Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be...

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Veröffentlicht in:Haematologica (Roma) 2022-08, Vol.107 (8), p.1902-1913
Hauptverfasser: Lacey, Joanne, Webster, Simon J, Heath, Paul R, Hill, Chris J, Nicholson-Goult, Lucinda, Wagner, Bart E, Khan, Abdullah O, Morgan, Neil V, Makris, Michael, Daly, Martina E
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Sprache:eng
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Zusammenfassung:Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.
ISSN:0390-6078
1592-8721
DOI:10.3324/haematol.2021.279636