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Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors.sup.1-6. LEPR.sup.+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors.sup.7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin.sup.13,14, distinguishes peri-arteriolar LEPR.sup.+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR.sup.+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR.sup.+osteolectin.sup.+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin.sup.+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin.sup.+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin.sup.+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin.sup.+ cells depleted osteolectin.sup.+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing. A peri-arteriolar niche in the bone marrow for osteogenesis and lymphopoiesis is maintained by mechanical stimulation and is depleted during ageing., Author(s): Bo Shen [sup.1] , Alpaslan Tasdogan [sup.1] , Jessalyn M. Ubellacker [sup.1] , Jingzhu Zhang [sup.1] , Elena D. Nosyreva [sup.2] , Liming Du [sup.1] , Malea M. Murphy [...]

A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration that pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), the expression of which is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of hypoxia-induced factor 1a (HIF1[alpha]), which is induced in the setting of muscle damage. Sustained HIF1[alpha] signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of HIF1[alpha] allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint that regulates the speed of myofiber maturation in response to Mfn2 and HIF1[alpha] activity., Introduction In response to injury, skeletal muscle undergoes a synchronized sequence of events over several days, including clearance of muscle debris, revascularization of the damaged region, activation and proliferation of [...]

Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial β-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.

The study of protein-protein interactions represents a key aspect of biological research. Identifying unknown protein binding partners using mass spectrometry (MS)-based proteomics has evolved into an indispensable strategy in drug discovery. The classic approach of immunoprecipitation with specific antibodies against the proteins of interest has limitations, such as the need for immunoprecipitation-qualified antibody. The biotin AP-tag pull-down system has the advantage of high specificity, ease of use, and no requirement for antibody. It is based on the high specificity, high affinity interaction between biotin and streptavidin. After pulldown, in-gel tryptic digestion and tandem mass spectrometry (MS/MS) analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein bands can be performed. In this work, we provide protocols that can be used for the identification of proteins that interact with FOXM1, a protein that has recently emerged as a potential biomarker and drug target in oncotherapy, as an example. We focus on the pull-down procedure and assess the efficacy of the pulldown with known FOXM1 interactors such as β-catenin. We use a high performance LTQ Orbitrap MSn system that combines rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap analysis to identify the interacting proteins.