In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field
SUMMARY Despite great advances in understanding the mechanisms underlying blood production, lineage specification at the level of multipotent progenitors (MPPs) remains poorly understood. Here, we show that MPP2 and MPP3 are distinct myeloid-biased MPP subsets that work together with lymphoid-primed MPP4 cells to control blood production. We find that all MPPs are produced in parallel by hematopoietic stem cells (HSCs), but with different kinetics and at variable levels depending on hematopoietic demands. We also show that the normally rare myeloid-biased MPPs are transiently overproduced by HSCs in regenerating conditions, hence supporting myeloid amplification to rebuild the hematopoietic system. This shift is accompanied by a reduction in self-renewal activity in regenerating HSCs and reprogramming of MPP4 fate towards the myeloid lineage. Our results support a dynamic model of blood development in which HSCs convey lineage specification through independent production of distinct lineage-biased MPP subsets that, in turn, support lineage expansion and differentiation.
While many aspects of blood production are now well understood, the spatial organization of myeloid differentiation in the bone marrow (BM) remains unknown. Here, we use imaging to track granulocyte/macrophage progenitor (GMP) behavior during emergency and leukemic myelopoiesis. At steady state, we find individual GMPs scattered throughout the BM. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. We describe how the timed release of important BM niche signals (SCF, IL-1β, G-CSF, TGF-β, CXCL4) and activation of an inducible Irf8/β-catenin progenitor self-renewal network controls the transient formation of regenerating GMP clusters. In leukemia, we show that GMP clusters are constantly produced due to persistent activation of the self-renewal network and lack of termination cytokines that normally restore stem cell quiescence. Our results uncover a previously unrecognized dynamic behavior of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukemia.
Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts.DOI: http://dx.doi.org/10.7554/eLife.17877.001
While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors.
Targeting commonly altered mechanisms in leukemia can provide additional treatment options. Here, we show that an inducible pathway of myeloid regeneration involving the remodeling of the multipotent progenitor (MPP) compartment downstream of hematopoietic stem cells (HSCs) is commonly hijacked in myeloid malignancies. We establish that differential regulation of Notch and Wnt signaling transiently triggers myeloid regeneration from HSCs in response to stress, and that constitutive low Notch and high Wnt activity in leukemic stem cells (LSCs) maintains this pathway activated in malignancies. We also identify compensatory crosstalk mechanisms between Notch and Wnt signaling that prevent damaging HSC function, MPP production, and blood output in conditions of high Notch and low Wnt activity. Finally, we demonstrate that restoring Notch and Wnt deregulated activity in LSCs attenuates disease progression. Our results uncover a mechanism that controls myeloid regeneration and early lineage decisions in HSCs and could be targeted in LSCs to normalize leukemic myeloid cell production.
The hematopoietic system is highly organized to maintain its functional integrity and to meet lifelong organismal demands. Hematopoietic stem cells (HSCs) must balance selfrenewal with differentiation and the regeneration of the blood system. It is a complex balancing act between these competing HSC functions. Although highly quiescent at steady state, HSCs become activated in response to inflammatory cytokines and regenerative challenges. This activation phase leads to many intrinsic stresses such as replicative, metabolic, and oxidative stress, which can cause functional decline, impaired self-renewal, and exhaustion of HSCs. To cope with these insults, HSCs use both built-in and emergency-triggered stress-response mechanisms to maintain homeostasis and to defend against disease development. In this review, we discuss how the hematopoietic system operates in steady state and stress conditions, what strategies are used to maintain functional integrity, and how deregulation in the balance between self-renewal and regeneration can drive malignant transformation.
Liposome-complexed adenoviral gene transfer appears to depend on lipid composition and the level of liposome uptake by cancer cells, in addition to CAR levels. Our study suggest that these multiple factors should be considered in designing liposome-complexed adenoviral vectors to improve outcomes of current adenoviral cancer gene therapies.
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