Mitochondrial Regulation Is Essential for Erythroid Nuclear Clearance

The terminal steps of red blood cell (RBC) generation involve an extensive cellular remodeling. This encompasses alterations of cellular content through five erythroblast stages that result in the expulsion of the nucleus (enucleation) followed by loss of mitochondria and all other organelles, and a transition to anaerobic glycolysis. Whether there is any link between the erythroid removal of the nucleus and the function of any other organelle including mitochondria remains unknown. Here we show that mitochondria are essential for nuclear clearance. We first demonstrate through high-throughput single-cell imaging and confocal microscopy that as mouse bone marrow erythroblasts mature (Gate 3: TER119⁺,CD44^low, FSC^low), mitochondria migrate to one end of the cell, aggregate and trail behind the nucleus as it extrudes from the cell, a prerequisite for enucleation to complete. We further show that mitochondrial localization behind the nucleus has similar kinetics as nuclear polarization. This process is also conserved in mouse fetal liver erythroid cells as well as in primary human CD34⁺-derived erythroblasts in culture. Notably, kinesin inhibition disrupts mitochondrial motility and localization and reduces significantly erythroblast enucleation rate in the absence of any impact on dynein or tubulin. These results suggest that mitochondria function as necessary chaperones during erythroblast enucleation. Furthermore, mitochondrial activity distinguishes erythroblasts on the verge of enucleation from others at the same erythroblast stage (Gate 3). We show that active mitochondrial respiration facilitates nuclear condensation and is required for nuclear extrusion. To our surprise however, metabolite profiling revealed that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation primarily through extracellular pyruvate but independently of glucose oxidation or anapleorotic reactions of amino acids. ¹³C-labeled metabolite tracing also confirmed pyruvate incorporation into mitochondria while glycolysis was minimal in orthochromatic erythroblasts. Thus, we provide evidence for the first time of a link between erythroid enucleation and mitochondrial metabolism. The process described establishes a model of mitochondrial compartmentalization within the cell for providing essential metabolites in a precise spatial and temporal manner. These findings are likely to improve the in vitro production of RBC and might be relevant to anemias of congenital mitochondrial disorders and aging.