Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity

Abstract

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.

Megakaryocyte derived immune-stimulating cells regulate host-defense immunity against bacterial pathogens

“…Furthermore, CXCR4 high MDICs egressed into circulation and infiltrated into peripheral organs, such as the liver, spleen, and lung. This is consistent with previous reports that MKs egress and migrate to the pulmonary capillary 12,37,93 , and CXCR4 navigates cell migration upon chemotactic stimuli 8,70 . HSCs like MK progenitors induce emergency megakaryopoiesis to generate mature MKs for platelet production 87 .…”

“…Moreover, we seldom observed CD41 + hematopoietic stem and progenitor cells (HSPCs) with a tighter gating strategy because of lower CD41 levels in HSCs, consistent with single-cell analysis showing rarely expressed HSC marker genes in MKs 65-68 (Supplemental Figure 1P, 2C). To assign the immune response and modulatory functionality to potential clusters, we identified surface protein and cytokinin encoded marker genes: Cdh1 (encoding CD324/E-cadherin) and Il6 enriched in MDICs (MK5), Ccr2 enriched in MK4 and MDICs (MK5), and high Cxcr4 expression in MDICs, compared to low Cxcr4 level in classic MKs 69,70 (Figure 1G and I). Single cells expressed a high level of Cxcr4 also enriched cell migration, immune-modulatory, and inflammatory response pathways (Supplemental Figure 1Q).…”

“…No reuse allowed without permission. Cxcr4 expression in MDICs, compared to low Cxcr4 level in classic MKs 69,70 (Figure 1G and I). Single cells expressed a high level of Cxcr4 also enriched cell migration, immunemodulatory, and inflammatory response pathways (Supplemental Figure 1Q).…”

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

“…6c, d). Importantly, the efficacy of Dynole 34-2 irrespective of activating mutations of growth factor-induced signalling could be explained by the importance of Dynamin for the CXCR4/CXCL12-mediated response to the microenvironment, which promotes survival of LSCs through the activation of the MAPK and AKT signalling pathways in acute leukemia 15,19,70 . Our data support previous reports confirming the efficacy of Dynole 34-2 to inhibit internalization of signalling receptors and downstream signal transduction in different malignancies 35,71 .…”

The role of pericytes in brain disorders: from the periphery to the brain

“…These genes may regulate cell adhesion and migration (Deem and Cook‐Mills ; Suraneni et al . ), but the consequences of their expression changes in hypertension remain to be determined.…”