells (HSC) has uncovered a previously unrecognized key role of this molecular circuit in hematopoietic stem cell self-renewal and maintenance. Experimental transient reduction of the LIP, mimicking the reduction of free intracellular iron upon cell division, led to a significant two to three-fold increase in HSC numbers using in vivo and ex vivio models. Mechanistically, our study uncovered that a transient reduction of the LIP triggers a compensatory molecular response in HSCs which, besides upregulation of the transferrin receptor and induction of ferritinophagy to restore the LIP, also comprises lipolysis activation, increased fatty acid mobilization and oxidation. This cascade, in turn, reinforces molecular HSC states. Together, our study has uncovered a new iron-dependent rheostat mechanism aiding in sustaining HSC identify upon cell division.
Bone marrow resident and rarely dividing hematopoietic stem cells (HSC) harbor an extensive self-renewal capacity to sustain life-long blood formation, albeit their function declines during ageing. ...Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown.
We have previously uncovered that iron chelator exposure increases the number of functional HSC ex vivo and in vivo (Kao et al., Science Transl Med 2018). While ensuring a sufficient amount of redox active, readily available iron which is required in numerous electron transfer reactions governing fundamental cellular processes, cells tightly regulate the size of the intracellular labile iron pool (LIP) to limit adverse ROS generation. Perturbations in the ability to limit intracellular iron is detrimental for cells and known to compromise HSC maintenance and function via altered redox signaling and increased macromolecule oxidation and damage. The HSC stimulatory effects of iron chelator (IC) treatment and the well characterized central roles of redox active intracellular iron in sustaining basic cell function prompted us to examine a potential regulatory role of the LIP in controlling somatic stem cell function.
In this study, we quantified LIP in young and aged HSC and monitored iron homoeostasis pathway activation, hallmarked by the stabilization of transferrin receptor (Tfrc) mRNA, in stem cells for which we developed a single molecule RNA fluorescence in situ hybridization (smRNA FISH) assay enabling the quantification of Tfrc dynamics with unparalleled resolution and sensitivity. We have further used experimental LIP modulation in primary hematopoietic stem cell models to characterize the consequences of iron homeostasis pathway activation in young and aged stem cells; and employed integrated comparative quantitative transcriptomics (single cell RNA-seq) and proteomics along with genetic and pharmacological rescue models to identify the consequences and mechanisms of LIP size alterations. Our findings demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation.
Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent hematopoietic stem cell dysfunction in a wide range of degenerative and malignant hematologic pathologies.
D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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