Figure 5.
Figure 5. Effect of microtubule assembly inhibitors on proplatelet elaboration. (Ai-x) Phase-contrast micrographs of mouse megakaryocytes grown for 20 hours in the (ii, viii) absence or (iii-vi, ix-xii) presence of microtubule inhibitors. (i-vii) Freshly plated megakaryocytes lack proplatelet extensions. Megakaryocytes cultured in the presence of (iii) 100 nM, (iv-v) 250 nM, and (vi) 1 μm nocodazole, or (ix-xi) 16 nM and (xii) 50 nM vinblastine. In control cultures, the cells become decorated with long proplatelets in 20 hours. Proplatelets were elaborated normally when the cells were cultured in (iii) 100 nM nocodazole and some proplatelets were found on cells cultured in either (iv-v) 250 nM nocodazole or (ix-xi) 16 nM vinblastine, although extensions are shorter and thicker compared with those elaborated in the absence of the inhibitors. Proplatelet formation is completely inhibited by (vi) 1 μm nocodazole or (xii) 50 nM vinblastine. Scale bar, 25 μm. (Bi) Effect of increasing concentrations of nocodazole on tubulin polymer levels in megakaryocytes. The graph compares the percentage of tubulin polymerized into microtubules in freshly plated megakaryocytes lacking proplatelets (∼ 55%) to megakaryocytes plated for 20 hours in the absence (control) or presence of 100 nM, 250 nM, and 1 μm nocodazole. Culturing of megakaryocytes for 20 hours in the absence or presence of 100 nM nocodazole resulted in an increase of total tubulin polymer to approximately 85% (a 25.9% increase from freshly plated cells). Tubulin polymer levels in megakaryocytes cultured in the presence of 250 nM nocodazole remained stable relative to the initial value, showing that 250 nM nocodazole acts as a kinetic stabilizer of microtubules. The tubulin polymer content of cultured megakaryocytes was decreased to approximately 40% by 1 μm nocodazole. (Bii) Proplatelet elongation is unaffected by 250 nM nocodazole. The rate of elongation was studied in 6 proplatelets before and after treatment with 250 nM nocodazole. Nocodazole was added after 30 minutes (arrow). (Biii) Effect of increasing concentrations of vinblastine on tubulin polymer levels in megakaryocytes. The graph compares the percentage of total tubulin polymerized into microtubules in freshly plated megakaryocytes lacking proplatelets (65%) to megakaryocytes plated for 20 hours in the absence (control) or presence of 16 nM and 50 nM vinblastine. Culturing of megakaryocytes for 20 hours increased the total tubulin polymer content of cells to approximately 90%. Megakaryocytes cultured in the presence of 16 nM and 50 nM vinblastine were unable to increase their polymer content or had diminished tubulin polymer contents, respectively. (Biv) Proplatelet elongation is unaffected by 16 nM vinblastine. The rate of elongation was studied in 6 proplatelets before and after treatment with 16 nM vinblastine, added at time 30 minutes (arrow). Error bars indicate standard deviations.

Effect of microtubule assembly inhibitors on proplatelet elaboration. (Ai-x) Phase-contrast micrographs of mouse megakaryocytes grown for 20 hours in the (ii, viii) absence or (iii-vi, ix-xii) presence of microtubule inhibitors. (i-vii) Freshly plated megakaryocytes lack proplatelet extensions. Megakaryocytes cultured in the presence of (iii) 100 nM, (iv-v) 250 nM, and (vi) 1 μm nocodazole, or (ix-xi) 16 nM and (xii) 50 nM vinblastine. In control cultures, the cells become decorated with long proplatelets in 20 hours. Proplatelets were elaborated normally when the cells were cultured in (iii) 100 nM nocodazole and some proplatelets were found on cells cultured in either (iv-v) 250 nM nocodazole or (ix-xi) 16 nM vinblastine, although extensions are shorter and thicker compared with those elaborated in the absence of the inhibitors. Proplatelet formation is completely inhibited by (vi) 1 μm nocodazole or (xii) 50 nM vinblastine. Scale bar, 25 μm. (Bi) Effect of increasing concentrations of nocodazole on tubulin polymer levels in megakaryocytes. The graph compares the percentage of tubulin polymerized into microtubules in freshly plated megakaryocytes lacking proplatelets (∼ 55%) to megakaryocytes plated for 20 hours in the absence (control) or presence of 100 nM, 250 nM, and 1 μm nocodazole. Culturing of megakaryocytes for 20 hours in the absence or presence of 100 nM nocodazole resulted in an increase of total tubulin polymer to approximately 85% (a 25.9% increase from freshly plated cells). Tubulin polymer levels in megakaryocytes cultured in the presence of 250 nM nocodazole remained stable relative to the initial value, showing that 250 nM nocodazole acts as a kinetic stabilizer of microtubules. The tubulin polymer content of cultured megakaryocytes was decreased to approximately 40% by 1 μm nocodazole. (Bii) Proplatelet elongation is unaffected by 250 nM nocodazole. The rate of elongation was studied in 6 proplatelets before and after treatment with 250 nM nocodazole. Nocodazole was added after 30 minutes (arrow). (Biii) Effect of increasing concentrations of vinblastine on tubulin polymer levels in megakaryocytes. The graph compares the percentage of total tubulin polymerized into microtubules in freshly plated megakaryocytes lacking proplatelets (65%) to megakaryocytes plated for 20 hours in the absence (control) or presence of 16 nM and 50 nM vinblastine. Culturing of megakaryocytes for 20 hours increased the total tubulin polymer content of cells to approximately 90%. Megakaryocytes cultured in the presence of 16 nM and 50 nM vinblastine were unable to increase their polymer content or had diminished tubulin polymer contents, respectively. (Biv) Proplatelet elongation is unaffected by 16 nM vinblastine. The rate of elongation was studied in 6 proplatelets before and after treatment with 16 nM vinblastine, added at time 30 minutes (arrow). Error bars indicate standard deviations.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal