The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation

Abstract

Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments.Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liverderived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function. (Blood. 2009;114:5532-5540) IntroductionPlatelets develop from megakaryocytes (MKs), large polyploid cells localized in the bone marrow. Mature MKs rearrange their entire cytoplasm into pseudopod protrusions (designated proplatelets), a process that is driven by and dependent on microtubules (MTs). 1,2 MT filaments emanate from the cell cortex into the proplatelet tips, where they coil into a loop and later build the platelet marginal band. In wild-type platelets, this MT loop is normally coiled 8 to 12 times. 3 Ultrastructural data previously suggested that the platelet marginal bundle contains a single coiled filament, [3][4][5][6] and isolation of MT coils by platelet extraction affirmed this idea. However, the study by Patel-Hett et al 7 on living platelets revealed up to 7 free plus-ends, suggesting that several shorter MT filaments associate dynamically with one longer filament, forming a bipolar array.Each MT filament is composed of 13 protofilament heteropolymers of ␣-and ␤-tubulins. Mammalian genomes encode 6 ␣-tubulin and 5 ␤-tubulin genes. ␤1-tubulin (also referred to as class VI) is the most diverse isotype 8 and is found exclusively in cells that harbor a marginal band: MKs, platelets, and erythroblasts. 9 More than 90% of proplatelet MT filaments are composed of this ␤-tubulin isotype, 10 whose expression depends on the MK-specific transcription factor NF-E2. Genetically engineered mice that lack a functional Tubb1 gene are thrombocytopenic, and Tubb1 Ϫ/Ϫ platelets lack lentiform shape; the marginal band consists of only 2 to 3 coils, resulting in platelet spherocytosis. 11,12 Although ubiquitously expressed tubulin isotypes ␤2 and ␤5 are up-regulated, the equilibrium is shifted toward monomer...

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…Protein expression in platelets and megakaryocytes was studied by immuoblotting according to recently described protocols (Cherpokova et al, 2015;Kunert et al, 2009). …”

“…The area covered by at least 100 individual megakaryocytes was quantified for each group. Ploidy was determined as described previously (Kunert et al, 2009). The fraction of megakaryocytes that undergo proplatelet formation was determined by analyzing 80 to 120 cells per well at the indicated time points.…”

“…Fetal liver cells were differentiated as recently described (Kunert et al, 2009). Megakaryocytes were preincubated with antibodies against GPVI (JAQ1; Emfret Analytics) or anti-α2-integrin (LEN/B; Shida et al, 2014) at 10 μg/ml (10 min, 37°C) prior to seeding onto coated culture surfaces.…”

Platelet defects in congenital variant of Rett syndrome patients with FOXG1 mutations or reduced expression due to a position effect at 14q12

“…Alternatively, the presence of rounder and often larger platelets could also point to cytoskeletal defects, given the importance of the microtubule ring to maintain the characteristic platelet discoid shape. 22,47,48 However, cytoskeletal changes typically also affect platelet secretion. 26,47 Interestingly, an important role for the PI3K/AKT pathway has been shown to stimulate FOXO3A and FOXG1 phosphorylation via epinephrine or IGF1, respectively.…”

Getting in shape with RanBP10

“…4 Nearly half of all megakaryocytes isolated from these mice demonstrated shortened, discontinuous microtubulin filaments. Proplatelet formation in RanBP10 Ϫ/Ϫ megakaryocytes cultured in vitro was slightly impaired.…”