Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished β1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.
Cdc42 interacting protein 4 (CIP4) is a membrane-associated BAR protein, which also forms a complex via its SH3 domain with the dynamins (DNMs) and Wiskott-Aldrich Syndrome (WAS) protein. Thus, CIP4 remodels the plasma membrane and cortical actin cytoskeleton. To determine its physiological function, we generated CIP4-null mice. They displayed thrombocytopenia similar to that of WAS-null mice and have abnormal megakaryocytes (MKs) with decreased proplatelet formation and underdeveloped demarcation membrane system (DMS) (Chen et al, Blood 2013). The DMS is an extensive network of membrane tubules which serves as a membrane reservoir for proplatelet formation. The membranes are enriched for polyphosphoinositides that are docking sites for BAR proteins and for pleckstrin homology domain-containing proteins such as the dynamins. Still, the formation of the DMS is poorly understood. Dynamins are cell vesicle trafficking proteins that possess a GTPase domain. They induce neck vesicle constriction and scission from the plasma membrane. When the GTPase activity is abrogated, vesicle scission does not occur; instead, the plasma membrane invagination induced by the BAR proteins results in deep plasma membrane tubulations. Of the three dynamin isoforms, DNM3 participates in MK development including DMS formation (Reems et al, Exp Hematol 2008; Wang et al, Stem Cells Dev 2011). Moreover, a recent genome-wide association study suggested that an MK-specific DNM3 isoform might play a role in human platelet size determination (Nürnberg et al, Blood 2012). However the exact mechanism for dynamin’s participation in DMS formation is unclear. A double knockout for dynamin 1 and dynamin 3 in neurons causes accumulation of long invaginations from the plasma membrane (Ferguson and De Camilli, Nat Rev Mol Cell Biol 2012). We initially hypothesized that CIP4’s association with DNM3 contributes to the DMS development during platelet biogenesis and wanted to test for functional redundancy with other dynamins present in MKs and platelets. To determine if CIP4 interacts with dynamin in the MK lineage, we found that following either phorbol ester (PMA) or fibronectin stimulation in the human MK cell line CHRF-288, CIP4 co-precipitated with DNM3 and colocalized by confocal microscopy. To determine dynamin’s effect on membrane biophysical properties, we measured the fluorescence anisotropy, which reflects the disorder of membrane lipids due to movement and indicate membrane rigidity. Compared with controls in CHRF-288 cells, shRNA-mediated knockdown (KD) of DNM2 or DNM3 resulted in higher membrane rigidity in response to PMA. The strongest effect was seen in double KD cells with decreased fluidity by 2.6 ± 0.3%, which is similar to what was observed with CIP4 KD and is physiologically significant (Chen et al Blood 2013). KD of DNM2 resulted in aberrant morphology, greater cell diameter, and electron microscopy (EM) showed formation of new multivesicular bodies (MVBs) which are sorting compartments during α- and dense granules formation. Single DNM3 KD cells had no observable phenotype. EM imaging of DNM2 and DNM3 double KD cells revealed plasma membrane tubulation that resembles the DMS. While control CHRF-288 cells, with high DNM3 protein expression, do not have a DMS at baseline, MK cell lines Meg-01 and L8057, with respectively lower or no dynamin-3 protein expression, both have a DMS (Battinelli et al PNAS 2001; Ishida Y et al, Exp Hematol 1993). Platelet microparticles (MPs) are known to mediate a prothrombotic state in patients. Having previously found that CIP4-null mice show reduced levels of platelet MPs, we measured MPs in dynamin knockdown cell supernatant by flow cytometry and CD41/Annexin V staining. Surprisingly, we found that microparticle levels were increased 2.9-fold in DNM2 KD cells and 3.8-fold in double DNM2 and DNM3 KD cells. Our findings suggest that: 1) there is only partial functional redundancy between DNM2 and DNM3 in platelet biogenesis, 2) DNM2 controls MVB formation and MP release in MK cells, and 3) the CIP4-dynamin pathway contributes to DMS formation. It is possible that CIP4’s interaction with dynamins restrains their spatial and temporal activity to allow for long invaginations to accumulate in the DMS. Dynamin depletion might also increase surface membrane availability for MP formation. Dynamins are thus potential targets to modulate thrombotic state and platelet biogenesis. Disclosures: No relevant conflicts of interest to declare.
Megakaryocytes (MKs) undergo directional migration from the proliferative osteoblastic niche within the bone marrow (BM) environment to the capillary-rich vascular niche for platelet production and release into the pulmonary circulation. This process is regulated, in part by dynamins, large GTPase proteins that regulate cellular functions such as endocytosis, vesicle transport and cell migration. Additional functions of dynamins include the formation of actin-rich structures, such as lamellipodia and dorsal membrane ruffles, invadopodia and podosomes. Previous studies have shown that mutations in Dynamin 2 (DNM2) cause thrombocytopenia in humans. To explore the function of dynamins in megakaryocyte migration and platelet production in more depth, we monitored the response of cells to chemotaxis SDF1α gradient signal by a microfluidic device-based approach. We observed an impaired directional migration by both human megakaryocytic cell lines and primary cells treated either with dynasore, a small molecule inhibitor of dynamins, or shRNA knockdown of Dynamin 2 and 3 (DNM2, DNM3). Since directional cell migration is tightly regulated by actin cytoskeletal rearrangements, we next measured actin polymerization and RhoA activity. We observed a profound decrease in the F-actin and Rho GTPase activity upon loss of DNM2 and DNM3 function. Next, since the response to chemoattractant signal is navigated by SDF1 through its receptor CXCR4, we explored the CXCR4 receptor response to ligand in dynamin defective megakaryocytes. Interestingly we observed an increase in CXCR4 expression in the dynasore treated primary human cells. Additionally, combined inhibition of DNM2 and DNM3 or over expression of dominant negative Dnm2-K44A or GTPase-defective DNM3 decreased the active β1- integrin (ITGB1) activity, which indicates a decrease in the integrin mediated endo/exocytic cycling during cell migration. Finally, to understand the role of dynamin in endosome recycling, we assayed the distribution of Rab11, a marker of recycling endosomes. We noticed an abnormal clustered staining pattern of Rab11 in dynasore-treated MKs which is indicative of a disruption in recycling pathways. This observation suggests decreased recruitment of the recycling pathway in dynasore-treated cells. Altogether, in this study we demonstrate that dynamins regulate MKs directional migration towards the SDF1α chemotaxis signal in the bone marrow and governs endocytosis and cell receptor trafficking. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.