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I Spy With My “Nano” Eye…

December 6, 2020


Dr. Xu presents his talk
“Phase Resolution in Erythropoiesis.”

Producing red cells on a large scale for clinical use has been fraught with challenges, necessitating further elucidation of the processes. A further understanding of the ultrastructure of both the mature erythrocyte cytoskeleton and the various stages of erythropoiesis is needed. The Scientific Program session “Location, Location, Location” (live Q&A Sunday, December 6, at 9:30 a.m. Pacific time) beautifully combines the new revelations in cytoskeletal ultrastructure using super-resolution microscopy, the molecular and structural basis for events of enucleation, and the interplay of transcriptional programs regulating the developmental waves of erythropoiesis. From advances in erythrocyte skeletal structure using confocal microscopy, electron microscopy, and atomic force microscopy, the speakers present a new characterization of the native ultrastructure using super resolution microscopy (3D STORM). In her talk, Dr. Velia Fowler suggests that using an approach almost akin to “reverse engineering” the location of protein complexes within the cell membrane, their ultrastructure, spatial positional relationships, and transcription factor activity, may reveal missing clues to further close the current knowledge gaps during erythropoiesis. Dr. Miguel R. Abboud chairs this very exciting and thought-provoking session.

Dr. Ke Xu presents the exciting recent delineation of native ultrastructure of the erythrocyte cytoskeleton using super-resolution microscopy. “The erythrocyte is a textbook prototype for the study of the submembrane of metazoan cells. It is anucleate and devoid of other organelles, extremely abundant, and easily accessible, and thus easy to study,” he explained. “Used as a prototype and the basis for a host of mathematically derived models, critical resolution and accuracy are therefore of utmost importance.” Current models of the erythrocyte cytoskeleton depict it as a two-dimensional structure made up of spectrin tetramers that connect at junctional complexes, consisting of actin filaments, tropomodulin, adducing, and protein 4.1.1 Mathematical estimates of the distance between the edges of the meshwork, suggested based on copy numbers of spectrin/actin, is approximately 70-80 nm, stated Dr. Xu. However, using prior methods of both electron microscopy and atomic force microscopy, that distance is reported as 200 nm.1 Dr. Xu and colleagues suggest that this distance is calculated based on the “stretched or extended” membrane cytoskeleton that occurs during preparation. “With advances in super-resolution fluorescence microscopy, we have new opportunities to probe intracellular structures at approximately 20 nm resolution with excellent molecular specificity and minimal sample processing,” said Dr, Xu. They report that the actual spacing is indeed 70-80 nm, not 200 nm, when studied in its native, relaxed state. Dr. Xu suggests a simple take-home message: “Higher resolution can give further insight and detailed structure. This application is relatively new to red cell rheology, and I think it is worth pursuing as it may have a broad clinical applicability.” I think we in the audience will agree.

Dr. Johan Flygare contributes a discussion of both published and unpublished work on reprogramming skin fibroblasts using transcription factors into erythroid cells. This system provides insight into the key transcription factors for erythropoiesis and sheds additional light on the obstacles in the scaling of ex vivo red cell production for transfusions. “Can we synchronize erythroid differentiation post-enucleation in a sustainable way?” he asks rhetorically.

In her presentation, Dr. Fowler critically appraises the molecular and structural basis for events of enucleation, with an eye on providing strategies for optimizing red cell production in vitro. She will discuss how the biogenesis of mammalian red blood cells is a highly orchestrated process of terminal differentiation with a series of cell divisions coupled to dramatic changes in cell and nuclear morphology, culminating in cell cycle exit and nuclear expulsion (enucleation). While enucleation has been assumed to be a type of asymmetric cell division, differences in cell polarity control and nanoscale organization of cytoskeletal structures indicate otherwise. Using updates in microscopy, Dr. Fowler draws our attention once again to “location, location, location.” She proposes that cell architecture controls the role and function. From the information provided by imaging, we may learn how they “work.” We can apply this thought process to the stages of erythropoiesis, in particular, how the orthochromic normoblast stage enucleates to the reticulocyte. Dr. Fowler’s enthusiasm for what is currently known in the field, and the yet unknown is glaring. While she admits that there is a variety of proposed models on the process of the erythrocyte enucleation, there is still a lot to learn. She leaves the audience with more thought-provoking suggestions and questions. First, we can glean a lot from looking. Improving imaging techniques will continue to provide higher resolutions of architectural detail. New detail in cellular ultrastructure will provide more functional clues. Second, Dr. Fowler encourages proposing questions — hypothesis generation — as a starting point, prior to seeking out “location.” Finally, she concludes, “look at how much we still do not know!”

As in the popular childhood game, “I spy, with my little eye,” we may take clues from revelations in ultramicroscopy. Advances in technology do beget discovery, hopefully thrusting the scientific community toward exponentially greater feats. They invite clinicians interested in inherited conditions of red cell membrane defects such as hereditary spherocytosis, ellipotcytosis, hemoglobinopathies, disorders of maturation arrest, and more, to ask questions and garner excitement for potential collaborations in various disease pathologies.

  1. Pan L, Yan R, Li W, et al. Super-resolution microscopy reveals the native ultrastructure of the erythrocyte cytoskeleton. Cell Rep. 2018;22:1151-1158.

 

Dr. Saah indicated no relevant conflicts of interest.

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