The May-Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the Rho-ROCK pathway -- Chen et al. 110 (1): 171 Data Supplement -

Supplemental materials for: Chen et al -- Blood

Blood, Vol. 110, Issue 1, 171-179, July 1, 2007

The May-Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the Rho-ROCK pathway
Blood Chen et al. 110: 171

Supplemental materials for: Chen et al

Files in this Data Supplement:

Figure S1. Study of myosin-IIA function in MK maturation by ES cell differentiation (JPG, 53 KB) -
(A) Methylcellulose colony formation by progenitors derived from Myh9^+/− (red) and Myh9^−/− (gray) embryoid bodies (EBs) on differentiation day 6. Myh9^−/− ES cells show reduced myeloid differentiation and increased formation of secondary EBs. (B) Schematic representation of myosin-IIA constructs used in our studies. (C) Tubulin immunostaining of platelet-like particles released by Myh9^+/− ES differentiated MKs, showing the substantial variation in size and shape that precludes application of the model to study defects in platelet form per se. (D) Flow cytometry analysis of platelet-like particles released by wild type or Myh9-transduced fetal liver MKs. PLT, circulating platelets; WT, wild type; GF, GFP-full length Myh9; GX, GFP-R1933X. Particles that are positive for both CD61 and GFP (in GF, GX and GFP) or positive for both CD61 and CD41 (in WT, PLT) were used to back-gate the original scatter plot. The resulting size distributions of such double-positive particles and platelets are shown. The gate drawn to encompass 99.5% of circulating platelets is also applied to each of the other analyses.
Figure S2. Effects of exogenous myosin-IIA expression on proplatelet morphology (JPG, 93.8 KB) -
(A) Left, Assessment of full length myosin-IIA expression by retroviral transduction of pMIB-MYH9 in Myh9^−/− ES cells. Cells surviving blasticidin selection were expanded and lysates from equal numbers of Myh9^+/−, Myh9^−/− and transduced Myh9^−/− ES cells were compared by immunoblotting. Right, myosin-IIA immunoblot analysis to assess exogenous expression of GFP-tagged myosin-IIA rod domain in primary fetal liver-derived MKs. Shown is a representative result for total cell lysates from a culture in which 33% of MKs showed a GFP signal by flow cytometry. Correction of the signal density (NIH Image) for this transduction efficiency indicated ∼7-fold average overexpression compared to endogenous levels of myosin-IIA. (B) Exogenous expression of full length and R1933X in fetal liver MKs. Representative cells are shown, with GFP and phase-contrast images from the same microscopic field; a small area in each panel from the center row is enlarged in the row below to highlight characteristic morphology. Long, beaded projections from MKs expressing GFP contrast with grape-like clusters wrapping near the cell body when MKs express excess full-length myosin-IIA. A few such cells appear in MKs expressing the R1933X myosin-IIA mutant form identified in some patients with the May-Hegglin anomaly, whereas other MKs in the same cultures elaborate normal proplatelets.
Figure S3. Functional differences between myosin-IIA constructs assessed in HUVEC (JPG, 89.1 KB) -
GFP tagged myosin-IIA constructs were introduced in HUVEC by retroviral infection and their expression followed by green fluorescence. Actin stress fibers were stained by Alexa594-labeled phalloidin (red) and nuclei with DAPI (blue). Full-length myosin-IIA and the R1933X mutant seemed to incorporate normally into actin stress fibers. In contrast, stress fibers were reduced in cells expressing GFP-fused rod domain; neighboring untransduced cells showed intact actin stress fibers (red, arrows).
Figure S4. Regulation of PPF by MLC and Rho (JPG, 52.8 KB) -
(A) Assessment of exogenous MLC expression in fetal liver MKs by immunoblot analysis. In this representative experiment, flow cytometry estimated transduction efficiencies for D₁₈D₁₉-MLC and A₁₈A₁₉-MLC at 44% and 28%, respectively, resulting in average ∼1.7-fold overexpression of each. (B) Rescue of PPF in RhoAv14-expressing MKs by co-expression of A₁₈A₁₉-MLC. Compared to control GFP-transduced cells (arrowhead points to a representative proplatelet), mature RhoAv14-expressing MKs showed greatly reduced PPF. Proplatelets were restored upon double infection with RFP-A₁₈A₁₉-MLC and RhoAV14, as indicated again by arrowheads.
Figure S5. Regulation of platelet biogenesis by the Rho-ROCK-MLC-myosin pathway (JPG, 26.5 KB) -
A tentative model to illustrate the presumptively competing effects of extracellular factors such as collagen I (activating for Rho) and Sdf-1 (inhibitory toward Rho) on a signaling pathway that converges on myosin IIA-mediated restraints on PPF. Platelet release would be constitutively repressed early in MK ontogeny and enabled in the face of suitable cell maturation, when the Rho-ROCK-myosin pathway is inhibited. Our study helps define the intracellular pathway but candidate ligands and receptors are derived from the published literature or results that require further investigation.
Figure S6. Morphologic criteria for scoring normal and abnormal PPF (JPG, 59.4 KB) -
(A-C) Representative morphologies of abnormal PPF. A, MK expressing GFP-tagged full length myosin-IIA, showing a ruffled surface without proplatelets. B, MK expressing GFP-tagged wild type MLC, revealing short proplatelets and enlarged apparent diameter resulting from grape-like clusters surrounding the cell body. C, view of a larger microscope field showing multiple GFP-MLC expressing MKs with abnormal PPF similar to those seen in panel B. (D-F) Representative morphologies of normal PPF. D-E, GFP-expressing MKs showing typical long and thin proplatelet filaments with branches. F, view of a larger microscope field with multiple examples of MKs with typical PPF.